Structured Review

Roche immunoprecipitation buffer
CNTNAP2 interacts with CASK at the plasma membrane in cortical GABAergic interneurons (a) Only yeast cells co-expressing CNTNAP2 bait and CASK prey constructs grow on high stringency yeast plates (QDO/X/A). (b–c) Cropped western blots of <t>co-immunoprecipitation</t> experiments with CASK and CNTNAP2 in mouse cortex. (d) Cropped western blots showing co-immunoprecipitation of various FLAG-CNTNAP2 truncation mutants ( Supplementary Figure 5a ; red lines) with untagged, full-length CASK in HEK293T cells. (e) Representative cropped western blots of membrane/cytosol fractions of HEK293T cells expressing pCS2-FLAG + CASK-mCherry (CASK), FLAG-CNTNAP2 + CASK-mCherry (CNTNAP2 + CASK), or FLAG-CNTNAP2 + CASKΔPDZ-mCherry (CNTNAP2 + CASKΔPDZ) and subsequent quantification of protein localization (CASK vs. CNTNAP2 + CASK vs. CNTNAP2 + CASKΔPDZ: 6 independent experiments; CASK alone vs. CASKΔPDZ alone: 3 independent experiments). Percentages were calculated by dividing the densitometry value of CASK/CNTNAP2 in either membrane or cytosol fraction by the summation of both. (f) Representative SIM image of endogenous CNTNAP2 and CASK co-localization (white) on a GFP-transfected interneuronal dendrite (scale bar = 5 μm). (g) Histogram showing distribution of CASK/CNTNAP2 co-localized puncta relative to the dendrite’s lateral edge from (f) (CASK colocalized with CNTNAP2: n = 79 puncta from 3 cultures; CNTNAP2 colocalized with CASK: n = 70 puncta from 3 cultures). (h) Representative confocal image showing PLA signal from endogenous CASK/CNTNAP2 staining, which occurs only when CASK and CNTNAP2 primary antibodies are both applied (scale bar = 1 μm). (i) Cropped immunoblots of subcellular fractionations from adult mouse forebrain probed with CNTNAP2, CASK, and β-tubulin. CNTNAP2 and CASK, but not β-tubulin, are found in the washed membrane fraction (S5; red box). (j) Cropped western blot of time course and (k) quantification of CASK expression in cultured cortical neurons (n = 3 independent experiments). Values are means ± SEM. * P≤0.05, ** P≤0.01, *** P≤0.001; one-way ANOVA with Bonferroni’s correction (k, top graph; e). Student’s t-test (middle and bottom graphs; e).
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Images

1) Product Images from "CNTNAP2 stabilizes interneuron dendritic arbors through CASK"

Article Title: CNTNAP2 stabilizes interneuron dendritic arbors through CASK

Journal: Molecular psychiatry

doi: 10.1038/s41380-018-0027-3

CNTNAP2 interacts with CASK at the plasma membrane in cortical GABAergic interneurons (a) Only yeast cells co-expressing CNTNAP2 bait and CASK prey constructs grow on high stringency yeast plates (QDO/X/A). (b–c) Cropped western blots of co-immunoprecipitation experiments with CASK and CNTNAP2 in mouse cortex. (d) Cropped western blots showing co-immunoprecipitation of various FLAG-CNTNAP2 truncation mutants ( Supplementary Figure 5a ; red lines) with untagged, full-length CASK in HEK293T cells. (e) Representative cropped western blots of membrane/cytosol fractions of HEK293T cells expressing pCS2-FLAG + CASK-mCherry (CASK), FLAG-CNTNAP2 + CASK-mCherry (CNTNAP2 + CASK), or FLAG-CNTNAP2 + CASKΔPDZ-mCherry (CNTNAP2 + CASKΔPDZ) and subsequent quantification of protein localization (CASK vs. CNTNAP2 + CASK vs. CNTNAP2 + CASKΔPDZ: 6 independent experiments; CASK alone vs. CASKΔPDZ alone: 3 independent experiments). Percentages were calculated by dividing the densitometry value of CASK/CNTNAP2 in either membrane or cytosol fraction by the summation of both. (f) Representative SIM image of endogenous CNTNAP2 and CASK co-localization (white) on a GFP-transfected interneuronal dendrite (scale bar = 5 μm). (g) Histogram showing distribution of CASK/CNTNAP2 co-localized puncta relative to the dendrite’s lateral edge from (f) (CASK colocalized with CNTNAP2: n = 79 puncta from 3 cultures; CNTNAP2 colocalized with CASK: n = 70 puncta from 3 cultures). (h) Representative confocal image showing PLA signal from endogenous CASK/CNTNAP2 staining, which occurs only when CASK and CNTNAP2 primary antibodies are both applied (scale bar = 1 μm). (i) Cropped immunoblots of subcellular fractionations from adult mouse forebrain probed with CNTNAP2, CASK, and β-tubulin. CNTNAP2 and CASK, but not β-tubulin, are found in the washed membrane fraction (S5; red box). (j) Cropped western blot of time course and (k) quantification of CASK expression in cultured cortical neurons (n = 3 independent experiments). Values are means ± SEM. * P≤0.05, ** P≤0.01, *** P≤0.001; one-way ANOVA with Bonferroni’s correction (k, top graph; e). Student’s t-test (middle and bottom graphs; e).
Figure Legend Snippet: CNTNAP2 interacts with CASK at the plasma membrane in cortical GABAergic interneurons (a) Only yeast cells co-expressing CNTNAP2 bait and CASK prey constructs grow on high stringency yeast plates (QDO/X/A). (b–c) Cropped western blots of co-immunoprecipitation experiments with CASK and CNTNAP2 in mouse cortex. (d) Cropped western blots showing co-immunoprecipitation of various FLAG-CNTNAP2 truncation mutants ( Supplementary Figure 5a ; red lines) with untagged, full-length CASK in HEK293T cells. (e) Representative cropped western blots of membrane/cytosol fractions of HEK293T cells expressing pCS2-FLAG + CASK-mCherry (CASK), FLAG-CNTNAP2 + CASK-mCherry (CNTNAP2 + CASK), or FLAG-CNTNAP2 + CASKΔPDZ-mCherry (CNTNAP2 + CASKΔPDZ) and subsequent quantification of protein localization (CASK vs. CNTNAP2 + CASK vs. CNTNAP2 + CASKΔPDZ: 6 independent experiments; CASK alone vs. CASKΔPDZ alone: 3 independent experiments). Percentages were calculated by dividing the densitometry value of CASK/CNTNAP2 in either membrane or cytosol fraction by the summation of both. (f) Representative SIM image of endogenous CNTNAP2 and CASK co-localization (white) on a GFP-transfected interneuronal dendrite (scale bar = 5 μm). (g) Histogram showing distribution of CASK/CNTNAP2 co-localized puncta relative to the dendrite’s lateral edge from (f) (CASK colocalized with CNTNAP2: n = 79 puncta from 3 cultures; CNTNAP2 colocalized with CASK: n = 70 puncta from 3 cultures). (h) Representative confocal image showing PLA signal from endogenous CASK/CNTNAP2 staining, which occurs only when CASK and CNTNAP2 primary antibodies are both applied (scale bar = 1 μm). (i) Cropped immunoblots of subcellular fractionations from adult mouse forebrain probed with CNTNAP2, CASK, and β-tubulin. CNTNAP2 and CASK, but not β-tubulin, are found in the washed membrane fraction (S5; red box). (j) Cropped western blot of time course and (k) quantification of CASK expression in cultured cortical neurons (n = 3 independent experiments). Values are means ± SEM. * P≤0.05, ** P≤0.01, *** P≤0.001; one-way ANOVA with Bonferroni’s correction (k, top graph; e). Student’s t-test (middle and bottom graphs; e).

Techniques Used: Expressing, Construct, Western Blot, Immunoprecipitation, Transfection, Proximity Ligation Assay, Staining, Cell Culture

2) Product Images from "Sorting nexin-21 is a scaffold for the endosomal recruitment of huntingtin"

Article Title: Sorting nexin-21 is a scaffold for the endosomal recruitment of huntingtin

Journal: Journal of Cell Science

doi: 10.1242/jcs.211672

Co-immunoprecipitation of Htt with SNX21 requires negatively charged residues in the SNX21 N-terminus, but does not require SNX21 to be endosomally localised. (A) Adaptation of SNX20 and SNX21 protein alignment previously generated by Clairfeuille and colleagues ( Clairfeuille et al., 2015 ) . Green boxed regions represent clusters of negatively charged amino acids not present in the SNX20 N-terminal extension. Red boxes denote conserved amino acids within SNX20 and SNX21 sequences and asterisks count every ten residues starting with the fist methionine of SNX20. (B) HEK293-T cells were transiently transfected to express GFP, GFP-tagged full-length SNX21 and two truncation mutants representing the two halves of the N-terminal region of SNX21. Precipitates from the GFP-nanotrap-isolated variants were analysed by western blotting and demonstrate the necessity for a full N-terminal extension in order to facilitate Htt binding. Data are representative of three biological replicates. (C) Site-directed mutagenesis was used to engineer a variety of charge swap mutants targeting the negatively charged clusters of amino acids, prior to probing for Htt binding as above. Two aspartic acid residues in the first N-terminal cluster are essential for precipitation of Htt with SNX21. (D) Both the point mutated GFP-SNX21 and truncation variants were expressed in HeLa cells prior to fixation and immunolabelling with anti-EEA1. Except for the N-terminal 1-129 construct, which lacks a PX domain, all mutants retained an endosomal localisation. Scale bars: 20 µm.
Figure Legend Snippet: Co-immunoprecipitation of Htt with SNX21 requires negatively charged residues in the SNX21 N-terminus, but does not require SNX21 to be endosomally localised. (A) Adaptation of SNX20 and SNX21 protein alignment previously generated by Clairfeuille and colleagues ( Clairfeuille et al., 2015 ) . Green boxed regions represent clusters of negatively charged amino acids not present in the SNX20 N-terminal extension. Red boxes denote conserved amino acids within SNX20 and SNX21 sequences and asterisks count every ten residues starting with the fist methionine of SNX20. (B) HEK293-T cells were transiently transfected to express GFP, GFP-tagged full-length SNX21 and two truncation mutants representing the two halves of the N-terminal region of SNX21. Precipitates from the GFP-nanotrap-isolated variants were analysed by western blotting and demonstrate the necessity for a full N-terminal extension in order to facilitate Htt binding. Data are representative of three biological replicates. (C) Site-directed mutagenesis was used to engineer a variety of charge swap mutants targeting the negatively charged clusters of amino acids, prior to probing for Htt binding as above. Two aspartic acid residues in the first N-terminal cluster are essential for precipitation of Htt with SNX21. (D) Both the point mutated GFP-SNX21 and truncation variants were expressed in HeLa cells prior to fixation and immunolabelling with anti-EEA1. Except for the N-terminal 1-129 construct, which lacks a PX domain, all mutants retained an endosomal localisation. Scale bars: 20 µm.

Techniques Used: Immunoprecipitation, Generated, Transfection, Isolation, Western Blot, Binding Assay, Mutagenesis, Construct

Co-immunoprecipitation of septins with SNX21 requires a surface exposed leucine in the PXB domain. (A) HEK293-T cells were transiently transfected with constructs encoding GFP, GFP-SNX20, GFP-SNX21 and various SNX21 point mutants. After GFP-nanotrap immunoisolation, precipitates were analysed by SDS-PAGE and western blotting. GFP-SNX21 precipitates both septin 2 and septin 7, an interaction that occurs via the SNX21 PXB domain and appears to require the endosomal localisation of SNX21. Data are representative of three biological replicates. (B) Amino acid residues mutated in the current study mapped onto the published structure of the mouse SNX21 ( Clairfeuille et al., 2015 ). (C) Site-directed mutagenesis of the SNX21 PXB domain, targeting predicted surface exposed residues. Constructs encoding GFP-tag chimeras of the various SNX21 mutants were transiently expressed in HEK293-T cells prior to GFP-nanotrap, SDS-PAGE and western blotting. Mutation of an evolutionarily conserved leucine (L363A) and a neighbouring lysine (K364E) is sufficient to perturb association with both septin 2 and septin 7. Data are representative of three biological replicates. (D) GFP-SNX21 mutants were expressed in HeLa cells prior to fixation and immunolabelling with anti-EEA1. Each of the mutants analysed retained an endosomal localisation in accordance with the wild-type protein. Scale bars: 20 µm.
Figure Legend Snippet: Co-immunoprecipitation of septins with SNX21 requires a surface exposed leucine in the PXB domain. (A) HEK293-T cells were transiently transfected with constructs encoding GFP, GFP-SNX20, GFP-SNX21 and various SNX21 point mutants. After GFP-nanotrap immunoisolation, precipitates were analysed by SDS-PAGE and western blotting. GFP-SNX21 precipitates both septin 2 and septin 7, an interaction that occurs via the SNX21 PXB domain and appears to require the endosomal localisation of SNX21. Data are representative of three biological replicates. (B) Amino acid residues mutated in the current study mapped onto the published structure of the mouse SNX21 ( Clairfeuille et al., 2015 ). (C) Site-directed mutagenesis of the SNX21 PXB domain, targeting predicted surface exposed residues. Constructs encoding GFP-tag chimeras of the various SNX21 mutants were transiently expressed in HEK293-T cells prior to GFP-nanotrap, SDS-PAGE and western blotting. Mutation of an evolutionarily conserved leucine (L363A) and a neighbouring lysine (K364E) is sufficient to perturb association with both septin 2 and septin 7. Data are representative of three biological replicates. (D) GFP-SNX21 mutants were expressed in HeLa cells prior to fixation and immunolabelling with anti-EEA1. Each of the mutants analysed retained an endosomal localisation in accordance with the wild-type protein. Scale bars: 20 µm.

Techniques Used: Immunoprecipitation, Transfection, Construct, SDS Page, Western Blot, Mutagenesis

3) Product Images from "Regulation of Sufu activity by p66β and Mycbp provides new insight into vertebrate Hedgehog signaling"

Article Title: Regulation of Sufu activity by p66β and Mycbp provides new insight into vertebrate Hedgehog signaling

Journal: Genes & Development

doi: 10.1101/gad.249425.114

Hedgehog signaling leads to reduced nuclear Sufu protein levels and Sufu dissociation from Gli2 and Gli3 primarily in the nucleus. ( A ) Western blot analysis and quantification of Sufu protein levels in the nuclear and cytoplasmic fractions derived from MEFs treated with Shh-conditioned medium. Cytoplasmic tubulin and nuclear lamin A were used to assess the purity of cytoplasmic and nuclear fractions. Nuclear Sufu protein levels were reduced by 60% upon Hh pathway activation, while cytoplasmic Sufu levels were largely unaltered. ( B ) Western blot analysis and quantification of Sufu protein levels in the nuclear and cytoplasmic fractions in the presence of Shh-conditioned medium and proteasome inhibitor MG132. Nuclear Sufu protein levels were notably restored upon MG132 addition. ( C ) Western blot analysis and quantification of immunoprecipitated Sufu, Gli2, and Gli3 using lysates from the nuclear and cytoplasmic fractions derived from MEFs expressing Flag-tagged Sufu. The amount of coimmunoprecipitated Gli2 and Gli3 by Sufu was significantly reduced in the nuclear fraction but only marginally decreased in the cytoplasmic fraction at indicated time points after Hh stimulation. (In) Input; (IP) immunoprecipitation. (*) P
Figure Legend Snippet: Hedgehog signaling leads to reduced nuclear Sufu protein levels and Sufu dissociation from Gli2 and Gli3 primarily in the nucleus. ( A ) Western blot analysis and quantification of Sufu protein levels in the nuclear and cytoplasmic fractions derived from MEFs treated with Shh-conditioned medium. Cytoplasmic tubulin and nuclear lamin A were used to assess the purity of cytoplasmic and nuclear fractions. Nuclear Sufu protein levels were reduced by 60% upon Hh pathway activation, while cytoplasmic Sufu levels were largely unaltered. ( B ) Western blot analysis and quantification of Sufu protein levels in the nuclear and cytoplasmic fractions in the presence of Shh-conditioned medium and proteasome inhibitor MG132. Nuclear Sufu protein levels were notably restored upon MG132 addition. ( C ) Western blot analysis and quantification of immunoprecipitated Sufu, Gli2, and Gli3 using lysates from the nuclear and cytoplasmic fractions derived from MEFs expressing Flag-tagged Sufu. The amount of coimmunoprecipitated Gli2 and Gli3 by Sufu was significantly reduced in the nuclear fraction but only marginally decreased in the cytoplasmic fraction at indicated time points after Hh stimulation. (In) Input; (IP) immunoprecipitation. (*) P

Techniques Used: Western Blot, Derivative Assay, Activation Assay, Immunoprecipitation, Expressing

A proteomic approach to identify Sufu-interacting proteins uncovers p66β and Mycbp. ( A ) Coomassie Blue-stained gel of control and Sufu immunoprecipitates treated with mock- or Shh-conditioned medium. Distinct bands were detected and were candidates for new Sufu-interacting proteins. Numbers at the right indicate locations of protein size standards. Large-scale immunoprecipitation (IP) and mass spectrometry were performed to identify new Sufu-interacting proteins and Sufu phosphorylation sites. Mass spectrometric analysis was performed directly on immunoprecipitates or specific bands cut out from SDS-PAGE gels. Immunoprecipitation and mass spectrometric analysis were repeated multiple times to eliminate nonspecific Sufu-binding proteins. ( B ) Western blot analysis of Sufu immunoprecipitates probed with anti-Sufu, anti-Gli2, and Gli3 antibodies. Endogenous Gli2 and Gli3 were detected in Sufu immunoprecipitates (but not in the control), suggesting that a physiologically relevant protein complex was pulled down. ( C ). ( D ) Western blot analysis of proteins pulled down by Sufu from lysates expressing Sufu and the indicated proteins, which were epitope-tagged. Both p66β and Mycbp physically interacted with Sufu by coimmunoprecipitation. Fu and Prc1 served as negative controls. ( E , top panels) Western blot analysis of Sufu immunoprecipitates using lysates derived from MEFs expressing Flag-tagged Sufu. Endogenous p66β and HDAC1 were coimmunoprecipitated. In contrast, HDAC2 and RBBP7/4 could not be detected in Sufu immunoprecipitates. ( Bottom panels) Western blot analysis of endogenous Sufu immunoprecipitated by an anti-Sufu antibody. p66β was coimmunoprecipitated by Sufu in wild-type MEFs but not in Sufu -deficient MEFs. p66β/Sufu interaction was not altered by Hh stimulation (Supplemental Fig. S6). ( F–H ) Immunofluorescence studies to assess the subcellular distribution of p66β and Mycbp. MEFs were transfected or transduced with p66β- and Mycbp-expressing constructs. p66β and Mycbp localized to the nucleus (marked by DAPI) of Hh-responsive cells. Cytoplasmic expression of Mycbp was also detected. Acetylated (Ac)-tubulin marks the primary cilium. Interestingly, Mycbp immunoreactivity can also be detected at the base of the cilium (white arrow).
Figure Legend Snippet: A proteomic approach to identify Sufu-interacting proteins uncovers p66β and Mycbp. ( A ) Coomassie Blue-stained gel of control and Sufu immunoprecipitates treated with mock- or Shh-conditioned medium. Distinct bands were detected and were candidates for new Sufu-interacting proteins. Numbers at the right indicate locations of protein size standards. Large-scale immunoprecipitation (IP) and mass spectrometry were performed to identify new Sufu-interacting proteins and Sufu phosphorylation sites. Mass spectrometric analysis was performed directly on immunoprecipitates or specific bands cut out from SDS-PAGE gels. Immunoprecipitation and mass spectrometric analysis were repeated multiple times to eliminate nonspecific Sufu-binding proteins. ( B ) Western blot analysis of Sufu immunoprecipitates probed with anti-Sufu, anti-Gli2, and Gli3 antibodies. Endogenous Gli2 and Gli3 were detected in Sufu immunoprecipitates (but not in the control), suggesting that a physiologically relevant protein complex was pulled down. ( C ). ( D ) Western blot analysis of proteins pulled down by Sufu from lysates expressing Sufu and the indicated proteins, which were epitope-tagged. Both p66β and Mycbp physically interacted with Sufu by coimmunoprecipitation. Fu and Prc1 served as negative controls. ( E , top panels) Western blot analysis of Sufu immunoprecipitates using lysates derived from MEFs expressing Flag-tagged Sufu. Endogenous p66β and HDAC1 were coimmunoprecipitated. In contrast, HDAC2 and RBBP7/4 could not be detected in Sufu immunoprecipitates. ( Bottom panels) Western blot analysis of endogenous Sufu immunoprecipitated by an anti-Sufu antibody. p66β was coimmunoprecipitated by Sufu in wild-type MEFs but not in Sufu -deficient MEFs. p66β/Sufu interaction was not altered by Hh stimulation (Supplemental Fig. S6). ( F–H ) Immunofluorescence studies to assess the subcellular distribution of p66β and Mycbp. MEFs were transfected or transduced with p66β- and Mycbp-expressing constructs. p66β and Mycbp localized to the nucleus (marked by DAPI) of Hh-responsive cells. Cytoplasmic expression of Mycbp was also detected. Acetylated (Ac)-tubulin marks the primary cilium. Interestingly, Mycbp immunoreactivity can also be detected at the base of the cilium (white arrow).

Techniques Used: Staining, Immunoprecipitation, Mass Spectrometry, SDS Page, Binding Assay, Western Blot, Expressing, Derivative Assay, Immunofluorescence, Transfection, Transduction, Construct

4) Product Images from "The Capsicum annuum class IV chitinase ChitIV interacts with receptor-like cytoplasmic protein kinase PIK1 to accelerate PIK1-triggered cell death and defence responses"

Article Title: The Capsicum annuum class IV chitinase ChitIV interacts with receptor-like cytoplasmic protein kinase PIK1 to accelerate PIK1-triggered cell death and defence responses

Journal: Journal of Experimental Botany

doi: 10.1093/jxb/erv001

CaPIK1 interacts with CaChitIV in yeast and in planta. (A) Interaction of CaPIK1 with CaChitIV in a GAL4-based yeast two-hybrid system. Negative control: combination of human lamin C (BD/Lam) and SV40 large T antigen (AD/SV40-T) fusion constructs. Positive control: combination of murine p53 (BD/p53) and SV40 large T antigen (AD/SV40-T) fusion constructs. BD, GAL4 DNA-binding domain; AD, GAL4 activation domain; SD-LT, synthetic dropout medium lacking leucine (L) and tryptophan (T); SD-AHLT X-α-gal, synthetic dropout medium lacking adenine (A), histidine (H), leucine (L), and tryptophan (T) supplemented with X-α-Gal to monitor reporter gene expression. (B) Bimolecular fluorescent complementation (BiFC) analysis of the CaPIK1–CaChitIV interaction in Nicotiana benthamiana . Confocal images were taken from leaf epidermal cells; bZIP63 -YFP N and bZIP63 -YFP C constructs were used as positive controls. Scale bars=50 μm. (C) Co-immunoprecipitation (Co-IP) and immunoblot (IB) analyses of CaChitIV-cMyc and CaPIK1-HA co-expressed in N. benthamiana leaves. Protein loading is shown by Coomassie brilliant blue (CBB) staining. α-cMyc, cMyc antibody; α-HA, HA antibody. (This figure is available in colour at JXB online.)
Figure Legend Snippet: CaPIK1 interacts with CaChitIV in yeast and in planta. (A) Interaction of CaPIK1 with CaChitIV in a GAL4-based yeast two-hybrid system. Negative control: combination of human lamin C (BD/Lam) and SV40 large T antigen (AD/SV40-T) fusion constructs. Positive control: combination of murine p53 (BD/p53) and SV40 large T antigen (AD/SV40-T) fusion constructs. BD, GAL4 DNA-binding domain; AD, GAL4 activation domain; SD-LT, synthetic dropout medium lacking leucine (L) and tryptophan (T); SD-AHLT X-α-gal, synthetic dropout medium lacking adenine (A), histidine (H), leucine (L), and tryptophan (T) supplemented with X-α-Gal to monitor reporter gene expression. (B) Bimolecular fluorescent complementation (BiFC) analysis of the CaPIK1–CaChitIV interaction in Nicotiana benthamiana . Confocal images were taken from leaf epidermal cells; bZIP63 -YFP N and bZIP63 -YFP C constructs were used as positive controls. Scale bars=50 μm. (C) Co-immunoprecipitation (Co-IP) and immunoblot (IB) analyses of CaChitIV-cMyc and CaPIK1-HA co-expressed in N. benthamiana leaves. Protein loading is shown by Coomassie brilliant blue (CBB) staining. α-cMyc, cMyc antibody; α-HA, HA antibody. (This figure is available in colour at JXB online.)

Techniques Used: Negative Control, Laser Capture Microdissection, Construct, Positive Control, Binding Assay, Activation Assay, Expressing, Bimolecular Fluorescence Complementation Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Staining

5) Product Images from "Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity"

Article Title: Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.2548-14.2015

NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.
Figure Legend Snippet: NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.

Techniques Used: Western Blot, Immunoprecipitation, Molecular Weight, Recombinant, Transfection, Immunofluorescence, Confocal Microscopy, Isolation, Negative Control, Marker, Cell Culture

6) Product Images from "Identification of a Novel Recycling Sequence in the C-tail of FPR2/ALX Receptor"

Article Title: Identification of a Novel Recycling Sequence in the C-tail of FPR2/ALX Receptor

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.612630

Attenuated recycling leads to lysosomal targeting and receptor down-regulation. A , HEK293 cells expressing N-terminally FLAG-tagged N333-stop and EGFP-tagged β-Arr2 were fed with anti-FLAG M1 antibody as in Fig. 2 , treated with W peptide, and then fixed, permeabilized, and incubated with secondary antibody and visualized using confocal microscopy. Representative images are shown with scale bars equal to 20 μm, and arrows indicate examples of co-localization. B , quantification of β-Arr2 association with FPR2 and N333-stop ( N333 ) at 30-min W peptide treatment expressed as Pearson's correlation. Data are represented as the mean co-localization of at least 30 cells performed on three separate occasions and analyzed using one-way ANOVA with Bonferroni's t test where ****, p ≤ 0.001 when compared with WT FPR2. E rror bars indicate mean ± S.D. C , HEK293 cells stably expressing FPR2 or N333-stop were labeled with anti-FLAG M1 antibody and LysoTracker and incubated for 90 min with 500 n m W peptide. Representative confocal images are shown with scale bars equal to 20 μm, and arrows indicate the lysosomal compartment. D , HEK293 cells stably expressing FPR2, N333-stop ( N333 ), P342-stop ( P343 ), or T346-stop ( T346 ) were surface-biotinylated and either untreated or stimulated with 500 n m W peptide for 30, 90, or 180 min. Receptor fate was assessed after immunoprecipitation with anti-FLAG M2 antibody, subsequent separation by SDS-PAGE electrophoresis, and streptavidin overlay. The 100% lane shows total surface receptor labeling, and the STRIP lane indicates the efficiency of the biotin cleavage. NT , non-treatment. Representative immunoblots are shown.
Figure Legend Snippet: Attenuated recycling leads to lysosomal targeting and receptor down-regulation. A , HEK293 cells expressing N-terminally FLAG-tagged N333-stop and EGFP-tagged β-Arr2 were fed with anti-FLAG M1 antibody as in Fig. 2 , treated with W peptide, and then fixed, permeabilized, and incubated with secondary antibody and visualized using confocal microscopy. Representative images are shown with scale bars equal to 20 μm, and arrows indicate examples of co-localization. B , quantification of β-Arr2 association with FPR2 and N333-stop ( N333 ) at 30-min W peptide treatment expressed as Pearson's correlation. Data are represented as the mean co-localization of at least 30 cells performed on three separate occasions and analyzed using one-way ANOVA with Bonferroni's t test where ****, p ≤ 0.001 when compared with WT FPR2. E rror bars indicate mean ± S.D. C , HEK293 cells stably expressing FPR2 or N333-stop were labeled with anti-FLAG M1 antibody and LysoTracker and incubated for 90 min with 500 n m W peptide. Representative confocal images are shown with scale bars equal to 20 μm, and arrows indicate the lysosomal compartment. D , HEK293 cells stably expressing FPR2, N333-stop ( N333 ), P342-stop ( P343 ), or T346-stop ( T346 ) were surface-biotinylated and either untreated or stimulated with 500 n m W peptide for 30, 90, or 180 min. Receptor fate was assessed after immunoprecipitation with anti-FLAG M2 antibody, subsequent separation by SDS-PAGE electrophoresis, and streptavidin overlay. The 100% lane shows total surface receptor labeling, and the STRIP lane indicates the efficiency of the biotin cleavage. NT , non-treatment. Representative immunoblots are shown.

Techniques Used: Expressing, Incubation, Confocal Microscopy, Stable Transfection, Labeling, Immunoprecipitation, SDS Page, Electrophoresis, Stripping Membranes, Western Blot

7) Product Images from "Trithorax Group Protein Oryza sativa Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] [OPEN]"

Article Title: Trithorax Group Protein Oryza sativa Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] [OPEN]

Journal: Plant Physiology

doi: 10.1104/pp.113.228049

transiently expressing OsTrx1-HA and Ehd3-Myc. Extracts were immunoprecipitated with anti-Myc antibody (B) or anti-HA antibody (C), and signals were detected by SDS-PAGE using anti-HA antibody (B) or anti-Myc antibody (C). Inputs are extracts before immunoprecipitation, and IP indicates elutes from agarose beads after immunoprecipitation. Plus and minus signs indicate constructs introduced into protoplasts. D, Identification of the OsTrx1 motif that interacts with Ehd3. Expression of truncated OsTrx1-HA (top), expression of Ehd3-Myc (middle), and interaction between two proteins after coimmunoprecipitation (bottom).
Figure Legend Snippet: transiently expressing OsTrx1-HA and Ehd3-Myc. Extracts were immunoprecipitated with anti-Myc antibody (B) or anti-HA antibody (C), and signals were detected by SDS-PAGE using anti-HA antibody (B) or anti-Myc antibody (C). Inputs are extracts before immunoprecipitation, and IP indicates elutes from agarose beads after immunoprecipitation. Plus and minus signs indicate constructs introduced into protoplasts. D, Identification of the OsTrx1 motif that interacts with Ehd3. Expression of truncated OsTrx1-HA (top), expression of Ehd3-Myc (middle), and interaction between two proteins after coimmunoprecipitation (bottom).

Techniques Used: Expressing, Immunoprecipitation, SDS Page, Construct

8) Product Images from "Homodimerization of RBPMS2 through a new RRM-interaction motif is necessary to control smooth muscle plasticity"

Article Title: Homodimerization of RBPMS2 through a new RRM-interaction motif is necessary to control smooth muscle plasticity

Journal: Nucleic Acids Research

doi: 10.1093/nar/gku692

The mutation of Leucine 49 into Glutamic acid (L49E) in the RRM of human RBPMS2 inhibits homodimerization, but does not alter binding to NOGGIN mRNA. ( A ) Analysis of the interaction of Myc-RBPMS2 with HA-RBPMS2 by Duolink PLA in DF-1 cells that co-express Myc-RBPMS2 or Myc-RBPMS2-L49E and HA-RBPMS2, or Myc-RBPMS2 and HA-TC10. Ha-tagged proteins were detected with anti-mouse HA antibodies (in green) and Myc-tagged proteins with anti-rabbit Myc antibodies (in red). Protein interactions were detected with Duolink PLA labeled in magenta. Images were collected by confocal microscopy. Bars, 10 μm. ( B ) Immunoprecipitation of RBPMS2 homodimers. Protein lysates from DF-1 cells that express HA-RBPMS2 and Myc - RBPMS2 (lanes 2 and 3) or Myc-RBPMS2-L49E (lanes 4 and 5) were immunoprecipitated with rabbit anti-Myc antibodies (lanes 3 and 5) or without (lanes 2 and 4). Lane 1: 10% of total cellular extracts from cells that express HA-RBPMS2 alone. Co-immunoprecipitation was monitored by immunoblotting with mouse anti-HA antibodies (upper panel). Immunoprecipitation efficiency was monitored by immunoblotting with rabbit anti-Myc antibodies (lower panel). ( C ) Subcellular localization of human RBPMS2 and RBPMS2-L49E. HEK293 cells that express Myc-RBPMS2 or Myc-RBPMS2-L49E were detected with anti-EiF3n (eukaryotic translation initiation factor 3n is present in stress granule) and rabbit anti-Myc antibodies. Myc-RBPMS2 and Myc-RBPMS2-L49E show similar cytoplasmic localization. ( D ) Experimental small-angle X-ray scattering curve (logarithm of intensity in arbitrary units as a function of the momentum transfer range s in Å −1 ) for RBPMS2-Nter-L49E measured at 1.1 mg/ml (green crosses), with its fitting theoretical curve (red continuous line) back-calculated from the RBPMS2-Nter-L49E NMR structure (Supplementary Figure S2). Blue dots represent the relative error bound. The χ 2 value of the fit is 1.096. ( E ) EMSA binding assays using a fixed high concentration of 161-nt NOGGIN RNA (100 nM) were performed with increasing concentrations of RBPMS2-Nter and RBPMS2-Nter-L49E ranging from 0.1 to 5 μM on a same gel and detected with SYBR ® Green EMSA nucleic acid gel stain. Note that RBPMS2-Nter forms defined RNA/protein complex as soon as 1 μM and two complexes at 5 μM (complexes I and II), whereas RBPMS2-Nter-L49E forms very diffuse bands (bracket and arrow). Free 161-nt RNA is indicated with a red arrow.
Figure Legend Snippet: The mutation of Leucine 49 into Glutamic acid (L49E) in the RRM of human RBPMS2 inhibits homodimerization, but does not alter binding to NOGGIN mRNA. ( A ) Analysis of the interaction of Myc-RBPMS2 with HA-RBPMS2 by Duolink PLA in DF-1 cells that co-express Myc-RBPMS2 or Myc-RBPMS2-L49E and HA-RBPMS2, or Myc-RBPMS2 and HA-TC10. Ha-tagged proteins were detected with anti-mouse HA antibodies (in green) and Myc-tagged proteins with anti-rabbit Myc antibodies (in red). Protein interactions were detected with Duolink PLA labeled in magenta. Images were collected by confocal microscopy. Bars, 10 μm. ( B ) Immunoprecipitation of RBPMS2 homodimers. Protein lysates from DF-1 cells that express HA-RBPMS2 and Myc - RBPMS2 (lanes 2 and 3) or Myc-RBPMS2-L49E (lanes 4 and 5) were immunoprecipitated with rabbit anti-Myc antibodies (lanes 3 and 5) or without (lanes 2 and 4). Lane 1: 10% of total cellular extracts from cells that express HA-RBPMS2 alone. Co-immunoprecipitation was monitored by immunoblotting with mouse anti-HA antibodies (upper panel). Immunoprecipitation efficiency was monitored by immunoblotting with rabbit anti-Myc antibodies (lower panel). ( C ) Subcellular localization of human RBPMS2 and RBPMS2-L49E. HEK293 cells that express Myc-RBPMS2 or Myc-RBPMS2-L49E were detected with anti-EiF3n (eukaryotic translation initiation factor 3n is present in stress granule) and rabbit anti-Myc antibodies. Myc-RBPMS2 and Myc-RBPMS2-L49E show similar cytoplasmic localization. ( D ) Experimental small-angle X-ray scattering curve (logarithm of intensity in arbitrary units as a function of the momentum transfer range s in Å −1 ) for RBPMS2-Nter-L49E measured at 1.1 mg/ml (green crosses), with its fitting theoretical curve (red continuous line) back-calculated from the RBPMS2-Nter-L49E NMR structure (Supplementary Figure S2). Blue dots represent the relative error bound. The χ 2 value of the fit is 1.096. ( E ) EMSA binding assays using a fixed high concentration of 161-nt NOGGIN RNA (100 nM) were performed with increasing concentrations of RBPMS2-Nter and RBPMS2-Nter-L49E ranging from 0.1 to 5 μM on a same gel and detected with SYBR ® Green EMSA nucleic acid gel stain. Note that RBPMS2-Nter forms defined RNA/protein complex as soon as 1 μM and two complexes at 5 μM (complexes I and II), whereas RBPMS2-Nter-L49E forms very diffuse bands (bracket and arrow). Free 161-nt RNA is indicated with a red arrow.

Techniques Used: Mutagenesis, Binding Assay, Proximity Ligation Assay, Labeling, Confocal Microscopy, Immunoprecipitation, Nuclear Magnetic Resonance, Concentration Assay, SYBR Green Assay, Staining

RBPMS2 dimers are formed in vitro and in vivo independently of RBPMS2 interaction with RNA. ( A ) Schematic representation of the different RBPMS2 clones isolated by Y2H screening with human RBPMS2 as bait. The RRM domain of RBPMS2 is between amino acids 32 and 105. ( B ) Immunoprecipitation with rabbit anti-Myc antibodies (lanes 3 and 6) or without (lanes 2 and 5) of protein lysates from DF-1 cells that express human HA-RBPMS2 or HA-TC10 and Myc-RBPMS2 or not. Lanes 1 and 4: 10% of total protein extracts from cells that express only HA-RBPMS2 or HA-TC10. Co-immunoprecipitation of HA-RBPMS2 was monitored by immunoblotting with mouse anti-HA antibodies (upper panel). The efficiency of immunoprecipitation was monitored by immunoblotting with rabbit anti-Myc antibodies (lower panel). ( C ) Co-immunoprecipitation of HA-RBPMS2 and Myc-RBPMS2 dimers in the absence of RNA. Protein lysates from DF-1 cells that express HA-RBPMS2 alone or with Myc-RBPMS2 (lanes 2–5) were incubated with 50-μg/ml RNase A at room temperature for 30 min (lanes 4 and 5) or left untreated (lanes 1–3) and then immunoprecipitated with rabbit anti-Myc antibodies (lanes 3 and 5) or without (lanes 2 and 4). Lane 1: 10% of total cell extracts from cells that express only HA-RBPMS2. Co-immunoprecipitation was monitored by immunoblotting with mouse anti-HA antibodies (upper panel). The immunoprecipitation efficiency was monitored by immunoblotting with rabbit anti-Myc antibodies (lower panel). ( D ) Analysis of the interaction of Myc-RBPMS2 with HA-RBPMS2 by proximity ligation assays (PLAs) in DF-1 cells that express Myc-RBPMS2 with HA-RBPMS2 or HA-TC10, or Myc-NICD and HA-RBPMS2, or Myc-RBPMS2 alone. HA-tagged proteins were detected with anti-mouse HA antibodies (in green) and Myc-tagged proteins with anti-rabbit Myc antibodies (in red). Interactions between proteins were detected with Duolink PLA labeled in magenta. Images were collected by confocal microscopy. Bars, 10 μm.
Figure Legend Snippet: RBPMS2 dimers are formed in vitro and in vivo independently of RBPMS2 interaction with RNA. ( A ) Schematic representation of the different RBPMS2 clones isolated by Y2H screening with human RBPMS2 as bait. The RRM domain of RBPMS2 is between amino acids 32 and 105. ( B ) Immunoprecipitation with rabbit anti-Myc antibodies (lanes 3 and 6) or without (lanes 2 and 5) of protein lysates from DF-1 cells that express human HA-RBPMS2 or HA-TC10 and Myc-RBPMS2 or not. Lanes 1 and 4: 10% of total protein extracts from cells that express only HA-RBPMS2 or HA-TC10. Co-immunoprecipitation of HA-RBPMS2 was monitored by immunoblotting with mouse anti-HA antibodies (upper panel). The efficiency of immunoprecipitation was monitored by immunoblotting with rabbit anti-Myc antibodies (lower panel). ( C ) Co-immunoprecipitation of HA-RBPMS2 and Myc-RBPMS2 dimers in the absence of RNA. Protein lysates from DF-1 cells that express HA-RBPMS2 alone or with Myc-RBPMS2 (lanes 2–5) were incubated with 50-μg/ml RNase A at room temperature for 30 min (lanes 4 and 5) or left untreated (lanes 1–3) and then immunoprecipitated with rabbit anti-Myc antibodies (lanes 3 and 5) or without (lanes 2 and 4). Lane 1: 10% of total cell extracts from cells that express only HA-RBPMS2. Co-immunoprecipitation was monitored by immunoblotting with mouse anti-HA antibodies (upper panel). The immunoprecipitation efficiency was monitored by immunoblotting with rabbit anti-Myc antibodies (lower panel). ( D ) Analysis of the interaction of Myc-RBPMS2 with HA-RBPMS2 by proximity ligation assays (PLAs) in DF-1 cells that express Myc-RBPMS2 with HA-RBPMS2 or HA-TC10, or Myc-NICD and HA-RBPMS2, or Myc-RBPMS2 alone. HA-tagged proteins were detected with anti-mouse HA antibodies (in green) and Myc-tagged proteins with anti-rabbit Myc antibodies (in red). Interactions between proteins were detected with Duolink PLA labeled in magenta. Images were collected by confocal microscopy. Bars, 10 μm.

Techniques Used: In Vitro, In Vivo, Clone Assay, Isolation, Immunoprecipitation, Incubation, Ligation, Proximity Ligation Assay, Labeling, Confocal Microscopy

9) Product Images from "Mechanism of suppressors of cytokine signaling 1 inhibition of epithelial-mesenchymal transition signaling through ROS regulation in colon cancer cells: suppression of Src leading to thioredoxin up-regulation"

Article Title: Mechanism of suppressors of cytokine signaling 1 inhibition of epithelial-mesenchymal transition signaling through ROS regulation in colon cancer cells: suppression of Src leading to thioredoxin up-regulation

Journal: Oncotarget

doi: 10.18632/oncotarget.11537

ROS-induced Jak and Src participate in EMT signaling, which are regulated by SOCS1 HCT116 p53 +/+ cells were treated with H 2 O 2 at 200 μM for different durations and analyzed for Jak and Src activation by Western blot A. HCT116 p53 +/+ cells were treated with H 2 O 2 at 200 μM for 2 h with or without pretreatment of JAK-inhibitor AG490 B. , or Src-inhibitor PP1 C. and changes in EMT markers and kinases were analyzed. HCT116 p53 +/+ sh sh-SOCS1 cells were incubated in the absence or presence NAC, AG490 and PP1. Changes in EMT associated proteins or kinase were analyzed by Western blot D. To examine the molecular interaction of Src and SOCS1, HCT116 p53+/+ cells over-expressing SOCS1 were subject to co-immunoprecipitation assay. Cell lysates were incubated with rabbit monoclonal c-Src Ab or control rabbit IgG followed by protein A/G-agarose beads. The immunoprecipitated pellets were separated from the supernatant, which were resolved on SDS-PAGE along with total lysates, and subjected to immunoblotting to reveal SOCS1 and Src protein bands E.
Figure Legend Snippet: ROS-induced Jak and Src participate in EMT signaling, which are regulated by SOCS1 HCT116 p53 +/+ cells were treated with H 2 O 2 at 200 μM for different durations and analyzed for Jak and Src activation by Western blot A. HCT116 p53 +/+ cells were treated with H 2 O 2 at 200 μM for 2 h with or without pretreatment of JAK-inhibitor AG490 B. , or Src-inhibitor PP1 C. and changes in EMT markers and kinases were analyzed. HCT116 p53 +/+ sh sh-SOCS1 cells were incubated in the absence or presence NAC, AG490 and PP1. Changes in EMT associated proteins or kinase were analyzed by Western blot D. To examine the molecular interaction of Src and SOCS1, HCT116 p53+/+ cells over-expressing SOCS1 were subject to co-immunoprecipitation assay. Cell lysates were incubated with rabbit monoclonal c-Src Ab or control rabbit IgG followed by protein A/G-agarose beads. The immunoprecipitated pellets were separated from the supernatant, which were resolved on SDS-PAGE along with total lysates, and subjected to immunoblotting to reveal SOCS1 and Src protein bands E.

Techniques Used: Activation Assay, Western Blot, Incubation, Expressing, Co-Immunoprecipitation Assay, Immunoprecipitation, SDS Page

10) Product Images from "Palmitoylation mediates membrane association of hepatitis E virus ORF3 protein and is required for infectious particle secretion"

Article Title: Palmitoylation mediates membrane association of hepatitis E virus ORF3 protein and is required for infectious particle secretion

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1007471

HEV ORF3 protein oligomerizes in mammalian cells. ( A ]. ( B ) ORF3 protein oligomerization was analyzed by FLAG immunoprecipitation. Lysates (Input) of U-2 OS cells transfected with pCMVORF3-HA and/or pCMVORF3-FLAG as well as immunoprecipitates (IP: FLAG) were subjected to immunoblot with either specific anti-FLAG or anti-HA antibodies. The presence of a strong signal for ORF3-HA after pull-down of ORF3-FLAG indicates oligomerization of ORF3 protein in cells. ( C ) FRET analyses reveal oligomerization of HEV ORF3. CFP (cyan fluorescent protein) or YFP (yellow fluorescent protein) fused to the C-termini of HEV ORF3 segments aa 1–113, 1–94, 1–70, 1–53, 28–113 or 53–113 were co-expressed in U-2 OS cells. FRET analyses were performed by the acceptor photobleaching method as described in the Materials and Methods section. The CFP-YFP fusion protein and cotransfection of unfused CFP and YFP served as positive and negative controls, respectively. Box-and-whisker plots represent the median FRET efficiency (FRETeff) values of 20 measurements (middle line), the values from the lower to the upper quartile (central box), and the minimum and maximum values (vertical line). The significance of the observed differences was assessed as described in Materials and Methods (*, P
Figure Legend Snippet: HEV ORF3 protein oligomerizes in mammalian cells. ( A ]. ( B ) ORF3 protein oligomerization was analyzed by FLAG immunoprecipitation. Lysates (Input) of U-2 OS cells transfected with pCMVORF3-HA and/or pCMVORF3-FLAG as well as immunoprecipitates (IP: FLAG) were subjected to immunoblot with either specific anti-FLAG or anti-HA antibodies. The presence of a strong signal for ORF3-HA after pull-down of ORF3-FLAG indicates oligomerization of ORF3 protein in cells. ( C ) FRET analyses reveal oligomerization of HEV ORF3. CFP (cyan fluorescent protein) or YFP (yellow fluorescent protein) fused to the C-termini of HEV ORF3 segments aa 1–113, 1–94, 1–70, 1–53, 28–113 or 53–113 were co-expressed in U-2 OS cells. FRET analyses were performed by the acceptor photobleaching method as described in the Materials and Methods section. The CFP-YFP fusion protein and cotransfection of unfused CFP and YFP served as positive and negative controls, respectively. Box-and-whisker plots represent the median FRET efficiency (FRETeff) values of 20 measurements (middle line), the values from the lower to the upper quartile (central box), and the minimum and maximum values (vertical line). The significance of the observed differences was assessed as described in Materials and Methods (*, P

Techniques Used: Immunoprecipitation, Transfection, Cotransfection, Whisker Assay

HEV ORF3 protein is palmitoylated. Protein lysates from S10-3 cells transfected with pCMVORF3, pCMVORF3 C1-4 , pCMVORF3 C45-8 or pCMVORF3_gt1 and from Hep293TT cells replicating the full-length p6 or 83–2 HEV clone were prepared 1 or 6 days post-transfection, respectively, and subjected to immunoprecipitation with either anti-ORF3 pAb (+) or non-relevant rabbit serum (-). After immunoprecipitation, the elution samples were separated by 17% SDS-PAGE and subjected to either immunoblot with anti-ORF3 pAb followed by chemiluminescence revelation or autoradiography (40 d of exposure).
Figure Legend Snippet: HEV ORF3 protein is palmitoylated. Protein lysates from S10-3 cells transfected with pCMVORF3, pCMVORF3 C1-4 , pCMVORF3 C45-8 or pCMVORF3_gt1 and from Hep293TT cells replicating the full-length p6 or 83–2 HEV clone were prepared 1 or 6 days post-transfection, respectively, and subjected to immunoprecipitation with either anti-ORF3 pAb (+) or non-relevant rabbit serum (-). After immunoprecipitation, the elution samples were separated by 17% SDS-PAGE and subjected to either immunoblot with anti-ORF3 pAb followed by chemiluminescence revelation or autoradiography (40 d of exposure).

Techniques Used: Transfection, Immunoprecipitation, SDS Page, Autoradiography

11) Product Images from "MyoD Stimulates RB Promoter Activity via the CREB/p300 Nuclear Transduction Pathway †"

Article Title: MyoD Stimulates RB Promoter Activity via the CREB/p300 Nuclear Transduction Pathway †

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.8.2893-2906.2003

In vivo association between MyoD and CREB. C3H10T1/2 fibroblasts were transfected with CREB and/or FLAG-tagged MyoD expression constructs as indicated above the lanes. Transfected cells were cultured in either GM or DM for 48 h before being harvested. Aliquots of 500 μg of protein from each lysate were immunoprecipitated (IP) with anti-FLAG antibody M2. The precipitated proteins were fractionated by SDS-PAGE and revealed with antibodies specific for MyoD, CREB, or phospho-CREB (upper panels). P, phosphorylated. Western blot analysis of the input lysates used for immunoprecipitation (20-μg aliquot of each) is also shown (lower panels) (the anti-phospho-CREB antibody also detects the phosphorylated form of ATF1).
Figure Legend Snippet: In vivo association between MyoD and CREB. C3H10T1/2 fibroblasts were transfected with CREB and/or FLAG-tagged MyoD expression constructs as indicated above the lanes. Transfected cells were cultured in either GM or DM for 48 h before being harvested. Aliquots of 500 μg of protein from each lysate were immunoprecipitated (IP) with anti-FLAG antibody M2. The precipitated proteins were fractionated by SDS-PAGE and revealed with antibodies specific for MyoD, CREB, or phospho-CREB (upper panels). P, phosphorylated. Western blot analysis of the input lysates used for immunoprecipitation (20-μg aliquot of each) is also shown (lower panels) (the anti-phospho-CREB antibody also detects the phosphorylated form of ATF1).

Techniques Used: In Vivo, Transfection, Expressing, Construct, Cell Culture, Immunoprecipitation, SDS Page, Western Blot

12) Product Images from "Modular Structure of PACT: Distinct Domains for Binding and Activating PKR"

Article Title: Modular Structure of PACT: Distinct Domains for Binding and Activating PKR

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.21.6.1908-1920.2001

Weak interactions of domain 3 with PKR at a physiological salt concentration. (A) Failure of Δ1,2 to interact with PKR in vivo. Coimmunoprecipitation of PKR in transfected HT1080 cells with anti-FLAG agarose as described in Materials and Methods. Lane 1, PKR; lane 2, Δ1,2; lane 3, PKR and Δ1,2; lane 4, PKR and wt PACT. (B) Δ1,2 interacts weakly with PKR under physiological salt concentrations in vitro. 35 S-labeled PKR and FLAG-Δ1,2 mutant or FLAG-wt PACT were synthesized independently in vitro. A total of 3 μl of the reticulocyte lysate containing PKR was mixed with 3 μl of the lysates containing wt PACT or Δ1,2. PACT was immunoprecipitated from the lysate using anti-FLAG (M2) agarose in high (physiological)-salt buffer or low-salt buffer, and the proteins coimmunoprecipitating with it were analyzed. Lanes 1 and 2 show all proteins in the mixture before immunoprecipitation, and lanes 3 to 6 represent immunoprecipitated proteins. Proteins in lanes 3 and 4 were immunoprecipitated and washed in low-salt buffer, while proteins in lanes 5 and 6 were immunoprecipitated and washed in high-salt buffer. Lanes 1, 3, and 5, PKR and Δ1,2; lanes 2, 4, and 6, PKR and wt PACT. (C) MBP-3 can be coimmunoprecipitated with PKR in low-salt but not in high-salt conditions. MBP, MBP-3, and wt PACT were tested for PKR binding in conditions of low or high salt. Equal amounts of purified MBP, MBP-3, or wt PACT were added to an extract from HT1080 cells treated with 1,000 U of IFN β per ml for 24 h. PKR was immunoprecipitated with anti-PKR monoclonal antibody and washed in conditions of high or low salt. The proteins which remained bound to the beads after washing were analyzed by Western blotting with anti-PACT polyclonal antibody. Lanes 1 to 3 show immunoprecipitated proteins in high salt conditions, and lanes 4 to 5 show immunoprecipitated proteins in low-salt conditions. Lanes 1 and 4, MBP (1 μg); lanes 2 and 5, MBP-3 (1 μg); lanes 3 and 6, wt PACT (1 μg). (D) MBP-3 cannot bind to DD. The conditions were the same as for panel C, except that purified DD was used instead of cell extracts containing PKR. The same PKR antibody immunoprecipitated DD and its associated proteins.
Figure Legend Snippet: Weak interactions of domain 3 with PKR at a physiological salt concentration. (A) Failure of Δ1,2 to interact with PKR in vivo. Coimmunoprecipitation of PKR in transfected HT1080 cells with anti-FLAG agarose as described in Materials and Methods. Lane 1, PKR; lane 2, Δ1,2; lane 3, PKR and Δ1,2; lane 4, PKR and wt PACT. (B) Δ1,2 interacts weakly with PKR under physiological salt concentrations in vitro. 35 S-labeled PKR and FLAG-Δ1,2 mutant or FLAG-wt PACT were synthesized independently in vitro. A total of 3 μl of the reticulocyte lysate containing PKR was mixed with 3 μl of the lysates containing wt PACT or Δ1,2. PACT was immunoprecipitated from the lysate using anti-FLAG (M2) agarose in high (physiological)-salt buffer or low-salt buffer, and the proteins coimmunoprecipitating with it were analyzed. Lanes 1 and 2 show all proteins in the mixture before immunoprecipitation, and lanes 3 to 6 represent immunoprecipitated proteins. Proteins in lanes 3 and 4 were immunoprecipitated and washed in low-salt buffer, while proteins in lanes 5 and 6 were immunoprecipitated and washed in high-salt buffer. Lanes 1, 3, and 5, PKR and Δ1,2; lanes 2, 4, and 6, PKR and wt PACT. (C) MBP-3 can be coimmunoprecipitated with PKR in low-salt but not in high-salt conditions. MBP, MBP-3, and wt PACT were tested for PKR binding in conditions of low or high salt. Equal amounts of purified MBP, MBP-3, or wt PACT were added to an extract from HT1080 cells treated with 1,000 U of IFN β per ml for 24 h. PKR was immunoprecipitated with anti-PKR monoclonal antibody and washed in conditions of high or low salt. The proteins which remained bound to the beads after washing were analyzed by Western blotting with anti-PACT polyclonal antibody. Lanes 1 to 3 show immunoprecipitated proteins in high salt conditions, and lanes 4 to 5 show immunoprecipitated proteins in low-salt conditions. Lanes 1 and 4, MBP (1 μg); lanes 2 and 5, MBP-3 (1 μg); lanes 3 and 6, wt PACT (1 μg). (D) MBP-3 cannot bind to DD. The conditions were the same as for panel C, except that purified DD was used instead of cell extracts containing PKR. The same PKR antibody immunoprecipitated DD and its associated proteins.

Techniques Used: Concentration Assay, In Vivo, Transfection, In Vitro, Labeling, Mutagenesis, Synthesized, Immunoprecipitation, Binding Assay, Purification, Western Blot

PKR and dsRNA binding by deletion mutants of PACT. (A) Maps of human wt PACT protein and deletion constructs. wt PACT contains three putative dsRNA-protein or protein-protein interaction domains indicated by the large numbers 1, 2, and 3. Small numbers indicate the amino acid residue number. Δ1 is missing PACT amino acid residues 35 to 99; Δ2 is missing PACT amino acid residues 127 to 192; Δ3 is missing PACT amino acid residues 240 to 305; Δ1,2 is missing PACT amino acid residues 35 to 99 and 127 to 192. (B) PACT and its mutant proteins each interact with PKR in vitro and in vivo. In vitro and in vivo coimmunoprecipitation of PKR with FLAG-tagged PACT and its mutants. (In vitro) 35 S-labeled PKR (K296R), FLAG-wt PACT, and FLAG-PACT mutants were synthesized independently. A total of 3 μl of the reticulocyte lysate containing PKR (K296R) was mixed with 3 μl of the lysates containing FLAG-wt PACT, FLAG-Δ1, FLAG-Δ2, FLAG-Δ3. PACT was immunoprecipitated using anti-FLAG (M2) agarose, and the proteins coimmunoprecipitating with it were analyzed. Lanes 1 to 5 show all proteins in the mixture before immunoprecipitation, and lanes 6 to 10 represent immunoprecipitated proteins. All lanes contain PKR (K296R). Lanes 1 and 6, wt PACT; lanes 2 and 7, Δ1, lanes 3 and 8, Δ2; lanes 4 and 9, Δ3; lanes 5 and 10, only PKR. (In vivo) Coimmunoprecipitation of PKR in transfected HT1080 cells with anti-FLAG agarose as described in Materials and Methods. A total of 5 μg each of CMV-PKR (K296R) and FLAG-tagged PACT construct was transfected unless indicated otherwise below. Lane 1, PKR (K296R) alone (10 μg, transfected); lane 2, wt PACT alone (10 μg, transfected); lane 3, PKR and wt PACT; lane 4, PKR and Δ1; lane 5, PKR and Δ2; lane 6, PKR and Δ3. (C) PACT and its mutant proteins bind dsRNA. wt PACT and its mutant proteins were tested for poly(I-C) agarose binding activity as described in Materials and Methods. Equal amounts of the total translation mix were loaded for all samples. PhosphorImager analysis was done to quantify the binding activity. The fraction of bound protein was calculated as the radioactivity in the bound protein band/total radioactivity assayed. The dsRNA binding of wt PACT was considered 100%, and values for other PACT mutant proteins or luciferase are presented as a percentage of that value. Forty percent of input wt PACT bound to the resin.
Figure Legend Snippet: PKR and dsRNA binding by deletion mutants of PACT. (A) Maps of human wt PACT protein and deletion constructs. wt PACT contains three putative dsRNA-protein or protein-protein interaction domains indicated by the large numbers 1, 2, and 3. Small numbers indicate the amino acid residue number. Δ1 is missing PACT amino acid residues 35 to 99; Δ2 is missing PACT amino acid residues 127 to 192; Δ3 is missing PACT amino acid residues 240 to 305; Δ1,2 is missing PACT amino acid residues 35 to 99 and 127 to 192. (B) PACT and its mutant proteins each interact with PKR in vitro and in vivo. In vitro and in vivo coimmunoprecipitation of PKR with FLAG-tagged PACT and its mutants. (In vitro) 35 S-labeled PKR (K296R), FLAG-wt PACT, and FLAG-PACT mutants were synthesized independently. A total of 3 μl of the reticulocyte lysate containing PKR (K296R) was mixed with 3 μl of the lysates containing FLAG-wt PACT, FLAG-Δ1, FLAG-Δ2, FLAG-Δ3. PACT was immunoprecipitated using anti-FLAG (M2) agarose, and the proteins coimmunoprecipitating with it were analyzed. Lanes 1 to 5 show all proteins in the mixture before immunoprecipitation, and lanes 6 to 10 represent immunoprecipitated proteins. All lanes contain PKR (K296R). Lanes 1 and 6, wt PACT; lanes 2 and 7, Δ1, lanes 3 and 8, Δ2; lanes 4 and 9, Δ3; lanes 5 and 10, only PKR. (In vivo) Coimmunoprecipitation of PKR in transfected HT1080 cells with anti-FLAG agarose as described in Materials and Methods. A total of 5 μg each of CMV-PKR (K296R) and FLAG-tagged PACT construct was transfected unless indicated otherwise below. Lane 1, PKR (K296R) alone (10 μg, transfected); lane 2, wt PACT alone (10 μg, transfected); lane 3, PKR and wt PACT; lane 4, PKR and Δ1; lane 5, PKR and Δ2; lane 6, PKR and Δ3. (C) PACT and its mutant proteins bind dsRNA. wt PACT and its mutant proteins were tested for poly(I-C) agarose binding activity as described in Materials and Methods. Equal amounts of the total translation mix were loaded for all samples. PhosphorImager analysis was done to quantify the binding activity. The fraction of bound protein was calculated as the radioactivity in the bound protein band/total radioactivity assayed. The dsRNA binding of wt PACT was considered 100%, and values for other PACT mutant proteins or luciferase are presented as a percentage of that value. Forty percent of input wt PACT bound to the resin.

Techniques Used: Binding Assay, Construct, Mutagenesis, In Vitro, In Vivo, Labeling, Synthesized, Immunoprecipitation, Transfection, Activity Assay, Radioactivity, Luciferase

13) Product Images from "A protein secreted by the respiratory pathogen Chlamydia pneumoniae impairs IL-17 signaling via interaction with human Act1"

Article Title: A protein secreted by the respiratory pathogen Chlamydia pneumoniae impairs IL-17 signaling via interaction with human Act1

Journal: Cellular microbiology

doi: 10.1111/j.1462-5822.2009.01290.x

CP0236 interacts with Act1. (A) Yeast grown on synthetic medium (SC) without Leu, Trp, or on SC-TDO (without Leu, Trp, His, + 30 mM 3AT). (1) pCP0236 101- 279 -DBD + pAct1 306-565 -AD, (2) pYpkA 442-733 ), (3) pCP0236 101- 279 -DBD + pDEST22-AD (negative control), (4) pDEST32 + pDEST22 (negative control). (B) Co-immunoprecipitation of FLAG-tagged proteins with endogenous Act1. Act1 was immunoprecipitated from HeLa cells expressing 3xFLAG-CP0236, 3xFLAG-CT694, or empty vector. Purified material was probed with anti-Act1 or anti-FLAG.
Figure Legend Snippet: CP0236 interacts with Act1. (A) Yeast grown on synthetic medium (SC) without Leu, Trp, or on SC-TDO (without Leu, Trp, His, + 30 mM 3AT). (1) pCP0236 101- 279 -DBD + pAct1 306-565 -AD, (2) pYpkA 442-733 ), (3) pCP0236 101- 279 -DBD + pDEST22-AD (negative control), (4) pDEST32 + pDEST22 (negative control). (B) Co-immunoprecipitation of FLAG-tagged proteins with endogenous Act1. Act1 was immunoprecipitated from HeLa cells expressing 3xFLAG-CP0236, 3xFLAG-CT694, or empty vector. Purified material was probed with anti-Act1 or anti-FLAG.

Techniques Used: Negative Control, Immunoprecipitation, Expressing, Plasmid Preparation, Purification

14) Product Images from "Cellular RelB interacts with the transactivator Tat and enhance HIV-1 expression"

Article Title: Cellular RelB interacts with the transactivator Tat and enhance HIV-1 expression

Journal: Retrovirology

doi: 10.1186/s12977-018-0447-9

Interaction between HIV-1 Tat and RelB. a , b Myc-Tat (3 μg) was transfected into HEK 293T cells (4 × 10 6 ) together with empty vectors (control) (3 μg) or pFlag-RelB (3 μg) Co-immunoprecipitation was performed with anti-Flag ( a ) or anti-Myc ( b ) antibodies. Samples of both cell lysates and immunoprecipitates were subjected to western blotting and probed with rabbit anti-Myc and anti-Flag antibodies. c Co-IP of endogenous RelB and ectopically expressed Tat. The lysate from HA-Tat-expressing HeLa cells (4 × 10 6 ) was immunoprecipitated with mouse anti-HA antibodies, and the precipitated proteins were examined with western blotting. d Effect of RNases on the association of endogenous RelB and ectopically expressed Tat. Lysates (in the presence or absence of RNase A [5 μg/ml]) of HA-Tat expressing HeLa cells (4 × 10 6 ) were immunoprecipitated with control rabbit IgG or rabbit anti-RelB antibodies. Samples from cell lysates and immunoprecipitates were subjected to western blotting. e Tat partially co-localizes with RelB. HeLa cells (0.1 × 10 6 ) were transfected with HA-Tat (200 ng) and Flag-RelB (200 ng) plasmid DNA. Indirect IFA was performed to detect HA-Tat (with rabbit anti-HA antibody and TRITC-conjugated goat anti rabbit secondary antibody) and Flag-RelB (with mouse anti-Flag antibody and FITC-conjugated goat anti mouse secondary antibody). Nuclei were visualized with DAPI staining. Representative images are shown. The inset shows a higher magnification of the boxed area
Figure Legend Snippet: Interaction between HIV-1 Tat and RelB. a , b Myc-Tat (3 μg) was transfected into HEK 293T cells (4 × 10 6 ) together with empty vectors (control) (3 μg) or pFlag-RelB (3 μg) Co-immunoprecipitation was performed with anti-Flag ( a ) or anti-Myc ( b ) antibodies. Samples of both cell lysates and immunoprecipitates were subjected to western blotting and probed with rabbit anti-Myc and anti-Flag antibodies. c Co-IP of endogenous RelB and ectopically expressed Tat. The lysate from HA-Tat-expressing HeLa cells (4 × 10 6 ) was immunoprecipitated with mouse anti-HA antibodies, and the precipitated proteins were examined with western blotting. d Effect of RNases on the association of endogenous RelB and ectopically expressed Tat. Lysates (in the presence or absence of RNase A [5 μg/ml]) of HA-Tat expressing HeLa cells (4 × 10 6 ) were immunoprecipitated with control rabbit IgG or rabbit anti-RelB antibodies. Samples from cell lysates and immunoprecipitates were subjected to western blotting. e Tat partially co-localizes with RelB. HeLa cells (0.1 × 10 6 ) were transfected with HA-Tat (200 ng) and Flag-RelB (200 ng) plasmid DNA. Indirect IFA was performed to detect HA-Tat (with rabbit anti-HA antibody and TRITC-conjugated goat anti rabbit secondary antibody) and Flag-RelB (with mouse anti-Flag antibody and FITC-conjugated goat anti mouse secondary antibody). Nuclei were visualized with DAPI staining. Representative images are shown. The inset shows a higher magnification of the boxed area

Techniques Used: Transfection, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Expressing, Plasmid Preparation, Immunofluorescence, Staining

15) Product Images from "Transcriptional Activity of Erythroid Kruppel-like Factor (EKLF/KLF1) Modulated by PIAS3 (Protein Inhibitor of Activated STAT3) *"

Article Title: Transcriptional Activity of Erythroid Kruppel-like Factor (EKLF/KLF1) Modulated by PIAS3 (Protein Inhibitor of Activated STAT3) *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.610246

EKLF interacts with PIAS3. 293T cells were cotransfected with constructs encoding EKLF and Flag-tagged PIAS3. A and B , lysates were subjected to immunoprecipitation ( IP ) with anti-EKLF antibody (6B3), blotted, and probed with anti-Flag antibody ( A ) or
Figure Legend Snippet: EKLF interacts with PIAS3. 293T cells were cotransfected with constructs encoding EKLF and Flag-tagged PIAS3. A and B , lysates were subjected to immunoprecipitation ( IP ) with anti-EKLF antibody (6B3), blotted, and probed with anti-Flag antibody ( A ) or

Techniques Used: Construct, Immunoprecipitation

The EKLF transactivation proline and ZnF DNA-binding domains interact with multiple domains of PIAS3. A , 293T cells were cotransfected with constructs encoding full-length EKLF or its proline domain and Flag-tagged PIAS3. Lysates were subjected to immunoprecipitation
Figure Legend Snippet: The EKLF transactivation proline and ZnF DNA-binding domains interact with multiple domains of PIAS3. A , 293T cells were cotransfected with constructs encoding full-length EKLF or its proline domain and Flag-tagged PIAS3. Lysates were subjected to immunoprecipitation

Techniques Used: Binding Assay, Construct, Immunoprecipitation

16) Product Images from "Sorting nexin-21 is a scaffold for the endosomal recruitment of huntingtin"

Article Title: Sorting nexin-21 is a scaffold for the endosomal recruitment of huntingtin

Journal: Journal of Cell Science

doi: 10.1242/jcs.211672

Co-immunoprecipitation of Htt with SNX21 requires negatively charged residues in the SNX21 N-terminus, but does not require SNX21 to be endosomally localised. ) . Green boxed regions represent clusters of negatively charged amino acids not present in the SNX20 N-terminal extension. Red boxes denote conserved amino acids within SNX20 and SNX21 sequences and asterisks count every ten residues starting with the fist methionine of SNX20. (B) HEK293-T cells were transiently transfected to express GFP, GFP-tagged full-length SNX21 and two truncation mutants representing the two halves of the N-terminal region of SNX21. Precipitates from the GFP-nanotrap-isolated variants were analysed by western blotting and demonstrate the necessity for a full N-terminal extension in order to facilitate Htt binding. Data are representative of three biological replicates. (C) Site-directed mutagenesis was used to engineer a variety of charge swap mutants targeting the negatively charged clusters of amino acids, prior to probing for Htt binding as above. Two aspartic acid residues in the first N-terminal cluster are essential for precipitation of Htt with SNX21. (D) Both the point mutated GFP-SNX21 and truncation variants were expressed in HeLa cells prior to fixation and immunolabelling with anti-EEA1. Except for the N-terminal 1-129 construct, which lacks a PX domain, all mutants retained an endosomal localisation. Scale bars: 20 µm.
Figure Legend Snippet: Co-immunoprecipitation of Htt with SNX21 requires negatively charged residues in the SNX21 N-terminus, but does not require SNX21 to be endosomally localised. ) . Green boxed regions represent clusters of negatively charged amino acids not present in the SNX20 N-terminal extension. Red boxes denote conserved amino acids within SNX20 and SNX21 sequences and asterisks count every ten residues starting with the fist methionine of SNX20. (B) HEK293-T cells were transiently transfected to express GFP, GFP-tagged full-length SNX21 and two truncation mutants representing the two halves of the N-terminal region of SNX21. Precipitates from the GFP-nanotrap-isolated variants were analysed by western blotting and demonstrate the necessity for a full N-terminal extension in order to facilitate Htt binding. Data are representative of three biological replicates. (C) Site-directed mutagenesis was used to engineer a variety of charge swap mutants targeting the negatively charged clusters of amino acids, prior to probing for Htt binding as above. Two aspartic acid residues in the first N-terminal cluster are essential for precipitation of Htt with SNX21. (D) Both the point mutated GFP-SNX21 and truncation variants were expressed in HeLa cells prior to fixation and immunolabelling with anti-EEA1. Except for the N-terminal 1-129 construct, which lacks a PX domain, all mutants retained an endosomal localisation. Scale bars: 20 µm.

Techniques Used: Immunoprecipitation, Transfection, Isolation, Western Blot, Binding Assay, Mutagenesis, Construct

Co-immunoprecipitation of septins with SNX21 requires a surface exposed leucine in the PXB domain. ). (C) Site-directed mutagenesis of the SNX21 PXB domain, targeting predicted surface exposed residues. Constructs encoding GFP-tag chimeras of the various SNX21 mutants were transiently expressed in HEK293-T cells prior to GFP-nanotrap, SDS-PAGE and western blotting. Mutation of an evolutionarily conserved leucine (L363A) and a neighbouring lysine (K364E) is sufficient to perturb association with both septin 2 and septin 7. Data are representative of three biological replicates. (D) GFP-SNX21 mutants were expressed in HeLa cells prior to fixation and immunolabelling with anti-EEA1. Each of the mutants analysed retained an endosomal localisation in accordance with the wild-type protein. Scale bars: 20 µm.
Figure Legend Snippet: Co-immunoprecipitation of septins with SNX21 requires a surface exposed leucine in the PXB domain. ). (C) Site-directed mutagenesis of the SNX21 PXB domain, targeting predicted surface exposed residues. Constructs encoding GFP-tag chimeras of the various SNX21 mutants were transiently expressed in HEK293-T cells prior to GFP-nanotrap, SDS-PAGE and western blotting. Mutation of an evolutionarily conserved leucine (L363A) and a neighbouring lysine (K364E) is sufficient to perturb association with both septin 2 and septin 7. Data are representative of three biological replicates. (D) GFP-SNX21 mutants were expressed in HeLa cells prior to fixation and immunolabelling with anti-EEA1. Each of the mutants analysed retained an endosomal localisation in accordance with the wild-type protein. Scale bars: 20 µm.

Techniques Used: Immunoprecipitation, Mutagenesis, Construct, SDS Page, Western Blot

17) Product Images from "NP1 Regulates Neuronal Activity-Dependent Accumulation of BAX in Mitochondria and Mitochondrial Dynamics"

Article Title: NP1 Regulates Neuronal Activity-Dependent Accumulation of BAX in Mitochondria and Mitochondrial Dynamics

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.4604-11.2012

BAX immunoprecipitates NP1 and NP1 colocalizes with active BAX in mitochondria. A–C , Representative Western blots of total protein lysate (TL), the eluate of BAX immunoprecipitation (IP; Bax), and the eluate of IgG immunoprecipitation controls (IgG). A , Immunoprecipitation of BAX from total brain lysates. Rat brains were homogenized, and protein lysates prepared as described in Materials and Methods. B , Immunoprecipitation of BAX from CGN lysates. CGNs were treated with low K + for 2 h. C , Immunoprecipitation of BAX from HEK 293T cell lysates transfected with recombinant rat Bax and NP1 cDNAs. D , Confocal microscopy studies were performed in mature CGNs in culture after 4 h of K + deprivation. Orthogonal sectioning shows colocalization between NP1 and active BAX in mitochondria. Colocalization between NP1 and MtDsRed is seen as yellow, between NP1 and BAX as cyan, and between BAX and MtDsRed as magenta, and colocalization between all three proteins is shown in white. Scale bar, 5 μm.
Figure Legend Snippet: BAX immunoprecipitates NP1 and NP1 colocalizes with active BAX in mitochondria. A–C , Representative Western blots of total protein lysate (TL), the eluate of BAX immunoprecipitation (IP; Bax), and the eluate of IgG immunoprecipitation controls (IgG). A , Immunoprecipitation of BAX from total brain lysates. Rat brains were homogenized, and protein lysates prepared as described in Materials and Methods. B , Immunoprecipitation of BAX from CGN lysates. CGNs were treated with low K + for 2 h. C , Immunoprecipitation of BAX from HEK 293T cell lysates transfected with recombinant rat Bax and NP1 cDNAs. D , Confocal microscopy studies were performed in mature CGNs in culture after 4 h of K + deprivation. Orthogonal sectioning shows colocalization between NP1 and active BAX in mitochondria. Colocalization between NP1 and MtDsRed is seen as yellow, between NP1 and BAX as cyan, and between BAX and MtDsRed as magenta, and colocalization between all three proteins is shown in white. Scale bar, 5 μm.

Techniques Used: Western Blot, Immunoprecipitation, Transfection, Recombinant, Confocal Microscopy

18) Product Images from "Functional Characterization of a WWP1/Tiul1 Tumor-derived Mutant Reveals a Paradigm of Its Constitutive Activation in Human Cancer *"

Article Title: Functional Characterization of a WWP1/Tiul1 Tumor-derived Mutant Reveals a Paradigm of Its Constitutive Activation in Human Cancer *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M115.642314

C2 and WW domains inhibit WWP1 autopolyubiquitination. A , HA-C2, HA-WW, and FLAG-Hect were transfected into HEK293 cells, and cell lysates were subjected to immunoprecipitation ( IP ) with anti-HA antibody before being analyzed by immunoblotting with anti-FLAG
Figure Legend Snippet: C2 and WW domains inhibit WWP1 autopolyubiquitination. A , HA-C2, HA-WW, and FLAG-Hect were transfected into HEK293 cells, and cell lysates were subjected to immunoprecipitation ( IP ) with anti-HA antibody before being analyzed by immunoblotting with anti-FLAG

Techniques Used: Transfection, Immunoprecipitation

19) Product Images from "Atypical parkinsonism–associated retromer mutant alters endosomal sorting of specific cargo proteins"

Article Title: Atypical parkinsonism–associated retromer mutant alters endosomal sorting of specific cargo proteins

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201604057

Missense VPS26A mutants do not perturb assembly of the retromer heterotrimer or its endosome association. (A) Fluorescence-based Western blot showing the expression levels of GFP-VPS26A and GFP-VPS26A mutants compared with endogenous VPS26A. Expression of GFP-VPS26A (p.K297X) cannot be seen because of the antibody recognizing the C terminus of VPS26A. (B) Fluorescence-based Western analysis after GFP-Trap immunoprecipitation of GFP-VPS26A and GFP-VPS26A mutants with endogenous VPS35 and VPS29. (C) Quantification of B from three (VPS35) and four (VPS29) independent experiments. Data expressed as a percentage of the GFP-VPS26A control. Error bars represent mean ± SEM. No statistically significant difference was observed. NS, not significant. (D) Immunofluorescence staining of VPS35, EEA1, LAMP1, and FAM21 in RPE-1 cells expressing GFP-VPS26A or the GFP-VPS26A mutants. Bars, 20 µm. (E) Colocalization of GFP-VPS26A or GFP-VPS26A mutants with VPS35, EEA1, LAMP1, or FAM21 from three independent experiments. Error bars represent mean ± SEM. No statistically significant difference was found.
Figure Legend Snippet: Missense VPS26A mutants do not perturb assembly of the retromer heterotrimer or its endosome association. (A) Fluorescence-based Western blot showing the expression levels of GFP-VPS26A and GFP-VPS26A mutants compared with endogenous VPS26A. Expression of GFP-VPS26A (p.K297X) cannot be seen because of the antibody recognizing the C terminus of VPS26A. (B) Fluorescence-based Western analysis after GFP-Trap immunoprecipitation of GFP-VPS26A and GFP-VPS26A mutants with endogenous VPS35 and VPS29. (C) Quantification of B from three (VPS35) and four (VPS29) independent experiments. Data expressed as a percentage of the GFP-VPS26A control. Error bars represent mean ± SEM. No statistically significant difference was observed. NS, not significant. (D) Immunofluorescence staining of VPS35, EEA1, LAMP1, and FAM21 in RPE-1 cells expressing GFP-VPS26A or the GFP-VPS26A mutants. Bars, 20 µm. (E) Colocalization of GFP-VPS26A or GFP-VPS26A mutants with VPS35, EEA1, LAMP1, or FAM21 from three independent experiments. Error bars represent mean ± SEM. No statistically significant difference was found.

Techniques Used: Fluorescence, Western Blot, Expressing, Immunoprecipitation, Immunofluorescence, Staining

VPS26A (p.K297X) leads to a loss of and enhancement of VPS26A’s association with specific components of its interactome. (A) Logarithmic graph showing the interactors identified from comparative SILAC proteomics of GFP-VPS26A versus GFP-VPS26A (p.K93E), GFP-VPS26A (p.M112V), or GFP-VPS26A (p.K297X). The SILAC ratio is the fold-enrichment of proteins in GFP-VPS26A mutant over GFP-VPS26A. Red circles indicate either a pronounced enhancement or loss of association with GFP-VPS26A (K297X). Data is mean of n = 2–3 independent experiments. (B and C) Fluorescence-based Western analysis after GFP-Trap immunoprecipitation of GFP-VPS26A, GFP-VPS26A (p.K93E), GFP-VPS26A (p.M112V), and GFP-VPS26A (p.K297X). (D) Quantification of data from three independent experiments. Data are expressed as a percentage of the GFP-VPS26A control and analyzed by a one-way ANOVA followed by a Dunnett posthoc test. Error bars represent mean ± SEM. NS, not significant; *, P
Figure Legend Snippet: VPS26A (p.K297X) leads to a loss of and enhancement of VPS26A’s association with specific components of its interactome. (A) Logarithmic graph showing the interactors identified from comparative SILAC proteomics of GFP-VPS26A versus GFP-VPS26A (p.K93E), GFP-VPS26A (p.M112V), or GFP-VPS26A (p.K297X). The SILAC ratio is the fold-enrichment of proteins in GFP-VPS26A mutant over GFP-VPS26A. Red circles indicate either a pronounced enhancement or loss of association with GFP-VPS26A (K297X). Data is mean of n = 2–3 independent experiments. (B and C) Fluorescence-based Western analysis after GFP-Trap immunoprecipitation of GFP-VPS26A, GFP-VPS26A (p.K93E), GFP-VPS26A (p.M112V), and GFP-VPS26A (p.K297X). (D) Quantification of data from three independent experiments. Data are expressed as a percentage of the GFP-VPS26A control and analyzed by a one-way ANOVA followed by a Dunnett posthoc test. Error bars represent mean ± SEM. NS, not significant; *, P

Techniques Used: Mutagenesis, Fluorescence, Western Blot, Immunoprecipitation

20) Product Images from "The Rho Target PRK2 Regulates Apical Junction Formation in Human Bronchial Epithelial Cells ▿"

Article Title: The Rho Target PRK2 Regulates Apical Junction Formation in Human Bronchial Epithelial Cells ▿

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01001-10

PRK2 function is RhoA-dependent, kinase-dependent, and requires its C2-like domain. (A) Domain organization of PRK2, including HR1 (homology region 1) GTPase-binding domains, a C2-like domain, and a serine-threonine kinase domain. (B) Wild-type mouse PRK2 (mPRK2) but not mPRK2(A66K,A155K) interacts with L63RhoA in coimmunoprecipitation experiments when overexpressed in HEK293T cells. WB, Western blotting; IP, immunoprecipitation. (C to E) 16HBE cells were infected with pBABE retroviral vectors expressing HA-tagged mPRK2, HA-tagged mPRK2(A66K,A155K), or HA-tagged mPRK2ΔC2 and selected in puromycin. Stable pools were transfected with PRK2 siRNA duplex1 or siControl and analyzed at 3 days posttransfection. (C) Western blot analysis of cell lysates with the indicated antibodies. Note that the PRK2 antibody does not recognize the mouse PRK2 constructs, whereas the phospho-PRK antibody has been raised against a site that is completely conserved between human and mouse PRK2. The phospho-PRK antibody also recognizes PRK1. Note that mPRK2ΔC2 runs at the same size as endogenous PRK1. (D) Cells were fixed and stained with anti-ZO-1 (green) and Hoechst stain (blue). Scale bar shows 20 μm for all images. (E) Quantification of apical junction formation (see Materials and Methods) from 3 independent experiments. Error bars, SEM; nsd, no significant difference; **, P
Figure Legend Snippet: PRK2 function is RhoA-dependent, kinase-dependent, and requires its C2-like domain. (A) Domain organization of PRK2, including HR1 (homology region 1) GTPase-binding domains, a C2-like domain, and a serine-threonine kinase domain. (B) Wild-type mouse PRK2 (mPRK2) but not mPRK2(A66K,A155K) interacts with L63RhoA in coimmunoprecipitation experiments when overexpressed in HEK293T cells. WB, Western blotting; IP, immunoprecipitation. (C to E) 16HBE cells were infected with pBABE retroviral vectors expressing HA-tagged mPRK2, HA-tagged mPRK2(A66K,A155K), or HA-tagged mPRK2ΔC2 and selected in puromycin. Stable pools were transfected with PRK2 siRNA duplex1 or siControl and analyzed at 3 days posttransfection. (C) Western blot analysis of cell lysates with the indicated antibodies. Note that the PRK2 antibody does not recognize the mouse PRK2 constructs, whereas the phospho-PRK antibody has been raised against a site that is completely conserved between human and mouse PRK2. The phospho-PRK antibody also recognizes PRK1. Note that mPRK2ΔC2 runs at the same size as endogenous PRK1. (D) Cells were fixed and stained with anti-ZO-1 (green) and Hoechst stain (blue). Scale bar shows 20 μm for all images. (E) Quantification of apical junction formation (see Materials and Methods) from 3 independent experiments. Error bars, SEM; nsd, no significant difference; **, P

Techniques Used: Binding Assay, Western Blot, Immunoprecipitation, Infection, Expressing, Transfection, Construct, Staining

21) Product Images from "Negative Regulation of the RalGAP Complex by 14-3-3 *"

Article Title: Negative Regulation of the RalGAP Complex by 14-3-3 *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.426106

RGC2 T715 phosphorylation is required for 14-3-3 binding. A , schematic of λ-phosphatase protection assay. IP , immunoprecipitation. B , λ-phosphatase protection assay. 293T cells were transfected with empty vector or HA-RGC1 and FLAG-RGC2.
Figure Legend Snippet: RGC2 T715 phosphorylation is required for 14-3-3 binding. A , schematic of λ-phosphatase protection assay. IP , immunoprecipitation. B , λ-phosphatase protection assay. 293T cells were transfected with empty vector or HA-RGC1 and FLAG-RGC2.

Techniques Used: Binding Assay, Immunoprecipitation, Transfection, Plasmid Preparation

14-3-3 binding does not affect RalGAP complex in vitro catalytic activity. A , 293A cells were transfected with empty vector or HA-RGC1 and FLAG-RGC2 as indicated. Two days after transfection, cell lysates were subjected to immunoprecipitation ( IP ) using
Figure Legend Snippet: 14-3-3 binding does not affect RalGAP complex in vitro catalytic activity. A , 293A cells were transfected with empty vector or HA-RGC1 and FLAG-RGC2 as indicated. Two days after transfection, cell lysates were subjected to immunoprecipitation ( IP ) using

Techniques Used: Binding Assay, In Vitro, Activity Assay, Transfection, Plasmid Preparation, Immunoprecipitation

14-3-3 interacts with the RalGAP complex. A , 293T cells were transfected with empty vector or HA-RGC1 and FLAG-RGC2 as indicated. Two days after transfection, cell lysates were subjected to immunoprecipitation ( IP ) using anti-FLAG antibody ( left panel
Figure Legend Snippet: 14-3-3 interacts with the RalGAP complex. A , 293T cells were transfected with empty vector or HA-RGC1 and FLAG-RGC2 as indicated. Two days after transfection, cell lysates were subjected to immunoprecipitation ( IP ) using anti-FLAG antibody ( left panel

Techniques Used: Transfection, Plasmid Preparation, Immunoprecipitation

22) Product Images from "PKCζ regulates Notch receptor routing and activity in a Notch signaling-dependent manner"

Article Title: PKCζ regulates Notch receptor routing and activity in a Notch signaling-dependent manner

Journal: Cell Research

doi: 10.1038/cr.2014.34

Notch1 interacts with PKCζ at the membrane. (A) Untransfected C2C12 cells undergoing differentiation were harvested at different time points and subjected to immunoprecipitation (IP) using a Notch1 antibody (C20 goat). Immunoblotting was performed
Figure Legend Snippet: Notch1 interacts with PKCζ at the membrane. (A) Untransfected C2C12 cells undergoing differentiation were harvested at different time points and subjected to immunoprecipitation (IP) using a Notch1 antibody (C20 goat). Immunoblotting was performed

Techniques Used: Immunoprecipitation

23) Product Images from "Phospho-regulation of soma-to-axon transcytosis of neurotrophin receptors"

Article Title: Phospho-regulation of soma-to-axon transcytosis of neurotrophin receptors

Journal: Developmental cell

doi: 10.1016/j.devcel.2017.08.009

PTP1B dephosphorylates endocytosed soma surface-derived Trk receptors (A) NGF-induced association of TrkA with PTP1B Y46F/D181A substrate trapping mutant, but not PTP1B WT , in sympathetic neurons. Adenoviruses under Tet-On regulation were used to express PTP1B WT -Myc or PTP1B Y46F/D181A -Myc in sympathetic neurons. No exogenous PTP1B-Myc expression is observed in absence of doxycycline (Dox). Immunoprecipitation was done with anti-Myc, and immunoblotting with anti-Myc or anti-TrkA antibodies. (B) NGF-induced tyrosine phosphorylation of TrkA is suppressed by PTP1B WT over-expression, and enhanced by PTP1B Y46F/D181A substrate-trapping mutant. (C) Densitometric quantification of P-TrkA levels normalized to total TrkA. **p
Figure Legend Snippet: PTP1B dephosphorylates endocytosed soma surface-derived Trk receptors (A) NGF-induced association of TrkA with PTP1B Y46F/D181A substrate trapping mutant, but not PTP1B WT , in sympathetic neurons. Adenoviruses under Tet-On regulation were used to express PTP1B WT -Myc or PTP1B Y46F/D181A -Myc in sympathetic neurons. No exogenous PTP1B-Myc expression is observed in absence of doxycycline (Dox). Immunoprecipitation was done with anti-Myc, and immunoblotting with anti-Myc or anti-TrkA antibodies. (B) NGF-induced tyrosine phosphorylation of TrkA is suppressed by PTP1B WT over-expression, and enhanced by PTP1B Y46F/D181A substrate-trapping mutant. (C) Densitometric quantification of P-TrkA levels normalized to total TrkA. **p

Techniques Used: Derivative Assay, Mutagenesis, Expressing, Immunoprecipitation, Over Expression

24) Product Images from "Study of Plasmodium falciparum DHHC palmitoyl transferases identifies a role for PfDHHC9 in gametocytogenesis) Study of Plasmodium falciparum DHHC palmitoyl transferases identifies a role for PfDHHC9 in gametocytogenesis"

Article Title: Study of Plasmodium falciparum DHHC palmitoyl transferases identifies a role for PfDHHC9 in gametocytogenesis) Study of Plasmodium falciparum DHHC palmitoyl transferases identifies a role for PfDHHC9 in gametocytogenesis

Journal: Cellular Microbiology

doi: 10.1111/cmi.12599

Immunoprecipitation of mutant PfSec22 and PfARO proteins using antibodies against the c‐Myc tag. Human embryonic kidney 293E cells were co‐transfected with plasmids coding for the expression of mutant c‐Myc‐tagged PfSec22 or PfARO (which had the predicted palmitoylated cysteine residues mutated to alanine residues), along with FLAG‐tagged PfDHHC5 or the control vector (CD4). The mutant PfSec22 and PfARO proteins were immunoprecipitated from cell lysates using α ‐c‐Myc antibody. The proteins were separated by SDS‐PAGE and visualized by immunoblot, using α ‐c‐Myc antibody from a different species. A. Immunoprecipitation of PfSec22 point mutants (PfSec22‐C2dA, PfSec22‐C8dA and PfSec22‐C2C8dA) along with wild‐type PfSec22. The amino acid sequence of the N‐terminal region of PfSec22 is also shown with the cysteine residues of interest highlighted in red. B. Immunoprecipitation of PfARO point mutants (PfARO‐C5dA, PfARO‐C6dA and PfARO‐C5C6dA) along with wild‐type PfARO. The amino acid sequence of the N‐terminal region of PfARO is also shown with the cysteine residues of interest highlighted in red.
Figure Legend Snippet: Immunoprecipitation of mutant PfSec22 and PfARO proteins using antibodies against the c‐Myc tag. Human embryonic kidney 293E cells were co‐transfected with plasmids coding for the expression of mutant c‐Myc‐tagged PfSec22 or PfARO (which had the predicted palmitoylated cysteine residues mutated to alanine residues), along with FLAG‐tagged PfDHHC5 or the control vector (CD4). The mutant PfSec22 and PfARO proteins were immunoprecipitated from cell lysates using α ‐c‐Myc antibody. The proteins were separated by SDS‐PAGE and visualized by immunoblot, using α ‐c‐Myc antibody from a different species. A. Immunoprecipitation of PfSec22 point mutants (PfSec22‐C2dA, PfSec22‐C8dA and PfSec22‐C2C8dA) along with wild‐type PfSec22. The amino acid sequence of the N‐terminal region of PfSec22 is also shown with the cysteine residues of interest highlighted in red. B. Immunoprecipitation of PfARO point mutants (PfARO‐C5dA, PfARO‐C6dA and PfARO‐C5C6dA) along with wild‐type PfARO. The amino acid sequence of the N‐terminal region of PfARO is also shown with the cysteine residues of interest highlighted in red.

Techniques Used: Immunoprecipitation, Mutagenesis, Transfection, Expressing, Plasmid Preparation, SDS Page, Sequencing

Palmitoyl‐transferase activity assay demonstrating the palmitoyl‐transferase activity of the PfDHHC proteins on PfSec22. A. Immunoprecipitation of PfSec22 co‐expressed with each PfDHHC protein. Human embryonic kidney 293E cells were co‐transfected with plasmids coding for the expression of c‐Myc‐tagged PfSec22, along with the indicated FLAG‐tagged PfDHHC proteins (PfDHHC3, 5, 7 and 9) or the control vector (CD4). Pfsec22 was immunoprecipitated from cell lysates using α ‐c‐Myc antibody. The proteins were separated by SDS‐PAGE and visualized by immunoblot, using α ‐c‐Myc antibody from a different species. B. PAT activity assay. Human embryonic kidney 293E cells were co‐transfected with plasmids expressing c‐Myc‐tagged PfSec22, along with the indicated FLAG‐tagged PfDHHC protein or the control vector (CD4). Cells were either treated with the metabolic label, 17‐octadecynoic acid (17‐ODYA) or mock‐treated with DMSO. Proteins were extracted and an aliquot of each lysate kept aside to confirm protein expression. The remaining lysates were put through click chemistry reactions to biotin‐azide, and 17‐ODYA‐labelled proteins were streptavidin affinity purified and eluted by boiling in SDS. Samples from the initial lysates and the click chemistry elutions were separated by SDS‐PAGE, and the presence of c‐Myc‐tagged PfSec22 in each of the samples was observed by immunoblot using antibodies against the c‐Myc tag.
Figure Legend Snippet: Palmitoyl‐transferase activity assay demonstrating the palmitoyl‐transferase activity of the PfDHHC proteins on PfSec22. A. Immunoprecipitation of PfSec22 co‐expressed with each PfDHHC protein. Human embryonic kidney 293E cells were co‐transfected with plasmids coding for the expression of c‐Myc‐tagged PfSec22, along with the indicated FLAG‐tagged PfDHHC proteins (PfDHHC3, 5, 7 and 9) or the control vector (CD4). Pfsec22 was immunoprecipitated from cell lysates using α ‐c‐Myc antibody. The proteins were separated by SDS‐PAGE and visualized by immunoblot, using α ‐c‐Myc antibody from a different species. B. PAT activity assay. Human embryonic kidney 293E cells were co‐transfected with plasmids expressing c‐Myc‐tagged PfSec22, along with the indicated FLAG‐tagged PfDHHC protein or the control vector (CD4). Cells were either treated with the metabolic label, 17‐octadecynoic acid (17‐ODYA) or mock‐treated with DMSO. Proteins were extracted and an aliquot of each lysate kept aside to confirm protein expression. The remaining lysates were put through click chemistry reactions to biotin‐azide, and 17‐ODYA‐labelled proteins were streptavidin affinity purified and eluted by boiling in SDS. Samples from the initial lysates and the click chemistry elutions were separated by SDS‐PAGE, and the presence of c‐Myc‐tagged PfSec22 in each of the samples was observed by immunoblot using antibodies against the c‐Myc tag.

Techniques Used: Activity Assay, Immunoprecipitation, Transfection, Expressing, Plasmid Preparation, SDS Page, Affinity Purification

Palmitoyl‐transferase activity assay demonstrating the palmitoyl‐transferase activity of the PfDHHC proteins on PfARO. A. Immunoprecipitation of PfARO co‐expressed with each PfDHHC protein. Human embryonic kidney 293E cells were co‐transfected with plasmids coding for the expression of c‐Myc‐tagged PfARO, along with the indicated FLAG‐tagged PfDHHC proteins (PfDHHC3, 5, 7 and 9) or the control vector (CD4). PfARO was immunoprecipitated from cell lysates using α ‐c‐Myc antibody. The proteins were separated by SDS‐PAGE and visualized by immunoblot, using α ‐c‐Myc antibody from a different species. B. PAT activity assay. Human embryonic kidney 293E cells were co‐transfected with plasmids expressing c‐Myc‐tagged PfARO, along with the indicated FLAG‐tagged PfDHHC protein or the control vector, CD4. Cells were either treated with the metabolic label, 17‐octadecynoic acid or mock‐treated with DMSO. Proteins were extracted, and an aliquot of each lysate kept aside to confirm protein expression. The remaining lysates were put through click chemistry reactions to biotin‐azide, and 17‐octadecynoic acid‐labelled proteins were streptavidin affinity purified and eluted by boiling in SDS. Samples from the initial lysates and the click chemistry elutions were separated by SDS‐PAGE, and the presence of c‐Myc‐tagged PfARO in each of the samples was observed by immunoblot using antibodies against the c‐Myc tag.
Figure Legend Snippet: Palmitoyl‐transferase activity assay demonstrating the palmitoyl‐transferase activity of the PfDHHC proteins on PfARO. A. Immunoprecipitation of PfARO co‐expressed with each PfDHHC protein. Human embryonic kidney 293E cells were co‐transfected with plasmids coding for the expression of c‐Myc‐tagged PfARO, along with the indicated FLAG‐tagged PfDHHC proteins (PfDHHC3, 5, 7 and 9) or the control vector (CD4). PfARO was immunoprecipitated from cell lysates using α ‐c‐Myc antibody. The proteins were separated by SDS‐PAGE and visualized by immunoblot, using α ‐c‐Myc antibody from a different species. B. PAT activity assay. Human embryonic kidney 293E cells were co‐transfected with plasmids expressing c‐Myc‐tagged PfARO, along with the indicated FLAG‐tagged PfDHHC protein or the control vector, CD4. Cells were either treated with the metabolic label, 17‐octadecynoic acid or mock‐treated with DMSO. Proteins were extracted, and an aliquot of each lysate kept aside to confirm protein expression. The remaining lysates were put through click chemistry reactions to biotin‐azide, and 17‐octadecynoic acid‐labelled proteins were streptavidin affinity purified and eluted by boiling in SDS. Samples from the initial lysates and the click chemistry elutions were separated by SDS‐PAGE, and the presence of c‐Myc‐tagged PfARO in each of the samples was observed by immunoblot using antibodies against the c‐Myc tag.

Techniques Used: Activity Assay, Immunoprecipitation, Transfection, Expressing, Plasmid Preparation, SDS Page, Affinity Purification

25) Product Images from "NP1 Regulates Neuronal Activity-Dependent Accumulation of BAX in Mitochondria and Mitochondrial Dynamics"

Article Title: NP1 Regulates Neuronal Activity-Dependent Accumulation of BAX in Mitochondria and Mitochondrial Dynamics

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.4604-11.2012

BAX immunoprecipitates NP1 and NP1 colocalizes with active BAX in mitochondria. A–C , Representative Western blots of total protein lysate (TL), the eluate of BAX immunoprecipitation (IP; Bax), and the eluate of IgG immunoprecipitation controls (IgG). A , Immunoprecipitation of BAX from total brain lysates. Rat brains were homogenized, and protein lysates prepared as described in Materials and Methods. B , Immunoprecipitation of BAX from CGN lysates. CGNs were treated with low K + for 2 h. C , Immunoprecipitation of BAX from HEK 293T cell lysates transfected with recombinant rat Bax and NP1 cDNAs. D , Confocal microscopy studies were performed in mature CGNs in culture after 4 h of K + deprivation. Orthogonal sectioning shows colocalization between NP1 and active BAX in mitochondria. Colocalization between NP1 and MtDsRed is seen as yellow, between NP1 and BAX as cyan, and between BAX and MtDsRed as magenta, and colocalization between all three proteins is shown in white. Scale bar, 5 μm.
Figure Legend Snippet: BAX immunoprecipitates NP1 and NP1 colocalizes with active BAX in mitochondria. A–C , Representative Western blots of total protein lysate (TL), the eluate of BAX immunoprecipitation (IP; Bax), and the eluate of IgG immunoprecipitation controls (IgG). A , Immunoprecipitation of BAX from total brain lysates. Rat brains were homogenized, and protein lysates prepared as described in Materials and Methods. B , Immunoprecipitation of BAX from CGN lysates. CGNs were treated with low K + for 2 h. C , Immunoprecipitation of BAX from HEK 293T cell lysates transfected with recombinant rat Bax and NP1 cDNAs. D , Confocal microscopy studies were performed in mature CGNs in culture after 4 h of K + deprivation. Orthogonal sectioning shows colocalization between NP1 and active BAX in mitochondria. Colocalization between NP1 and MtDsRed is seen as yellow, between NP1 and BAX as cyan, and between BAX and MtDsRed as magenta, and colocalization between all three proteins is shown in white. Scale bar, 5 μm.

Techniques Used: Western Blot, Immunoprecipitation, Transfection, Recombinant, Confocal Microscopy

26) Product Images from "The Fission Yeast Nup107-120 Complex Functionally Interacts with the Small GTPase Ran/Spi1 and Is Required for mRNA Export, Nuclear Pore Distribution, and Proper Cell Division"

Article Title: The Fission Yeast Nup107-120 Complex Functionally Interacts with the Small GTPase Ran/Spi1 and Is Required for mRNA Export, Nuclear Pore Distribution, and Proper Cell Division

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.24.14.6379-6392.2004

The S. pombe Nup107-120 NPC subcomplex biochemically purifies as two distinct entities. (A) Western blot analysis of immunoprecipitation experiments performed on S. pombe whole-cell lysates using an anti-GFP antibody. S. pombe strains expressing an HA-Nup133a, -Nup133b, or -Nup120 fusion in a Δ nup133a , Δ nup133b , or Δ nup120 background, respectively, and either no GFP fusion or a GFP-tagged nucleoporin (Nup107-GFP, GFP-Nup85, or GFP-Seh1) were grown at 30°C to log phase in supplemented liquid EMM in the presence of thiamine. Immunoprecipitation experiments were performed on these 12 different S. pombe whole-cell lysates using an anti-GFP antibody. Equivalent amounts of total protein extracts (T), depleted supernatants (S), and a 10-fold equivalent of the immune pellets (P) were analyzed by Western blotting using anti-GFP (a) or anti-HA (a′, b′, and c′) antibody. The results with the GFP antibody were similar in all experiments and are presented only for the strain expressing HA-Nup133a. Molecular mass markers are on the right in kilodaltons. (B) Silver staining of immunoprecipitates from control, GFP-SpNup85, GFP-Seh1, and Nup107-GFP cell extracts obtained with an anti-GFP antibody. The asterisks indicate the position of the GFP fusion in each lane. The dots indicate additional bands subsequently identified by mass spectrometry as Nup145-C (band 1) and Nup85 (band 2). (C) Schematic model of the Nup107-120 complex in S. pombe , based on the structural data obtained from S. cerevisiae ).
Figure Legend Snippet: The S. pombe Nup107-120 NPC subcomplex biochemically purifies as two distinct entities. (A) Western blot analysis of immunoprecipitation experiments performed on S. pombe whole-cell lysates using an anti-GFP antibody. S. pombe strains expressing an HA-Nup133a, -Nup133b, or -Nup120 fusion in a Δ nup133a , Δ nup133b , or Δ nup120 background, respectively, and either no GFP fusion or a GFP-tagged nucleoporin (Nup107-GFP, GFP-Nup85, or GFP-Seh1) were grown at 30°C to log phase in supplemented liquid EMM in the presence of thiamine. Immunoprecipitation experiments were performed on these 12 different S. pombe whole-cell lysates using an anti-GFP antibody. Equivalent amounts of total protein extracts (T), depleted supernatants (S), and a 10-fold equivalent of the immune pellets (P) were analyzed by Western blotting using anti-GFP (a) or anti-HA (a′, b′, and c′) antibody. The results with the GFP antibody were similar in all experiments and are presented only for the strain expressing HA-Nup133a. Molecular mass markers are on the right in kilodaltons. (B) Silver staining of immunoprecipitates from control, GFP-SpNup85, GFP-Seh1, and Nup107-GFP cell extracts obtained with an anti-GFP antibody. The asterisks indicate the position of the GFP fusion in each lane. The dots indicate additional bands subsequently identified by mass spectrometry as Nup145-C (band 1) and Nup85 (band 2). (C) Schematic model of the Nup107-120 complex in S. pombe , based on the structural data obtained from S. cerevisiae ).

Techniques Used: Western Blot, Immunoprecipitation, Expressing, Silver Staining, Mass Spectrometry

27) Product Images from "Membrane protein recycling from the vacuole/lysosome membrane"

Article Title: Membrane protein recycling from the vacuole/lysosome membrane

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201709162

Specific residues in the cytoplasmic tail of Atg27 are required for its retrograde traffic. (A) Schematic of Atg27 mutational analysis from Fig. S3 C. (B) Atg27-diL-GFP localization after 1 h of rapamycin treatment. (C) The percentage of cells with Atg27-diL-GFP on the vacuole in B. (D) Atg27-diL-GFP mutant localization in vps35 Δ cells after 1 h of rapamycin treatment. (E) The percentage of cells with Atg27-diL-GFP on the vacuole in D and Fig. S3 D. (F) Cells were treated with rapamycin for 1 h and Atg27-diL immunoprecipitated. Interacting Snx4-3XHA mutants were detected by immunoblotting. (G) Model for Atg27 trafficking. Atg27 is delivered to the vacuole membrane via the AP-3 pathway and then recycled from the vacuole membrane to the endosome by the Snx4 complex. Atg27 is then retrieved from the endosome by retromer. (H) Membrane trafficking pathways in yeast cells. For all quantification shown in this figure, at least 100 cells were measured, and the data from three independent experiments were used for statistical analysis; two-tailed Student’s t test. Error bars represent SD. IP, immunoprecipitation. Bars, 2 µm.
Figure Legend Snippet: Specific residues in the cytoplasmic tail of Atg27 are required for its retrograde traffic. (A) Schematic of Atg27 mutational analysis from Fig. S3 C. (B) Atg27-diL-GFP localization after 1 h of rapamycin treatment. (C) The percentage of cells with Atg27-diL-GFP on the vacuole in B. (D) Atg27-diL-GFP mutant localization in vps35 Δ cells after 1 h of rapamycin treatment. (E) The percentage of cells with Atg27-diL-GFP on the vacuole in D and Fig. S3 D. (F) Cells were treated with rapamycin for 1 h and Atg27-diL immunoprecipitated. Interacting Snx4-3XHA mutants were detected by immunoblotting. (G) Model for Atg27 trafficking. Atg27 is delivered to the vacuole membrane via the AP-3 pathway and then recycled from the vacuole membrane to the endosome by the Snx4 complex. Atg27 is then retrieved from the endosome by retromer. (H) Membrane trafficking pathways in yeast cells. For all quantification shown in this figure, at least 100 cells were measured, and the data from three independent experiments were used for statistical analysis; two-tailed Student’s t test. Error bars represent SD. IP, immunoprecipitation. Bars, 2 µm.

Techniques Used: Mutagenesis, Immunoprecipitation, Two Tailed Test

The Snx4 complex mediates Atg27 vacuole-to-endosome retrograde transport. (A) Schematic predicting Atg27 accumulation on the vacuole membrane in retromer and recycling double mutants. (B) Atg27-GFP localization in vps35 Δ snx4 Δ cells after 1 h of rapamycin treatment. (C) The percentage of cells with Atg27-GFP on the vacuole. (D) Atg27-GFP localization in vps35ts snx4 Δ mutants after shift to 37°C with rapamycin treatment for 1 h. (E) Quantification of Atg27-GFP fluorescence intensity on the vacuole from D and Fig. S2 C. (F) Cells were treated with rapamycin for 1 h, Atg27-GFP was immunoprecipitated, and interacting Snx4-3XHA was detected by immunoblot. For all quantification shown in this figure, at least 100 cells were measured, and the data from three independent experiments were used for statistical analysis; two-tailed Student’s t test. Error bars represent SD. IP, immunoprecipitation; n.s., not significant. Bar, 2 µm.
Figure Legend Snippet: The Snx4 complex mediates Atg27 vacuole-to-endosome retrograde transport. (A) Schematic predicting Atg27 accumulation on the vacuole membrane in retromer and recycling double mutants. (B) Atg27-GFP localization in vps35 Δ snx4 Δ cells after 1 h of rapamycin treatment. (C) The percentage of cells with Atg27-GFP on the vacuole. (D) Atg27-GFP localization in vps35ts snx4 Δ mutants after shift to 37°C with rapamycin treatment for 1 h. (E) Quantification of Atg27-GFP fluorescence intensity on the vacuole from D and Fig. S2 C. (F) Cells were treated with rapamycin for 1 h, Atg27-GFP was immunoprecipitated, and interacting Snx4-3XHA was detected by immunoblot. For all quantification shown in this figure, at least 100 cells were measured, and the data from three independent experiments were used for statistical analysis; two-tailed Student’s t test. Error bars represent SD. IP, immunoprecipitation; n.s., not significant. Bar, 2 µm.

Techniques Used: Fluorescence, Immunoprecipitation, Two Tailed Test

28) Product Images from "The RNA helicase MOV10L1 binds piRNA precursors to initiate piRNA processing"

Article Title: The RNA helicase MOV10L1 binds piRNA precursors to initiate piRNA processing

Journal: Genes & Development

doi: 10.1101/gad.254631.114

G4 antigenicity of piRNA precursor transcripts increases in Mov10l1 mutant mice. ( A ) RNA immunoprecipitation of piRNA precursor transcripts using the BG4 antibody that specifically recognizes RNA G4s using testis lysates from wild-type (WT) and Mov10l1
Figure Legend Snippet: G4 antigenicity of piRNA precursor transcripts increases in Mov10l1 mutant mice. ( A ) RNA immunoprecipitation of piRNA precursor transcripts using the BG4 antibody that specifically recognizes RNA G4s using testis lysates from wild-type (WT) and Mov10l1

Techniques Used: Mutagenesis, Mouse Assay, Immunoprecipitation

29) Product Images from "Differential TOR activation and cell proliferation in Arabidopsis root and shoot apexes"

Article Title: Differential TOR activation and cell proliferation in Arabidopsis root and shoot apexes

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1618782114

ROP2 mediated the light-auxin activation of TOR. ( A ) ROP2 directly interacted with TOR kinase as tested by immunoprecipitation from shoot apexes of seedlings and Western blot analysis. ( B ) CA-ROP2 activated TOR kinase. Protoplasts coexpressing S6K1 with
Figure Legend Snippet: ROP2 mediated the light-auxin activation of TOR. ( A ) ROP2 directly interacted with TOR kinase as tested by immunoprecipitation from shoot apexes of seedlings and Western blot analysis. ( B ) CA-ROP2 activated TOR kinase. Protoplasts coexpressing S6K1 with

Techniques Used: Activation Assay, Immunoprecipitation, Western Blot

30) Product Images from "Transcription factors RUNX1 and RUNX3 in the induction and suppressive function of Foxp3+ inducible regulatory T cells"

Article Title: Transcription factors RUNX1 and RUNX3 in the induction and suppressive function of Foxp3+ inducible regulatory T cells

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20090596

Binding of RUNX1 and RUNX3 proteins to the predicted binding sites in the FOXP3 promoter. (A) Mutated and wild-type oligonucleotides are shown. The predicted RUNX binding sites are accentuated (boxed and in green letters) and stars mark mutations introduced into the binding site of the control oligonucleotides. Nuclear extracts from HEK293T cells were incubated with biotinylated oligonucleotides. The precipitated oligonucleotide–transcription factor complexes were separated by SDS-PAGE and identified by Western blotting with anti-RUNX1 and anti-RUNX3 antibodies. A mixture of all three oligonucleotides with the predicted binding sites or with the inserted mutation into the predicted sites was used. Data shown are one representative of three independent experiments with similar results. (B) Promoter enzyme immunoassay using wild-type and mutated oligonucleotides within the FOXP3 promoter. Bars show mean ± SE of three independent experiments. (C) Chromatin immunoprecipitation assay results show binding of RUNX1 and RUNX3 complexes containing CBFβ to the human FOXP3 promoter in naive CD4 + T cells that were cultured with IL-2 together with anti-CD2/3/28 and TGF-β. There was no change in site occupancy in all immunoprecipitations when IGX1A negative control primers were used. The results are normalized to input and isotype control antibody. Bars show mean ± SE of three independent experiments. Statistical differences were verified by the paired Student's t test. *, P
Figure Legend Snippet: Binding of RUNX1 and RUNX3 proteins to the predicted binding sites in the FOXP3 promoter. (A) Mutated and wild-type oligonucleotides are shown. The predicted RUNX binding sites are accentuated (boxed and in green letters) and stars mark mutations introduced into the binding site of the control oligonucleotides. Nuclear extracts from HEK293T cells were incubated with biotinylated oligonucleotides. The precipitated oligonucleotide–transcription factor complexes were separated by SDS-PAGE and identified by Western blotting with anti-RUNX1 and anti-RUNX3 antibodies. A mixture of all three oligonucleotides with the predicted binding sites or with the inserted mutation into the predicted sites was used. Data shown are one representative of three independent experiments with similar results. (B) Promoter enzyme immunoassay using wild-type and mutated oligonucleotides within the FOXP3 promoter. Bars show mean ± SE of three independent experiments. (C) Chromatin immunoprecipitation assay results show binding of RUNX1 and RUNX3 complexes containing CBFβ to the human FOXP3 promoter in naive CD4 + T cells that were cultured with IL-2 together with anti-CD2/3/28 and TGF-β. There was no change in site occupancy in all immunoprecipitations when IGX1A negative control primers were used. The results are normalized to input and isotype control antibody. Bars show mean ± SE of three independent experiments. Statistical differences were verified by the paired Student's t test. *, P

Techniques Used: Binding Assay, Incubation, SDS Page, Western Blot, Mutagenesis, Enzyme-linked Immunosorbent Assay, Chromatin Immunoprecipitation, Cell Culture, Negative Control

31) Product Images from "Arrestin and the Multi-PDZ Domain-containing Protein MPZ-1 Interact with Phosphatase and Tensin Homolog (PTEN) and Regulate Caenorhabditis elegans Longevity * Longevity * ♦"

Article Title: Arrestin and the Multi-PDZ Domain-containing Protein MPZ-1 Interact with Phosphatase and Tensin Homolog (PTEN) and Regulate Caenorhabditis elegans Longevity * Longevity * ♦

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.104612

ARR-1 forms a complex with MPZ-1 and DAF-18. A , left panel , GST pulldown showing an interaction between MPZ-1 PDZ domains 6–10 and ARR-1. The input equals 0.5% of the lysate used in the binding experiment. Right panel , GST pulldown showing an interaction between MPZ-1 PDZ domains 6–10 and DAF-18 ( right panel ). The input equals 2% of the lysate used in the binding experiment. IB , immunoblot. B , co-immunoprecipitation ( IP ) assay showing that ARR-1, HA-tagged DAF-18, and FLAG-tagged MPZ-1 PDZ domains 6–10 precipitate together in a complex. The bottom blot represents protein expression in ∼4 μg of total protein lysate. C , co-immunoprecipitation assay showing an interaction between ARR-1 and HA-tagged DAF-18. The bottom blot represents protein expression in ∼4 μg of total protein lysate. D , proposed model illustrating the role of ARR-1 in the DAF-2 signaling pathway. DAF-2 activation leads to the phosphorylation and negative regulation of the transcription factor DAF-16, an event mediated by the kinase signaling cascade downstream of DAF-2, consisting of AGE-1, PDK-1, and SGK-1/AKT 1/2. ARR-1 forms a complex with MPZ-1 and DAF-18 within the DAF-2 signaling pathway, where ARR-1 and MPZ-1 act together to regulate DAF-18 and ultimately function to positively regulate DAF-2. In turn, this negatively regulates DAF-16, leading to an increase in longevity.
Figure Legend Snippet: ARR-1 forms a complex with MPZ-1 and DAF-18. A , left panel , GST pulldown showing an interaction between MPZ-1 PDZ domains 6–10 and ARR-1. The input equals 0.5% of the lysate used in the binding experiment. Right panel , GST pulldown showing an interaction between MPZ-1 PDZ domains 6–10 and DAF-18 ( right panel ). The input equals 2% of the lysate used in the binding experiment. IB , immunoblot. B , co-immunoprecipitation ( IP ) assay showing that ARR-1, HA-tagged DAF-18, and FLAG-tagged MPZ-1 PDZ domains 6–10 precipitate together in a complex. The bottom blot represents protein expression in ∼4 μg of total protein lysate. C , co-immunoprecipitation assay showing an interaction between ARR-1 and HA-tagged DAF-18. The bottom blot represents protein expression in ∼4 μg of total protein lysate. D , proposed model illustrating the role of ARR-1 in the DAF-2 signaling pathway. DAF-2 activation leads to the phosphorylation and negative regulation of the transcription factor DAF-16, an event mediated by the kinase signaling cascade downstream of DAF-2, consisting of AGE-1, PDK-1, and SGK-1/AKT 1/2. ARR-1 forms a complex with MPZ-1 and DAF-18 within the DAF-2 signaling pathway, where ARR-1 and MPZ-1 act together to regulate DAF-18 and ultimately function to positively regulate DAF-2. In turn, this negatively regulates DAF-16, leading to an increase in longevity.

Techniques Used: Binding Assay, Immunoprecipitation, Expressing, Co-Immunoprecipitation Assay, Activation Assay, Activated Clotting Time Assay

32) Product Images from "African Swine Fever Virus Polyproteins pp220 and pp62 Assemble into the Core Shell"

Article Title: African Swine Fever Virus Polyproteins pp220 and pp62 Assemble into the Core Shell

Journal: Journal of Virology

doi: 10.1128/JVI.76.24.12473-12482.2002

Gel mapping, stoichiometry, and relative abundances of ASFV polyprotein products in the virus particle. (A) 2-D gel electrophoresis of ASFV labeled with [ 35 S]methionine. Basic proteins were resolved by NEPHGE, while acidic proteins were separated by IEF. The positions of the major capsid protein p72; the polyprotein pp220-derived products p150, p37, p34, and p14; and the polyprotein pp62-derived products p35 and p15 are indicated. The migrations of molecular weight (MW) markers (in thousands) and of pI markers are indicated at the right and at the bottom of the 2-D gels, respectively. (B) 2-D mapping of proteins p35 and p15. Protein p15 was mapped in NEPHGE gels by immunoprecipitation, whereas p35 was identified in IEF gels by immunoblotting. (C) 1-D gel electrophoresis of ASFV particles labeled with [ 35 S]methionine or 14 C-amino acids or stained with Coomassie blue (C.B.). The positions of several structural proteins are indicated. (D) Quantitative analysis of the polyprotein products. Polyprotein products and, as a reference, capsid protein p72 were quantified by densitometry analysis of 2-D gels of [ 35 S]methionine-labeled proteins (two experiments) or of 1-D gels of ASFV proteins labeled with [ 35 S]methionine (three experiments) or 14 C-amino acids (two experiments) or stained with Coomassie blue (three experiments). Stoichiometry data were normalized by the number of methionine residues of each protein (for 35 S-labeled proteins) or by its molecular weight (for 14 C-labeled or Coomassie blue-stained proteins) and referred to protein p34. Relative abundances (percentages) were estimated from 1-D gels of 14 C-labeled or Coomassie blue-stained proteins. Standard deviations of the means were less than 25% of the means. Proteins p150 and p72 were not quantified in 2-D gels due to deficient focusing, while proteins p15 and p14 were not estimated in 1-D gels because of heterogeneity of the bands. The relative abundances of proteins p15 and p14 are values extrapolated by assuming an equimolar stoichiometry. Stoichiometry data for p37 and p14 [marked with asterisks in the 35 .
Figure Legend Snippet: Gel mapping, stoichiometry, and relative abundances of ASFV polyprotein products in the virus particle. (A) 2-D gel electrophoresis of ASFV labeled with [ 35 S]methionine. Basic proteins were resolved by NEPHGE, while acidic proteins were separated by IEF. The positions of the major capsid protein p72; the polyprotein pp220-derived products p150, p37, p34, and p14; and the polyprotein pp62-derived products p35 and p15 are indicated. The migrations of molecular weight (MW) markers (in thousands) and of pI markers are indicated at the right and at the bottom of the 2-D gels, respectively. (B) 2-D mapping of proteins p35 and p15. Protein p15 was mapped in NEPHGE gels by immunoprecipitation, whereas p35 was identified in IEF gels by immunoblotting. (C) 1-D gel electrophoresis of ASFV particles labeled with [ 35 S]methionine or 14 C-amino acids or stained with Coomassie blue (C.B.). The positions of several structural proteins are indicated. (D) Quantitative analysis of the polyprotein products. Polyprotein products and, as a reference, capsid protein p72 were quantified by densitometry analysis of 2-D gels of [ 35 S]methionine-labeled proteins (two experiments) or of 1-D gels of ASFV proteins labeled with [ 35 S]methionine (three experiments) or 14 C-amino acids (two experiments) or stained with Coomassie blue (three experiments). Stoichiometry data were normalized by the number of methionine residues of each protein (for 35 S-labeled proteins) or by its molecular weight (for 14 C-labeled or Coomassie blue-stained proteins) and referred to protein p34. Relative abundances (percentages) were estimated from 1-D gels of 14 C-labeled or Coomassie blue-stained proteins. Standard deviations of the means were less than 25% of the means. Proteins p150 and p72 were not quantified in 2-D gels due to deficient focusing, while proteins p15 and p14 were not estimated in 1-D gels because of heterogeneity of the bands. The relative abundances of proteins p15 and p14 are values extrapolated by assuming an equimolar stoichiometry. Stoichiometry data for p37 and p14 [marked with asterisks in the 35 .

Techniques Used: Nucleic Acid Electrophoresis, Labeling, Electrofocusing, Derivative Assay, Molecular Weight, Immunoprecipitation, Staining

33) Product Images from "Plant A20/AN1 protein serves as the important hub to mediate antiviral immunity"

Article Title: Plant A20/AN1 protein serves as the important hub to mediate antiviral immunity

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1007288

SA regulates Pha13 at post-translational level. (A) Total proteins were extracted from leaves of P . aphrodite without treatment (0 h), treated with H 2 O (Mock), or SA at different times (h), and used for in vivo immunoprecipitation (IP) assay using anti-Pha13 antibody. Samples after IP were analyzed by immunoblotting using anti-Pha13 or anti-ubiquitin antibody. (B) P . aphrodite was treated with H 2 O (Mock) or SA, and immediately followed by infiltration of DMSO (-) or MG132 (+). Total proteins were extracted from leaves of the treated samples at 3 h post-treatment, and were used for in vivo IP assay using anti-Pha13 antibodies. Samples after IP were analyzed by immunoblotting using anti-Pha13 antibody. (A and B) Extracted total proteins (input) stained by coomassie brilliant blue (CBB) served as a loading control.
Figure Legend Snippet: SA regulates Pha13 at post-translational level. (A) Total proteins were extracted from leaves of P . aphrodite without treatment (0 h), treated with H 2 O (Mock), or SA at different times (h), and used for in vivo immunoprecipitation (IP) assay using anti-Pha13 antibody. Samples after IP were analyzed by immunoblotting using anti-Pha13 or anti-ubiquitin antibody. (B) P . aphrodite was treated with H 2 O (Mock) or SA, and immediately followed by infiltration of DMSO (-) or MG132 (+). Total proteins were extracted from leaves of the treated samples at 3 h post-treatment, and were used for in vivo IP assay using anti-Pha13 antibodies. Samples after IP were analyzed by immunoblotting using anti-Pha13 antibody. (A and B) Extracted total proteins (input) stained by coomassie brilliant blue (CBB) served as a loading control.

Techniques Used: In Vivo, Immunoprecipitation, Staining

34) Product Images from "Regulation of Ischemic Neuronal Death by E2F4-p130 Protein Complexes *"

Article Title: Regulation of Ischemic Neuronal Death by E2F4-p130 Protein Complexes *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.574145

p130 and E2F4 protein levels are down-regulated following hypoxia/reoxygenation of CGNs in culture. A , E2F4 forms a complex with p130 in CGNs. Total cell lysates from CGN cultures were subjected to reciprocal immunoprecipitation ( IP ) with anti-E2F4 and
Figure Legend Snippet: p130 and E2F4 protein levels are down-regulated following hypoxia/reoxygenation of CGNs in culture. A , E2F4 forms a complex with p130 in CGNs. Total cell lysates from CGN cultures were subjected to reciprocal immunoprecipitation ( IP ) with anti-E2F4 and

Techniques Used: Immunoprecipitation

35) Product Images from "Palmitoylation mediates membrane association of hepatitis E virus ORF3 protein and is required for infectious particle secretion"

Article Title: Palmitoylation mediates membrane association of hepatitis E virus ORF3 protein and is required for infectious particle secretion

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1007471

HEV ORF3 protein oligomerizes in mammalian cells. ( A ) Sequence analysis of ORF3 protein. Amino acid sequences of ORF3 from HEV genotypes 1–8 (GenBank accession numbers AB740232, AF444002, M74506, AJ272108, AB573435, AB856243, KJ496144 and KX387866) were aligned using ClustalW [ 52 ]. Segment aa 30–53 predicted as transmembrane passage by TMPred is boxed in grey [ 53 ]. A consensus secondary structure was predicted using algorithms MLRC, DSC and PHD (available at https://npsa-prabi.ibcp.fr ) and is shown below the alignment (c, random coil; h, α-helix; e, extended strand;?, discrepant prediction). The degree of aa physicochemical conservation at each position is shown on the bottom line and can be inferred with the similarity index according to ClustalW convention (asterisk, invariant; colon, highly similar; dot, similar) [ 52 ]. ( B ) ORF3 protein oligomerization was analyzed by FLAG immunoprecipitation. Lysates (Input) of U-2 OS cells transfected with pCMVORF3-HA and/or pCMVORF3-FLAG as well as immunoprecipitates (IP: FLAG) were subjected to immunoblot with either specific anti-FLAG or anti-HA antibodies. The presence of a strong signal for ORF3-HA after pull-down of ORF3-FLAG indicates oligomerization of ORF3 protein in cells. ( C ) FRET analyses reveal oligomerization of HEV ORF3. CFP (cyan fluorescent protein) or YFP (yellow fluorescent protein) fused to the C-termini of HEV ORF3 segments aa 1–113, 1–94, 1–70, 1–53, 28–113 or 53–113 were co-expressed in U-2 OS cells. FRET analyses were performed by the acceptor photobleaching method as described in the Materials and Methods section. The CFP-YFP fusion protein and cotransfection of unfused CFP and YFP served as positive and negative controls, respectively. Box-and-whisker plots represent the median FRET efficiency (FRETeff) values of 20 measurements (middle line), the values from the lower to the upper quartile (central box), and the minimum and maximum values (vertical line). The significance of the observed differences was assessed as described in Materials and Methods (*, P
Figure Legend Snippet: HEV ORF3 protein oligomerizes in mammalian cells. ( A ) Sequence analysis of ORF3 protein. Amino acid sequences of ORF3 from HEV genotypes 1–8 (GenBank accession numbers AB740232, AF444002, M74506, AJ272108, AB573435, AB856243, KJ496144 and KX387866) were aligned using ClustalW [ 52 ]. Segment aa 30–53 predicted as transmembrane passage by TMPred is boxed in grey [ 53 ]. A consensus secondary structure was predicted using algorithms MLRC, DSC and PHD (available at https://npsa-prabi.ibcp.fr ) and is shown below the alignment (c, random coil; h, α-helix; e, extended strand;?, discrepant prediction). The degree of aa physicochemical conservation at each position is shown on the bottom line and can be inferred with the similarity index according to ClustalW convention (asterisk, invariant; colon, highly similar; dot, similar) [ 52 ]. ( B ) ORF3 protein oligomerization was analyzed by FLAG immunoprecipitation. Lysates (Input) of U-2 OS cells transfected with pCMVORF3-HA and/or pCMVORF3-FLAG as well as immunoprecipitates (IP: FLAG) were subjected to immunoblot with either specific anti-FLAG or anti-HA antibodies. The presence of a strong signal for ORF3-HA after pull-down of ORF3-FLAG indicates oligomerization of ORF3 protein in cells. ( C ) FRET analyses reveal oligomerization of HEV ORF3. CFP (cyan fluorescent protein) or YFP (yellow fluorescent protein) fused to the C-termini of HEV ORF3 segments aa 1–113, 1–94, 1–70, 1–53, 28–113 or 53–113 were co-expressed in U-2 OS cells. FRET analyses were performed by the acceptor photobleaching method as described in the Materials and Methods section. The CFP-YFP fusion protein and cotransfection of unfused CFP and YFP served as positive and negative controls, respectively. Box-and-whisker plots represent the median FRET efficiency (FRETeff) values of 20 measurements (middle line), the values from the lower to the upper quartile (central box), and the minimum and maximum values (vertical line). The significance of the observed differences was assessed as described in Materials and Methods (*, P

Techniques Used: Sequencing, Immunoprecipitation, Transfection, Cotransfection, Whisker Assay

HEV ORF3 protein is palmitoylated. Protein lysates from S10-3 cells transfected with pCMVORF3, pCMVORF3 C1-4 , pCMVORF3 C45-8 or pCMVORF3_gt1 and from Hep293TT cells replicating the full-length p6 or 83–2 HEV clone were prepared 1 or 6 days post-transfection, respectively, and subjected to immunoprecipitation with either anti-ORF3 pAb (+) or non-relevant rabbit serum (-). After immunoprecipitation, the elution samples were separated by 17% SDS-PAGE and subjected to either immunoblot with anti-ORF3 pAb followed by chemiluminescence revelation or autoradiography (40 d of exposure).
Figure Legend Snippet: HEV ORF3 protein is palmitoylated. Protein lysates from S10-3 cells transfected with pCMVORF3, pCMVORF3 C1-4 , pCMVORF3 C45-8 or pCMVORF3_gt1 and from Hep293TT cells replicating the full-length p6 or 83–2 HEV clone were prepared 1 or 6 days post-transfection, respectively, and subjected to immunoprecipitation with either anti-ORF3 pAb (+) or non-relevant rabbit serum (-). After immunoprecipitation, the elution samples were separated by 17% SDS-PAGE and subjected to either immunoblot with anti-ORF3 pAb followed by chemiluminescence revelation or autoradiography (40 d of exposure).

Techniques Used: Transfection, Immunoprecipitation, SDS Page, Autoradiography

36) Product Images from "Modulation of Vitamin D Receptor Activity by the Corepressor Hairless: Differential Effects of Hairless Isoforms"

Article Title: Modulation of Vitamin D Receptor Activity by the Corepressor Hairless: Differential Effects of Hairless Isoforms

Journal: Endocrinology

doi: 10.1210/en.2009-0358

Interactions of the HR isoforms with VDR and HDAC1. A, The HR isoforms interact with VDR. VDR was coexpressed with Flag-tagged HRs in COS-7 cells. Proteins were immunoprecipitated with anti-Flag antibody (IP:Flag) and detected by immunoblot with anti-VDR antibody (WB:VDR). B, Interactions of HDAC1 with the HR isoforms. HA-tagged HDAC1 was coexpressed with Flag-tagged HRs in COS-7 cells. Proteins were immunoprecipitated with anti-Flag antibody (IP:Flag) and detected by immunoblot with anti-HA antibody (WB:HA). In, Input; IP, immunoprecipitation; NS, nonspecific.
Figure Legend Snippet: Interactions of the HR isoforms with VDR and HDAC1. A, The HR isoforms interact with VDR. VDR was coexpressed with Flag-tagged HRs in COS-7 cells. Proteins were immunoprecipitated with anti-Flag antibody (IP:Flag) and detected by immunoblot with anti-VDR antibody (WB:VDR). B, Interactions of HDAC1 with the HR isoforms. HA-tagged HDAC1 was coexpressed with Flag-tagged HRs in COS-7 cells. Proteins were immunoprecipitated with anti-Flag antibody (IP:Flag) and detected by immunoblot with anti-HA antibody (WB:HA). In, Input; IP, immunoprecipitation; NS, nonspecific.

Techniques Used: Immunoprecipitation, Western Blot

Mutations in 55-amino acid region in the full-length HR disrupt HDAC1 binding. A, COS-7 cells were transfected with pSG5 and p3XFLAG-CMV-7.1 vectors (control), pSG5-VDR and p3XFLAG–CMV-7.1 expression vectors (VDR), pSG5-VDR and p3XFLAG-HRE1100A/E1101A-CMV-7.1 (HR2E2A), and the 24-hydroxylase promoter luciferase reporter. Cells were treated with graded concentrations of 1,25(OH) 2 D 3 for 24 h and then assayed for luciferase activity. The E1100A/E1101A double mutation in the HR cDNA abolishes corepressor activity and disrupts interaction with HDAC1. Inset is immunoblot of HR E1100A/E1101A protein (HR2E2A). B, HA-tagged HDAC1 was coexpressed with Flag-tagged full-length HR or HR E1100A/E1101A in COS-7 cells. Proteins were immunoprecipitated with anti-Flag antibody (IP:Flag) and detected by immunoblot with anti-HA antibody (WB:HA). In, Input; IP, immunoprecipitation; RLU, relative light units.
Figure Legend Snippet: Mutations in 55-amino acid region in the full-length HR disrupt HDAC1 binding. A, COS-7 cells were transfected with pSG5 and p3XFLAG-CMV-7.1 vectors (control), pSG5-VDR and p3XFLAG–CMV-7.1 expression vectors (VDR), pSG5-VDR and p3XFLAG-HRE1100A/E1101A-CMV-7.1 (HR2E2A), and the 24-hydroxylase promoter luciferase reporter. Cells were treated with graded concentrations of 1,25(OH) 2 D 3 for 24 h and then assayed for luciferase activity. The E1100A/E1101A double mutation in the HR cDNA abolishes corepressor activity and disrupts interaction with HDAC1. Inset is immunoblot of HR E1100A/E1101A protein (HR2E2A). B, HA-tagged HDAC1 was coexpressed with Flag-tagged full-length HR or HR E1100A/E1101A in COS-7 cells. Proteins were immunoprecipitated with anti-Flag antibody (IP:Flag) and detected by immunoblot with anti-HA antibody (WB:HA). In, Input; IP, immunoprecipitation; RLU, relative light units.

Techniques Used: Binding Assay, Transfection, Expressing, Luciferase, Activity Assay, Mutagenesis, Immunoprecipitation, Western Blot

37) Product Images from "Drosophila TRP channels require a protein with a distinctive motif encoded by the inaF locus"

Article Title: Drosophila TRP channels require a protein with a distinctive motif encoded by the inaF locus

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.0708368104

Association of HA-tagged INAF-PB with TRP channels. ( A ) Immunolocalization. A representative confocal section of retina, costained with anti-TRP and anti-HA, from adult flies bearing the tagged version of inaF-PB , is shown. To eliminate interference by eye pigment, the transgenic line also carried bw and st mutations. Within the ommatidium shown, signal from INAF-B is detected in rhabdomeres (and not their surrounding cell bodies) and colocalizes with signal from TRP. ( B–F ) Immunoprecipitation. The top line of the key for each panel shows the antibody used to precipitate dodecyl-β-maltoside extracts of fly heads: anti-HA (H), anti-TRP (T), anti-INAD (D), or a nonspecific control antibody (N). The second line of the key indicates the presence (+) or absence (−) of an HA tag on a transgenic copy of INAF-B. Where appropriate, the key also indicates the wild-type (+) or mutant (−) status of the locus whose genotype varies in the samples of the panel. Immunoprecipitates were electrophoresed and then probed with the antibody indicated at the left of each panel. The lanes with IP status marked (−) contain a sample (≈3%) of material used as input for the corresponding immunoprecipitations.
Figure Legend Snippet: Association of HA-tagged INAF-PB with TRP channels. ( A ) Immunolocalization. A representative confocal section of retina, costained with anti-TRP and anti-HA, from adult flies bearing the tagged version of inaF-PB , is shown. To eliminate interference by eye pigment, the transgenic line also carried bw and st mutations. Within the ommatidium shown, signal from INAF-B is detected in rhabdomeres (and not their surrounding cell bodies) and colocalizes with signal from TRP. ( B–F ) Immunoprecipitation. The top line of the key for each panel shows the antibody used to precipitate dodecyl-β-maltoside extracts of fly heads: anti-HA (H), anti-TRP (T), anti-INAD (D), or a nonspecific control antibody (N). The second line of the key indicates the presence (+) or absence (−) of an HA tag on a transgenic copy of INAF-B. Where appropriate, the key also indicates the wild-type (+) or mutant (−) status of the locus whose genotype varies in the samples of the panel. Immunoprecipitates were electrophoresed and then probed with the antibody indicated at the left of each panel. The lanes with IP status marked (−) contain a sample (≈3%) of material used as input for the corresponding immunoprecipitations.

Techniques Used: Transgenic Assay, Immunoprecipitation, Mutagenesis

38) Product Images from "Drosophila TRP channels require a protein with a distinctive motif encoded by the inaF locus"

Article Title: Drosophila TRP channels require a protein with a distinctive motif encoded by the inaF locus

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.0708368104

Association of HA-tagged INAF-PB with TRP channels. ( A ) Immunolocalization. A representative confocal section of retina, costained with anti-TRP and anti-HA, from adult flies bearing the tagged version of inaF-PB , is shown. To eliminate interference by eye pigment, the transgenic line also carried bw and st mutations. Within the ommatidium shown, signal from INAF-B is detected in rhabdomeres (and not their surrounding cell bodies) and colocalizes with signal from TRP. ( B–F ) Immunoprecipitation. The top line of the key for each panel shows the antibody used to precipitate dodecyl-β-maltoside extracts of fly heads: anti-HA (H), anti-TRP (T), anti-INAD (D), or a nonspecific control antibody (N). The second line of the key indicates the presence (+) or absence (−) of an HA tag on a transgenic copy of INAF-B. Where appropriate, the key also indicates the wild-type (+) or mutant (−) status of the locus whose genotype varies in the samples of the panel. Immunoprecipitates were electrophoresed and then probed with the antibody indicated at the left of each panel. The lanes with IP status marked (−) contain a sample (≈3%) of material used as input for the corresponding immunoprecipitations.
Figure Legend Snippet: Association of HA-tagged INAF-PB with TRP channels. ( A ) Immunolocalization. A representative confocal section of retina, costained with anti-TRP and anti-HA, from adult flies bearing the tagged version of inaF-PB , is shown. To eliminate interference by eye pigment, the transgenic line also carried bw and st mutations. Within the ommatidium shown, signal from INAF-B is detected in rhabdomeres (and not their surrounding cell bodies) and colocalizes with signal from TRP. ( B–F ) Immunoprecipitation. The top line of the key for each panel shows the antibody used to precipitate dodecyl-β-maltoside extracts of fly heads: anti-HA (H), anti-TRP (T), anti-INAD (D), or a nonspecific control antibody (N). The second line of the key indicates the presence (+) or absence (−) of an HA tag on a transgenic copy of INAF-B. Where appropriate, the key also indicates the wild-type (+) or mutant (−) status of the locus whose genotype varies in the samples of the panel. Immunoprecipitates were electrophoresed and then probed with the antibody indicated at the left of each panel. The lanes with IP status marked (−) contain a sample (≈3%) of material used as input for the corresponding immunoprecipitations.

Techniques Used: Transgenic Assay, Immunoprecipitation, Mutagenesis

39) Product Images from "Stress signaling by Tec tyrosine kinase in the ischemic myocardium"

Article Title: Stress signaling by Tec tyrosine kinase in the ischemic myocardium

Journal: American Journal of Physiology - Heart and Circulatory Physiology

doi: 10.1152/ajpheart.00273.2010

Proteomic dissection of Tec tyrosine kinase signaling. A : Tec-associated proteins were purified by FLAG tag-based chromatography ( left ) or immunoprecipitation (IP; right ). Cells transfected with nontagged Tec (NT) were used as a negative control. Proteins were separated by SDS-PAGE, and gels were stained with SYPRO. B : workflow for proteomic analyses. Human embryonic kidney-293 cells were transfected with pcDNA3 containing the Tec-COOH-FLAG insert (or pcDNA3 with untagged Tec as a control). Forty-eight hours later, cells were lysed and subjected to either FLAG IP using anti-FLAG-M2 antibody or column purification with FLAG affinity beads. Tec-associated proteins were separated by SDS-PAGE and stained with Coomassie blue. Protein bands were excised, digested with trypsin, and analyzed with liquid chromotography (LC)/mass spectrometry (MS)/MS on an Orbi-trap. Protein identification was carried out by database searching using the Sequest algorithm. Results from both purification processes were merged and then filtered.
Figure Legend Snippet: Proteomic dissection of Tec tyrosine kinase signaling. A : Tec-associated proteins were purified by FLAG tag-based chromatography ( left ) or immunoprecipitation (IP; right ). Cells transfected with nontagged Tec (NT) were used as a negative control. Proteins were separated by SDS-PAGE, and gels were stained with SYPRO. B : workflow for proteomic analyses. Human embryonic kidney-293 cells were transfected with pcDNA3 containing the Tec-COOH-FLAG insert (or pcDNA3 with untagged Tec as a control). Forty-eight hours later, cells were lysed and subjected to either FLAG IP using anti-FLAG-M2 antibody or column purification with FLAG affinity beads. Tec-associated proteins were separated by SDS-PAGE and stained with Coomassie blue. Protein bands were excised, digested with trypsin, and analyzed with liquid chromotography (LC)/mass spectrometry (MS)/MS on an Orbi-trap. Protein identification was carried out by database searching using the Sequest algorithm. Results from both purification processes were merged and then filtered.

Techniques Used: Dissection, Purification, FLAG-tag, Chromatography, Immunoprecipitation, Transfection, Negative Control, SDS Page, Staining, Mass Spectrometry

40) Product Images from "Nuclear Export and Import of Human Hepatitis B Virus Capsid Protein and Particles"

Article Title: Nuclear Export and Import of Human Hepatitis B Virus Capsid Protein and Particles

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1001162

Specific physical and functional interactions between a cellular TAP protein and HBc ARD were shown by experiments of co-immunoprecipitation, si-RNA treatment, and cotransfection with a CMV-TAP expression vector. Fig. 7A . Huh7 cells were transfected with expression vectors of a) SV40 LT, or b) SV40 LT-HBc ARD chimera. Transfected cell lysates were immunoprecipitated (IP) with anti-SV40 LT antibody. Equal amounts of immunoprecipitated cell lysates from a) or b) were loaded on SDS-PAGE, respectively, followed by Western blot analysis using anti-TAP or anti-SV40 LT antibodies. The 66 kDa TAP protein can be detected only in cell lysates transfected with SV40 LT-HBc ARD, but not transfected with wild type SV40 LT, indicating that TA P could bind to HBc ARD. This result was confirmed in a reciprocal experiment using an anti-TAP antibody for IP. Fig. 7B . Knockdown of endogenous TAP protein by siRNA against TAP ( panel b ) resulted in a more than 17-fold increase of nuclear accumulation of wild type HBc (N > C pattern shifted from 3% to 52%). In contrast, control siRNA (Nontarget) ( panel a ) did not affect the subcellular localization pattern of HBc. HBc (red), α-tubulin (green) and DAPI (blue). Fig. 7C . Treatment of Huh7 cells with TAP-specific siRNA resulted in appreciable reduction of TAP protein by Western blot analysis using anti-TAP antibody. * Non-specific bands served as an internal control. (cf Materials and Methods for detail). Fig. 7D . The nuclear predominant phenotype of wild type HBc in Huh7 cells treated with siTAP can be reverted efficiently to a cytoplasmic predominant phenotype by cotransfection with a CMV-TAP expression vector. Fig. 7E . Western blot analysis of TAP protein in Huh7 cells transfected with a vector only or a plasmid CMV-TAP. Fig. 7F . Upon treatment with siTAP, wild type HBV DNA synthesis in Huh7 cells was reduced approximately 7-fold by Southern blot analysis. RC: relaxed circle DNA, SS: single-strand DNA. While there was no difference in the results of MTT cytotoxicity assay using media from cell culture with or without siTAP treatment, the HBsAg level by ELISA was reduced by approximately 10-fold with siTAP treatment. The averaged values of DNA intensity, MTT assay, and HBsAg assay were processed from four independent experiments ( Materials and Methods ).
Figure Legend Snippet: Specific physical and functional interactions between a cellular TAP protein and HBc ARD were shown by experiments of co-immunoprecipitation, si-RNA treatment, and cotransfection with a CMV-TAP expression vector. Fig. 7A . Huh7 cells were transfected with expression vectors of a) SV40 LT, or b) SV40 LT-HBc ARD chimera. Transfected cell lysates were immunoprecipitated (IP) with anti-SV40 LT antibody. Equal amounts of immunoprecipitated cell lysates from a) or b) were loaded on SDS-PAGE, respectively, followed by Western blot analysis using anti-TAP or anti-SV40 LT antibodies. The 66 kDa TAP protein can be detected only in cell lysates transfected with SV40 LT-HBc ARD, but not transfected with wild type SV40 LT, indicating that TA P could bind to HBc ARD. This result was confirmed in a reciprocal experiment using an anti-TAP antibody for IP. Fig. 7B . Knockdown of endogenous TAP protein by siRNA against TAP ( panel b ) resulted in a more than 17-fold increase of nuclear accumulation of wild type HBc (N > C pattern shifted from 3% to 52%). In contrast, control siRNA (Nontarget) ( panel a ) did not affect the subcellular localization pattern of HBc. HBc (red), α-tubulin (green) and DAPI (blue). Fig. 7C . Treatment of Huh7 cells with TAP-specific siRNA resulted in appreciable reduction of TAP protein by Western blot analysis using anti-TAP antibody. * Non-specific bands served as an internal control. (cf Materials and Methods for detail). Fig. 7D . The nuclear predominant phenotype of wild type HBc in Huh7 cells treated with siTAP can be reverted efficiently to a cytoplasmic predominant phenotype by cotransfection with a CMV-TAP expression vector. Fig. 7E . Western blot analysis of TAP protein in Huh7 cells transfected with a vector only or a plasmid CMV-TAP. Fig. 7F . Upon treatment with siTAP, wild type HBV DNA synthesis in Huh7 cells was reduced approximately 7-fold by Southern blot analysis. RC: relaxed circle DNA, SS: single-strand DNA. While there was no difference in the results of MTT cytotoxicity assay using media from cell culture with or without siTAP treatment, the HBsAg level by ELISA was reduced by approximately 10-fold with siTAP treatment. The averaged values of DNA intensity, MTT assay, and HBsAg assay were processed from four independent experiments ( Materials and Methods ).

Techniques Used: Functional Assay, Immunoprecipitation, Cotransfection, Expressing, Plasmid Preparation, Transfection, SDS Page, Western Blot, DNA Synthesis, Southern Blot, MTT Assay, Cytotoxicity Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, HBsAg Assay

41) Product Images from "Cellular RelB interacts with the transactivator Tat and enhance HIV-1 expression"

Article Title: Cellular RelB interacts with the transactivator Tat and enhance HIV-1 expression

Journal: Retrovirology

doi: 10.1186/s12977-018-0447-9

Interaction between HIV-1 Tat and RelB. a , b Myc-Tat (3 μg) was transfected into HEK 293T cells (4 × 10 6 ) together with empty vectors (control) (3 μg) or pFlag-RelB (3 μg) Co-immunoprecipitation was performed with anti-Flag ( a ) or anti-Myc ( b ) antibodies. Samples of both cell lysates and immunoprecipitates were subjected to western blotting and probed with rabbit anti-Myc and anti-Flag antibodies. c Co-IP of endogenous RelB and ectopically expressed Tat. The lysate from HA-Tat-expressing HeLa cells (4 × 10 6 ) was immunoprecipitated with mouse anti-HA antibodies, and the precipitated proteins were examined with western blotting. d Effect of RNases on the association of endogenous RelB and ectopically expressed Tat. Lysates (in the presence or absence of RNase A [5 μg/ml]) of HA-Tat expressing HeLa cells (4 × 10 6 ) were immunoprecipitated with control rabbit IgG or rabbit anti-RelB antibodies. Samples from cell lysates and immunoprecipitates were subjected to western blotting. e Tat partially co-localizes with RelB. HeLa cells (0.1 × 10 6 ) were transfected with HA-Tat (200 ng) and Flag-RelB (200 ng) plasmid DNA. Indirect IFA was performed to detect HA-Tat (with rabbit anti-HA antibody and TRITC-conjugated goat anti rabbit secondary antibody) and Flag-RelB (with mouse anti-Flag antibody and FITC-conjugated goat anti mouse secondary antibody). Nuclei were visualized with DAPI staining. Representative images are shown. The inset shows a higher magnification of the boxed area
Figure Legend Snippet: Interaction between HIV-1 Tat and RelB. a , b Myc-Tat (3 μg) was transfected into HEK 293T cells (4 × 10 6 ) together with empty vectors (control) (3 μg) or pFlag-RelB (3 μg) Co-immunoprecipitation was performed with anti-Flag ( a ) or anti-Myc ( b ) antibodies. Samples of both cell lysates and immunoprecipitates were subjected to western blotting and probed with rabbit anti-Myc and anti-Flag antibodies. c Co-IP of endogenous RelB and ectopically expressed Tat. The lysate from HA-Tat-expressing HeLa cells (4 × 10 6 ) was immunoprecipitated with mouse anti-HA antibodies, and the precipitated proteins were examined with western blotting. d Effect of RNases on the association of endogenous RelB and ectopically expressed Tat. Lysates (in the presence or absence of RNase A [5 μg/ml]) of HA-Tat expressing HeLa cells (4 × 10 6 ) were immunoprecipitated with control rabbit IgG or rabbit anti-RelB antibodies. Samples from cell lysates and immunoprecipitates were subjected to western blotting. e Tat partially co-localizes with RelB. HeLa cells (0.1 × 10 6 ) were transfected with HA-Tat (200 ng) and Flag-RelB (200 ng) plasmid DNA. Indirect IFA was performed to detect HA-Tat (with rabbit anti-HA antibody and TRITC-conjugated goat anti rabbit secondary antibody) and Flag-RelB (with mouse anti-Flag antibody and FITC-conjugated goat anti mouse secondary antibody). Nuclei were visualized with DAPI staining. Representative images are shown. The inset shows a higher magnification of the boxed area

Techniques Used: Transfection, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Expressing, Plasmid Preparation, Immunofluorescence, Staining

42) Product Images from "Amyloid Beta A4 Precursor Protein-binding Family B Member 1 (FE65) Interactomics Revealed Synaptic Vesicle Glycoprotein 2A (SV2A) and Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase 2 (SERCA2) as New Binding Proteins in the Human Brain *"

Article Title: Amyloid Beta A4 Precursor Protein-binding Family B Member 1 (FE65) Interactomics Revealed Synaptic Vesicle Glycoprotein 2A (SV2A) and Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase 2 (SERCA2) as New Binding Proteins in the Human Brain *

Journal: Molecular & Cellular Proteomics : MCP

doi: 10.1074/mcp.M113.029280

FE65 and SERCA2 co-immunoprecipitate and FE65 knockdown sensitizes cells to the endoplasmic reticulum stressor thapsigargin. A , co-immunoprecipitation assays for FE65 (red) revealed the precipitation of SERCA2 (green) using antibodies against EGFP (IP GFP) or FE65 (IP FE65). FE65 was identified on the SV2A immunoprecipitates (IP SERCA2). Lanes labeled with FE65 and SERCA2 correspond to lysates of cells expressing the individual protein. Lane SN corresponds to the supernatant after immunoprecipitation of a control (without primary antibody) demonstrating the presence of both proteins in the supernatant. B–D , the knockdown of FE65 in HEK293 cells resulted in less cell viability following 8 μm thapsigargin (Tha) stimulation after 24 h ( B ) and following 3 μm and 8 μm Tha stimulation after 48 h ( C ). The proteasomal inhibitors epoxomicin (Epoxo) and MG132 revealed no differences in cell viability in FE65 knockdown cells versus controls ( D ).
Figure Legend Snippet: FE65 and SERCA2 co-immunoprecipitate and FE65 knockdown sensitizes cells to the endoplasmic reticulum stressor thapsigargin. A , co-immunoprecipitation assays for FE65 (red) revealed the precipitation of SERCA2 (green) using antibodies against EGFP (IP GFP) or FE65 (IP FE65). FE65 was identified on the SV2A immunoprecipitates (IP SERCA2). Lanes labeled with FE65 and SERCA2 correspond to lysates of cells expressing the individual protein. Lane SN corresponds to the supernatant after immunoprecipitation of a control (without primary antibody) demonstrating the presence of both proteins in the supernatant. B–D , the knockdown of FE65 in HEK293 cells resulted in less cell viability following 8 μm thapsigargin (Tha) stimulation after 24 h ( B ) and following 3 μm and 8 μm Tha stimulation after 48 h ( C ). The proteasomal inhibitors epoxomicin (Epoxo) and MG132 revealed no differences in cell viability in FE65 knockdown cells versus controls ( D ).

Techniques Used: Immunoprecipitation, Labeling, Expressing

SV2A modulates the localization of FE65 and interacts with the adapter protein in co-immunoprecipitation assays. A–C , co-transfection of SV2A significantly modified the localization of FE65 in HEK293 cells, but not if TIP60 was co-transfected as well ( D–F ). Similar results were obtained with co-transfection experiments in SH-SY5Y ( G–L ). Co-immunoprecipitation assays validated the interaction of SV2A with FE65. In SV2A precipitates ( M ), both FE65 (arrowhead) and SV2A (arrows, two isoforms) could be identified. The co-immunoprecipitation supernatant (SN) still contained both proteins, whereas no signal was evident in the first washing step (W1). Conversely, immunoprecipitation of FE65-EGFP using an anti-GFP antibody ( N ) revealed the co-immunoprecipitation of SV2A (arrow). Immunoprecipitation using an anti-FE65 WW antibody ( O ) did not co-precipitate SV2A. However, SV2A was present in the immunoprecipitation supernatant. Control experiments using beads without antibody ( P ) showed no SV2A or FE65 signal.
Figure Legend Snippet: SV2A modulates the localization of FE65 and interacts with the adapter protein in co-immunoprecipitation assays. A–C , co-transfection of SV2A significantly modified the localization of FE65 in HEK293 cells, but not if TIP60 was co-transfected as well ( D–F ). Similar results were obtained with co-transfection experiments in SH-SY5Y ( G–L ). Co-immunoprecipitation assays validated the interaction of SV2A with FE65. In SV2A precipitates ( M ), both FE65 (arrowhead) and SV2A (arrows, two isoforms) could be identified. The co-immunoprecipitation supernatant (SN) still contained both proteins, whereas no signal was evident in the first washing step (W1). Conversely, immunoprecipitation of FE65-EGFP using an anti-GFP antibody ( N ) revealed the co-immunoprecipitation of SV2A (arrow). Immunoprecipitation using an anti-FE65 WW antibody ( O ) did not co-precipitate SV2A. However, SV2A was present in the immunoprecipitation supernatant. Control experiments using beads without antibody ( P ) showed no SV2A or FE65 signal.

Techniques Used: Immunoprecipitation, Cotransfection, Modification, Transfection

43) Product Images from "Beclin1 restricts RNA virus infection in plants through suppression and degradation of the viral polymerase"

Article Title: Beclin1 restricts RNA virus infection in plants through suppression and degradation of the viral polymerase

Journal: Nature Communications

doi: 10.1038/s41467-018-03658-2

NbBeclin1 interacts with NIb. a Yeast-two hybrid (Y2H) assays for possible interactions between NbBeclin1 and each of the 11 TuMV proteins. NbBeclin1 and 11 viral proteins were fused with a GAL4 activation domain (AD-NbBeclin1) and a GAL4-binding domain (BD-P1, BD-HC-Pro, BD-P3, BD-P3N-PIPO, BD-6K1, BD-CI, BD-6K2, BD-NIa-VPg, BD-NIa-Pro, BD-NIb, BD-CP), respectively. Y2H Gold yeast cells co-transformed with the indicated plasmids were subjected to 10-fold serial dilutions and plated on synthetic dextrose (SD)/-Trp, -Leu, -His, -Ade or SD/-Trp, -Leu medium to screen for positive interactions at 3 days after transformation. Yeast co-transformed with AD-T7-T+BD-T7-53 serves as a positive control; yeast cells co-transformed with AD-NbBeclin1 and the empty BD or with the empty AD and BD-NIb are negative controls. b BiFC assays between NbBeclin1 and NIb in the leaves of H2B-RFP transgenic N . benthamiana . Confocal imaging was performed at 48 hpi. NbBeclin1 and NIb were fused to the N (YN) and C-terminal (YC) fragments of yellow fluorescent protein (YFP). The NbBeclin1-NIb interaction led to the reconstituted fluorescence-competent structure and restoration of yellow fluorescence (green). Nuclei of tobacco leaf epidermal cells are indicated by the expression of H2B-RFP transgene (red). Bars, 50 μm. c Co-localization of NIb-YFP with NbBeclin1-CFP in the leaf cells of H2B-RFP transgenic N . benthamiana by confocal microscopy at 48 hpi. Arrow indicates yellow fluorescence, which was produced from the overlapping of NIb-YFP (green) and NbBeclin1-CFP (red). Bars, 50 μm. d Co-immunoprecipitation (Co-IP) analysis of NbBeclin1-CFP and Myc-NIb in vivo. N . benthamiana leaves were co-infiltrated with A . tumefaciens cells harboring expression vectors to express NbBeclin1-CFP and Myc-NIb (Lane 1), NbBeclin1-CFP and Myc-P3N-PIPO (Lane 2), Myc-NIb and GFP (Lane 3), and GFP and Myc-P3N-PIPO (Lane 4). Leaf protein extracts were incubated with GFP-Trap®_MA magnetic agarose beads (ChromoTek). Samples before (Input) and after (IP) immunopurification were analyzed by immunoblotting using GFP or Myc antibody
Figure Legend Snippet: NbBeclin1 interacts with NIb. a Yeast-two hybrid (Y2H) assays for possible interactions between NbBeclin1 and each of the 11 TuMV proteins. NbBeclin1 and 11 viral proteins were fused with a GAL4 activation domain (AD-NbBeclin1) and a GAL4-binding domain (BD-P1, BD-HC-Pro, BD-P3, BD-P3N-PIPO, BD-6K1, BD-CI, BD-6K2, BD-NIa-VPg, BD-NIa-Pro, BD-NIb, BD-CP), respectively. Y2H Gold yeast cells co-transformed with the indicated plasmids were subjected to 10-fold serial dilutions and plated on synthetic dextrose (SD)/-Trp, -Leu, -His, -Ade or SD/-Trp, -Leu medium to screen for positive interactions at 3 days after transformation. Yeast co-transformed with AD-T7-T+BD-T7-53 serves as a positive control; yeast cells co-transformed with AD-NbBeclin1 and the empty BD or with the empty AD and BD-NIb are negative controls. b BiFC assays between NbBeclin1 and NIb in the leaves of H2B-RFP transgenic N . benthamiana . Confocal imaging was performed at 48 hpi. NbBeclin1 and NIb were fused to the N (YN) and C-terminal (YC) fragments of yellow fluorescent protein (YFP). The NbBeclin1-NIb interaction led to the reconstituted fluorescence-competent structure and restoration of yellow fluorescence (green). Nuclei of tobacco leaf epidermal cells are indicated by the expression of H2B-RFP transgene (red). Bars, 50 μm. c Co-localization of NIb-YFP with NbBeclin1-CFP in the leaf cells of H2B-RFP transgenic N . benthamiana by confocal microscopy at 48 hpi. Arrow indicates yellow fluorescence, which was produced from the overlapping of NIb-YFP (green) and NbBeclin1-CFP (red). Bars, 50 μm. d Co-immunoprecipitation (Co-IP) analysis of NbBeclin1-CFP and Myc-NIb in vivo. N . benthamiana leaves were co-infiltrated with A . tumefaciens cells harboring expression vectors to express NbBeclin1-CFP and Myc-NIb (Lane 1), NbBeclin1-CFP and Myc-P3N-PIPO (Lane 2), Myc-NIb and GFP (Lane 3), and GFP and Myc-P3N-PIPO (Lane 4). Leaf protein extracts were incubated with GFP-Trap®_MA magnetic agarose beads (ChromoTek). Samples before (Input) and after (IP) immunopurification were analyzed by immunoblotting using GFP or Myc antibody

Techniques Used: Activation Assay, Binding Assay, Transformation Assay, Positive Control, Bimolecular Fluorescence Complementation Assay, Transgenic Assay, Imaging, Fluorescence, Expressing, Confocal Microscopy, Produced, Immunoprecipitation, Co-Immunoprecipitation Assay, In Vivo, Incubation, Immu-Puri

44) Product Images from "Phosphorylation Controls the Localization and Activation of the Lumenal Carbonic Anhydrase in Chlamydomonas reinhardtii"

Article Title: Phosphorylation Controls the Localization and Activation of the Lumenal Carbonic Anhydrase in Chlamydomonas reinhardtii

Journal: PLoS ONE

doi: 10.1371/journal.pone.0049063

Immunoprecipitation and dephosphorylation experiments of extrinsic thylakoid polypeptides. Extraction of extrinsic thylakoid proteins was accomplished by washing the thylakoid membranes with a medium containing low concentrations (0.05%) of Triton X-100. ( A ) The 30-kDa extrinsic phosphoprotein immunoprecipitates with Cah3. Extrinsic thylakoid proteins released from thylakoid membranes of C. reinhardtii cells acclimated to low CO 2 for 2 h (C) were immunoprecipitated with affinity-purified antibodies against Cah3 and protein A-Sepharose CL-4-B beads. The Sepharose beads were washed and the immunoprecipitate (I) and the supernatant (SN) obtained after centrifugation were analysed by SDS-PAGE and immunoblot and probed with antibodies against Cah3 (left) and Thr(P) (right). ( B ) Effect of Alkaline phosphatase (AP) treatment on extrinsic proteins released from thylakoid membranes isolated from both high-CO 2 -grown cells (High) or cells acclimated to low CO 2 for 2 h (Low). All lanes were loaded with 10 µg protein.
Figure Legend Snippet: Immunoprecipitation and dephosphorylation experiments of extrinsic thylakoid polypeptides. Extraction of extrinsic thylakoid proteins was accomplished by washing the thylakoid membranes with a medium containing low concentrations (0.05%) of Triton X-100. ( A ) The 30-kDa extrinsic phosphoprotein immunoprecipitates with Cah3. Extrinsic thylakoid proteins released from thylakoid membranes of C. reinhardtii cells acclimated to low CO 2 for 2 h (C) were immunoprecipitated with affinity-purified antibodies against Cah3 and protein A-Sepharose CL-4-B beads. The Sepharose beads were washed and the immunoprecipitate (I) and the supernatant (SN) obtained after centrifugation were analysed by SDS-PAGE and immunoblot and probed with antibodies against Cah3 (left) and Thr(P) (right). ( B ) Effect of Alkaline phosphatase (AP) treatment on extrinsic proteins released from thylakoid membranes isolated from both high-CO 2 -grown cells (High) or cells acclimated to low CO 2 for 2 h (Low). All lanes were loaded with 10 µg protein.

Techniques Used: Immunoprecipitation, De-Phosphorylation Assay, Affinity Purification, Centrifugation, SDS Page, Isolation

45) Product Images from "Phospholipase C γ1 regulates early secretory trafficking and cell migration via interaction with p115"

Article Title: Phospholipase C γ1 regulates early secretory trafficking and cell migration via interaction with p115

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E15-03-0178

(A) Coimmunoprecipitation of PLCG1 (IP: PLCG1) and p115 (IP: p115) from HeLa cell lysate. Input, the input material of the immunoprecipitation (5%). Neg. Control, the negative control in which the immunoprecipitation was performed in the absence of antibody but in the presence of protein G–Sepharose beads. Immunoprecipitated PLCG1 or p115 was eluted and immunoblotted against p115 and PLCG1, respectively (upper gel). Blots were stripped and immunoblotted against PLCG1 and p115 (lower gel). (B) Schematic depiction of the domains in PLCG1. (C) HeLa cells were transfected with a plasmid encoding wild-type GFP-tagged PLCG1 (PLCG1-WT) or truncation mutants lacking 19, 39, 60, and 81 C-terminal amino acids (PLCG1-Δ19, -Δ39, -Δ60, and -Δ81, respectively). In addition, a truncation mutant lacking the C2 domain was also used (PLCG1-ΔC2). After 24 h, cells were lysed and the lysate subjected to immunoprecipitation with GFP-tap beads (ChromoTek). The immunoprecipitated material was subjected to SDS–PAGE and immunoblotted against p115. The blot was stripped and probed with an antibody against GFP (to detect PLCG1). (D) HeLa cells were transfected with the indicated siRNAs. After 48 h, cells were transfected with plasmids encoding either GFP or GFP-tagged PLCG1-ΔC2. After 8 h, cells were plated into ibidi migration inserts. Cell migration was initiated by removing the insert, and cells were allowed to migrate for 18 h. (E, F) HeLa cells were transfected with the indicated siRNAs. After 48 h, cells were transfected with plasmids encoding GFP or GFP-tagged PLCG1-ΔC2 (E) or PLCG1-Δ81 (F). Asterisks indicate statistically significant differences from control (* p
Figure Legend Snippet: (A) Coimmunoprecipitation of PLCG1 (IP: PLCG1) and p115 (IP: p115) from HeLa cell lysate. Input, the input material of the immunoprecipitation (5%). Neg. Control, the negative control in which the immunoprecipitation was performed in the absence of antibody but in the presence of protein G–Sepharose beads. Immunoprecipitated PLCG1 or p115 was eluted and immunoblotted against p115 and PLCG1, respectively (upper gel). Blots were stripped and immunoblotted against PLCG1 and p115 (lower gel). (B) Schematic depiction of the domains in PLCG1. (C) HeLa cells were transfected with a plasmid encoding wild-type GFP-tagged PLCG1 (PLCG1-WT) or truncation mutants lacking 19, 39, 60, and 81 C-terminal amino acids (PLCG1-Δ19, -Δ39, -Δ60, and -Δ81, respectively). In addition, a truncation mutant lacking the C2 domain was also used (PLCG1-ΔC2). After 24 h, cells were lysed and the lysate subjected to immunoprecipitation with GFP-tap beads (ChromoTek). The immunoprecipitated material was subjected to SDS–PAGE and immunoblotted against p115. The blot was stripped and probed with an antibody against GFP (to detect PLCG1). (D) HeLa cells were transfected with the indicated siRNAs. After 48 h, cells were transfected with plasmids encoding either GFP or GFP-tagged PLCG1-ΔC2. After 8 h, cells were plated into ibidi migration inserts. Cell migration was initiated by removing the insert, and cells were allowed to migrate for 18 h. (E, F) HeLa cells were transfected with the indicated siRNAs. After 48 h, cells were transfected with plasmids encoding GFP or GFP-tagged PLCG1-ΔC2 (E) or PLCG1-Δ81 (F). Asterisks indicate statistically significant differences from control (* p

Techniques Used: Immunoprecipitation, Negative Control, Transfection, Plasmid Preparation, Mutagenesis, SDS Page, Migration

46) Product Images from "HMMR acts in the PLK1-dependent spindle positioning pathway and supports neural development"

Article Title: HMMR acts in the PLK1-dependent spindle positioning pathway and supports neural development

Journal: eLife

doi: 10.7554/eLife.28672

HMMR enables the PLK1-dependent spindle pole positioning pathway. ( A ) Western blot analysis of mitotic HeLa extracts stably expressing DHC-GFP treated with scrambled (siScr) siRNA or siRNA targeting HMMR (siHMMR) subjected to immunoprecipitation with GFP antibody or control IgG and blotted with the indicated antibodies, including GFP (GFP-DHC), dynein intermediate chain (DIC), p150 glued , DYNLL1, HMMR or Actin (four replicates). ( B ) Localization of Flag-Dynein light chain (DYNLL1) in HeLa cells treated with siScr or siHMMR (three replicates). Scale bars, 10 μm. ( C ) Localization of CHICA in cells treated with siScr and siHMMR (three replicates). Scale bars, 10 μm. ( D ) Localization of phospho-PLK1 (pPLK1) in HeLa cells expressing siScr or siHMMR. Scale bars, 5 μm. ( E ) Quantification of pPLK1 intensity from pole to pole. Data represents mean (50 cells from three experiments). ( F ) Astral microtubules contact the cortex in cells treated with siScr, siHMMR, or BI2536 (Plk1 inhibitor). Yellow box indicates EB1 inset and white dotted line indicates region of quantification. Scale bar, 5 μm. ( G ) Quantification of EB1 at the cortex in cells treated with siScr, siHMMR, or BI2536. Data are represented as mean ±SD (***p
Figure Legend Snippet: HMMR enables the PLK1-dependent spindle pole positioning pathway. ( A ) Western blot analysis of mitotic HeLa extracts stably expressing DHC-GFP treated with scrambled (siScr) siRNA or siRNA targeting HMMR (siHMMR) subjected to immunoprecipitation with GFP antibody or control IgG and blotted with the indicated antibodies, including GFP (GFP-DHC), dynein intermediate chain (DIC), p150 glued , DYNLL1, HMMR or Actin (four replicates). ( B ) Localization of Flag-Dynein light chain (DYNLL1) in HeLa cells treated with siScr or siHMMR (three replicates). Scale bars, 10 μm. ( C ) Localization of CHICA in cells treated with siScr and siHMMR (three replicates). Scale bars, 10 μm. ( D ) Localization of phospho-PLK1 (pPLK1) in HeLa cells expressing siScr or siHMMR. Scale bars, 5 μm. ( E ) Quantification of pPLK1 intensity from pole to pole. Data represents mean (50 cells from three experiments). ( F ) Astral microtubules contact the cortex in cells treated with siScr, siHMMR, or BI2536 (Plk1 inhibitor). Yellow box indicates EB1 inset and white dotted line indicates region of quantification. Scale bar, 5 μm. ( G ) Quantification of EB1 at the cortex in cells treated with siScr, siHMMR, or BI2536. Data are represented as mean ±SD (***p

Techniques Used: Western Blot, Stable Transfection, Expressing, Immunoprecipitation

47) Product Images from "Epiprofin orchestrates epidermal keratinocyte proliferation and differentiation"

Article Title: Epiprofin orchestrates epidermal keratinocyte proliferation and differentiation

Journal: Journal of Cell Science

doi: 10.1242/jcs.156778

Epfn promotes HaCaT cell differentiation through regulating Notch1. (A) Expression of keratinocyte marker genes as revealed by qPCR in HaCaT cells transfected with either Mock or Epfn expression vectors. (B) Co-immunoprecipitation assay of Epfn and E2F1 in HaCaT cells transfected with E2F1 (HA tag) and Epfn (VSV tag) expression vectors. IP, immunoprecipitated; IB, immunoblotted. (C) E2F activity assay in HaCaT cells transfected with Mock and Epfn expression vectors. (D) RBP-Jk reporter activity in HaCaT cells transfected with either Mock or Epfn expression vectors. Quantitative data in A,C,D show the mean+s.e.m. (three independent experiments); ** P
Figure Legend Snippet: Epfn promotes HaCaT cell differentiation through regulating Notch1. (A) Expression of keratinocyte marker genes as revealed by qPCR in HaCaT cells transfected with either Mock or Epfn expression vectors. (B) Co-immunoprecipitation assay of Epfn and E2F1 in HaCaT cells transfected with E2F1 (HA tag) and Epfn (VSV tag) expression vectors. IP, immunoprecipitated; IB, immunoblotted. (C) E2F activity assay in HaCaT cells transfected with Mock and Epfn expression vectors. (D) RBP-Jk reporter activity in HaCaT cells transfected with either Mock or Epfn expression vectors. Quantitative data in A,C,D show the mean+s.e.m. (three independent experiments); ** P

Techniques Used: Cell Differentiation, Expressing, Marker, Real-time Polymerase Chain Reaction, Transfection, Co-Immunoprecipitation Assay, Immunoprecipitation, Activity Assay

48) Product Images from "CK2 Phosphorylates Sec31 and Regulates ER-To-Golgi Trafficking"

Article Title: CK2 Phosphorylates Sec31 and Regulates ER-To-Golgi Trafficking

Journal: PLoS ONE

doi: 10.1371/journal.pone.0054382

CK2 phosphorylates human Sec31. (A) FLAG-Sec31 expressed in HRK293 cells was immunoprecipitated and incubated with/without recombinant CK2 (recCK2) followed by western blotting with anti-phospho serine/threonine and anti-FLAG antibodies (IP’ed Sec31). The phospho-Sec31 levels were normalized to IP’ed Sec31 levels and expressed as the normalized ratio. (B) FLAG-Sec13 expressed in HRK293 cells was immunoprecipitated, treated and subjected to western blotting as described in (A). (C) Cells were first transfected with siRNA1 to CK2 alpha1 (RNAi +) or to eGFP (RNAi −). After 2 days incubation, cells were transfected with FLAG-tagged Sec31 transfection followed by immunoprecipitation and western blotting as described in (A). Total cell lysates were also analyzed for CK2 by western blotting to determine the depletion efficiency as shown in the bottom panel. (D) FLAG-Sec31 expressed in HRK293 cells treated with or without CK2 inhibitor was immunoprecipitated and subjected to western blotting as described in (A).
Figure Legend Snippet: CK2 phosphorylates human Sec31. (A) FLAG-Sec31 expressed in HRK293 cells was immunoprecipitated and incubated with/without recombinant CK2 (recCK2) followed by western blotting with anti-phospho serine/threonine and anti-FLAG antibodies (IP’ed Sec31). The phospho-Sec31 levels were normalized to IP’ed Sec31 levels and expressed as the normalized ratio. (B) FLAG-Sec13 expressed in HRK293 cells was immunoprecipitated, treated and subjected to western blotting as described in (A). (C) Cells were first transfected with siRNA1 to CK2 alpha1 (RNAi +) or to eGFP (RNAi −). After 2 days incubation, cells were transfected with FLAG-tagged Sec31 transfection followed by immunoprecipitation and western blotting as described in (A). Total cell lysates were also analyzed for CK2 by western blotting to determine the depletion efficiency as shown in the bottom panel. (D) FLAG-Sec31 expressed in HRK293 cells treated with or without CK2 inhibitor was immunoprecipitated and subjected to western blotting as described in (A).

Techniques Used: Immunoprecipitation, Incubation, Recombinant, Western Blot, Transfection

Dephosphorylation of Sec31 at Serines 527 and 799 increases its affinity to Sec23. (A) HEK293 cells were co-transfected with indicated FLAG-tagged Sec31 mutants and GFP-tagged Sec23. Sec23 was immunoprecipitated with anti-GFP beads and subjected to western blotting with anti-FLAG and anti-GFP antibodies. The “input 10% Sec31” is a western blot of 10% aliquots of total cell lysates. The normalized ratio of Sec31 bound to Sec23 was shown below. W985A is a known Sec23-binding defect mutant [52] . The most right lane is a negative control with GFP. The graph below the blots shows the average of the quantification of three independent experiments with SD. (B) FLAG-Sec31 and GFP-Sec13 (instead of GFP-Sec23) were co-expressed and their interactions detected by co-immunoprecipitation and western blotting following the procedures described in Figure 4A . The lane on right most is a negative control with GFP.
Figure Legend Snippet: Dephosphorylation of Sec31 at Serines 527 and 799 increases its affinity to Sec23. (A) HEK293 cells were co-transfected with indicated FLAG-tagged Sec31 mutants and GFP-tagged Sec23. Sec23 was immunoprecipitated with anti-GFP beads and subjected to western blotting with anti-FLAG and anti-GFP antibodies. The “input 10% Sec31” is a western blot of 10% aliquots of total cell lysates. The normalized ratio of Sec31 bound to Sec23 was shown below. W985A is a known Sec23-binding defect mutant [52] . The most right lane is a negative control with GFP. The graph below the blots shows the average of the quantification of three independent experiments with SD. (B) FLAG-Sec31 and GFP-Sec13 (instead of GFP-Sec23) were co-expressed and their interactions detected by co-immunoprecipitation and western blotting following the procedures described in Figure 4A . The lane on right most is a negative control with GFP.

Techniques Used: De-Phosphorylation Assay, Transfection, Immunoprecipitation, Western Blot, Binding Assay, Mutagenesis, Negative Control

49) Product Images from "ERG-associated protein with SET domain (ESET)-Oct4 interaction regulates pluripotency and represses the trophectoderm lineage"

Article Title: ERG-associated protein with SET domain (ESET)-Oct4 interaction regulates pluripotency and represses the trophectoderm lineage

Journal: Epigenetics & Chromatin

doi: 10.1186/1756-8935-2-12

ERG-associated protein with SET domain (ESET)-mediated histone 3 lysine 9 trimethylation (H3K9me3) represses Cdx2 in embryonic stem (ES) cells . (a) Chromatin immunoprecipitation (ChIP) primers C1 to C10 used to detect enrichment of H3K9me3 on Cdx2 promoter (left). Primers O1 of Oct4 promoter served as a negative control (right). The numbers below the bars indicate distance from transcription start site (TSS) in base pairs (bp). (b) Quantitative polymerase chain reaction (Q-PCR) analysis of the enrichment of H3K9me3 at different positions along Cdx2 promoter region relative to the Oct4 promoter region after normalising against H3 ChIP and IgG controls. Primers C4 and C6 were not suitable for Q-PCR analysis. Error bars, standard deviation (SD) of three technical replicates. (c) Carrier ChIP semiquantitative PCR analysis of H3K9me3 at the Cdx2 promoter and major satellite regions of fluorescence-activated cell sorting (FACS)-sorted Eset knockdown ES cells relative to control cells after normalising against their respective input and IgG controls. Primers C6 and C7 were not suitable for carrier ChIP analysis. Error bars, SD of three independent experiments. (d) Q-PCR analysis of the levels of H3K9me3 at region C1 and C2 of the Cdx2 promoter in Eset knockdown ES cells relative to control cells after normalising against their respective input and IgG controls. Error bars, SD of three independent experiments. (e) Q-PCR analysis of the enrichment of ESET on different positions along the Cdx2 promoter relative to the Oct4 promoter after normalising against their respective input and IgG controls. Error bars, SD of three technical replicates.
Figure Legend Snippet: ERG-associated protein with SET domain (ESET)-mediated histone 3 lysine 9 trimethylation (H3K9me3) represses Cdx2 in embryonic stem (ES) cells . (a) Chromatin immunoprecipitation (ChIP) primers C1 to C10 used to detect enrichment of H3K9me3 on Cdx2 promoter (left). Primers O1 of Oct4 promoter served as a negative control (right). The numbers below the bars indicate distance from transcription start site (TSS) in base pairs (bp). (b) Quantitative polymerase chain reaction (Q-PCR) analysis of the enrichment of H3K9me3 at different positions along Cdx2 promoter region relative to the Oct4 promoter region after normalising against H3 ChIP and IgG controls. Primers C4 and C6 were not suitable for Q-PCR analysis. Error bars, standard deviation (SD) of three technical replicates. (c) Carrier ChIP semiquantitative PCR analysis of H3K9me3 at the Cdx2 promoter and major satellite regions of fluorescence-activated cell sorting (FACS)-sorted Eset knockdown ES cells relative to control cells after normalising against their respective input and IgG controls. Primers C6 and C7 were not suitable for carrier ChIP analysis. Error bars, SD of three independent experiments. (d) Q-PCR analysis of the levels of H3K9me3 at region C1 and C2 of the Cdx2 promoter in Eset knockdown ES cells relative to control cells after normalising against their respective input and IgG controls. Error bars, SD of three independent experiments. (e) Q-PCR analysis of the enrichment of ESET on different positions along the Cdx2 promoter relative to the Oct4 promoter after normalising against their respective input and IgG controls. Error bars, SD of three technical replicates.

Techniques Used: Chromatin Immunoprecipitation, Negative Control, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Standard Deviation, Fluorescence, FACS

Oct4 and ERG-associated protein with SET domain (ESET) synergistically repress Cdx2 . (a) Quantitative polymerase chain reaction (Q-PCR) analysis of the enrichment of Oct4 on different positions along the Cdx2 promoter relative to the least enriched region (C3) after normalising against their respective input and IgG controls. Error bars, standard deviation (SD) of three technical replicates. (b) Western blot shows upregulation of Cdx2 and downregulation of ESET upon depletion of Oct4 at day 2 of tetracycline (Tc) treatment of ZHBTc4 embryonic stem (ES) cells. (c) Q-PCR analysis of the levels of Oct4 and ESET enrichment at region C7 of the Cdx2 promoter in ZHBTc4 ES cells treated with Tc for 1 day relative to untreated cells after normalising against their respective input. Error bars, SD of three independent experiments. (d) Carrier chromatin immunoprecipitation (ChIP) Q-PCR analysis of the levels of histone 3 lysine 9 trimethylation (H3K9me3) at region C1 and C2 of the Cdx2 promoter in ZHBTc4 ES cells treated with Tc for the indicated days relative to untreated cells after normalising against their respective input and IgG controls. Error bars, SD of three independent experiments. (e) ES cell lysates were immunoprecipitated (IP) with anti-ESET antibody (kind gift of HH Ng; see text) under mild conditions in digitonin-containing buffer and subjected to western blotting (WB) with the antibodies indicated. Rabbit IgG was used as a negative control.
Figure Legend Snippet: Oct4 and ERG-associated protein with SET domain (ESET) synergistically repress Cdx2 . (a) Quantitative polymerase chain reaction (Q-PCR) analysis of the enrichment of Oct4 on different positions along the Cdx2 promoter relative to the least enriched region (C3) after normalising against their respective input and IgG controls. Error bars, standard deviation (SD) of three technical replicates. (b) Western blot shows upregulation of Cdx2 and downregulation of ESET upon depletion of Oct4 at day 2 of tetracycline (Tc) treatment of ZHBTc4 embryonic stem (ES) cells. (c) Q-PCR analysis of the levels of Oct4 and ESET enrichment at region C7 of the Cdx2 promoter in ZHBTc4 ES cells treated with Tc for 1 day relative to untreated cells after normalising against their respective input. Error bars, SD of three independent experiments. (d) Carrier chromatin immunoprecipitation (ChIP) Q-PCR analysis of the levels of histone 3 lysine 9 trimethylation (H3K9me3) at region C1 and C2 of the Cdx2 promoter in ZHBTc4 ES cells treated with Tc for the indicated days relative to untreated cells after normalising against their respective input and IgG controls. Error bars, SD of three independent experiments. (e) ES cell lysates were immunoprecipitated (IP) with anti-ESET antibody (kind gift of HH Ng; see text) under mild conditions in digitonin-containing buffer and subjected to western blotting (WB) with the antibodies indicated. Rabbit IgG was used as a negative control.

Techniques Used: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Standard Deviation, Western Blot, Chromatin Immunoprecipitation, Immunoprecipitation, Negative Control

50) Product Images from "Membrane protein recycling from the vacuole/lysosome membrane"

Article Title: Membrane protein recycling from the vacuole/lysosome membrane

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201709162

Specific residues in the cytoplasmic tail of Atg27 are required for its retrograde traffic. (A) Schematic of Atg27 mutational analysis from Fig. S3 C. (B) Atg27-diL-GFP localization after 1 h of rapamycin treatment. (C) The percentage of cells with Atg27-diL-GFP on the vacuole in B. (D) Atg27-diL-GFP mutant localization in vps35 Δ cells after 1 h of rapamycin treatment. (E) The percentage of cells with Atg27-diL-GFP on the vacuole in D and Fig. S3 D. (F) Cells were treated with rapamycin for 1 h and Atg27-diL immunoprecipitated. Interacting Snx4-3XHA mutants were detected by immunoblotting. (G) Model for Atg27 trafficking. Atg27 is delivered to the vacuole membrane via the AP-3 pathway and then recycled from the vacuole membrane to the endosome by the Snx4 complex. Atg27 is then retrieved from the endosome by retromer. (H) Membrane trafficking pathways in yeast cells. For all quantification shown in this figure, at least 100 cells were measured, and the data from three independent experiments were used for statistical analysis; two-tailed Student’s t test. Error bars represent SD. IP, immunoprecipitation. Bars, 2 µm.
Figure Legend Snippet: Specific residues in the cytoplasmic tail of Atg27 are required for its retrograde traffic. (A) Schematic of Atg27 mutational analysis from Fig. S3 C. (B) Atg27-diL-GFP localization after 1 h of rapamycin treatment. (C) The percentage of cells with Atg27-diL-GFP on the vacuole in B. (D) Atg27-diL-GFP mutant localization in vps35 Δ cells after 1 h of rapamycin treatment. (E) The percentage of cells with Atg27-diL-GFP on the vacuole in D and Fig. S3 D. (F) Cells were treated with rapamycin for 1 h and Atg27-diL immunoprecipitated. Interacting Snx4-3XHA mutants were detected by immunoblotting. (G) Model for Atg27 trafficking. Atg27 is delivered to the vacuole membrane via the AP-3 pathway and then recycled from the vacuole membrane to the endosome by the Snx4 complex. Atg27 is then retrieved from the endosome by retromer. (H) Membrane trafficking pathways in yeast cells. For all quantification shown in this figure, at least 100 cells were measured, and the data from three independent experiments were used for statistical analysis; two-tailed Student’s t test. Error bars represent SD. IP, immunoprecipitation. Bars, 2 µm.

Techniques Used: Mutagenesis, Immunoprecipitation, Two Tailed Test

The Snx4 complex mediates Atg27 vacuole-to-endosome retrograde transport. (A) Schematic predicting Atg27 accumulation on the vacuole membrane in retromer and recycling double mutants. (B) Atg27-GFP localization in vps35 Δ snx4 Δ cells after 1 h of rapamycin treatment. (C) The percentage of cells with Atg27-GFP on the vacuole. (D) Atg27-GFP localization in vps35ts snx4 Δ mutants after shift to 37°C with rapamycin treatment for 1 h. (E) Quantification of Atg27-GFP fluorescence intensity on the vacuole from D and Fig. S2 C. (F) Cells were treated with rapamycin for 1 h, Atg27-GFP was immunoprecipitated, and interacting Snx4-3XHA was detected by immunoblot. For all quantification shown in this figure, at least 100 cells were measured, and the data from three independent experiments were used for statistical analysis; two-tailed Student’s t test. Error bars represent SD. IP, immunoprecipitation; n.s., not significant. Bar, 2 µm.
Figure Legend Snippet: The Snx4 complex mediates Atg27 vacuole-to-endosome retrograde transport. (A) Schematic predicting Atg27 accumulation on the vacuole membrane in retromer and recycling double mutants. (B) Atg27-GFP localization in vps35 Δ snx4 Δ cells after 1 h of rapamycin treatment. (C) The percentage of cells with Atg27-GFP on the vacuole. (D) Atg27-GFP localization in vps35ts snx4 Δ mutants after shift to 37°C with rapamycin treatment for 1 h. (E) Quantification of Atg27-GFP fluorescence intensity on the vacuole from D and Fig. S2 C. (F) Cells were treated with rapamycin for 1 h, Atg27-GFP was immunoprecipitated, and interacting Snx4-3XHA was detected by immunoblot. For all quantification shown in this figure, at least 100 cells were measured, and the data from three independent experiments were used for statistical analysis; two-tailed Student’s t test. Error bars represent SD. IP, immunoprecipitation; n.s., not significant. Bar, 2 µm.

Techniques Used: Fluorescence, Immunoprecipitation, Two Tailed Test

51) Product Images from "BAG3 Directly Interacts with Mutated alphaB-Crystallin to Suppress Its Aggregation and Toxicity"

Article Title: BAG3 Directly Interacts with Mutated alphaB-Crystallin to Suppress Its Aggregation and Toxicity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0016828

BAG3 recognizes folding status of αB-crystallin. (A) BAG3 binds to mutant type of αB-crystallin strongly. Wild type (WT) or mutant (R120G) αB-crystallin was expressed in HEK293 cells with Flag-tagged BAG3, and the cell lysate was mixed with anti-Flag antibody. Precipitated samples were analyzed by SDS-PAGE using anti-αB-crystallin (upper panel), Flag-tagged BAG3 (middle panel), and actin (lower panel). The sample before immunoprecipitation was also loaded to confirm protein expression (right four lanes). (B) Direct recognition of mutant αB-crystallin by BAG3. Purified GST or GST-fused BAG3 beads were incubated with purified αB-crystallin wild type (WT) or mutant (R120G), and a pull-down assay was performed. Precipitated αB-crystallin was detected with anti-αB-crystallin antibody (upper panel). The same membrane stained with Ponceau is shown below. Mutated αB-crystallin preferentially binds to BAG3. Purified GST, GST-fused αB-crystallin wild type (WT) or mutant (R120G) was mixed with purified BAG3 for a pull-down assay. The detection of BAG3 was achieved with anti-BAG3 antibody after SDS-PAGE. The same membrane was stained with Ponceau (lower panel).
Figure Legend Snippet: BAG3 recognizes folding status of αB-crystallin. (A) BAG3 binds to mutant type of αB-crystallin strongly. Wild type (WT) or mutant (R120G) αB-crystallin was expressed in HEK293 cells with Flag-tagged BAG3, and the cell lysate was mixed with anti-Flag antibody. Precipitated samples were analyzed by SDS-PAGE using anti-αB-crystallin (upper panel), Flag-tagged BAG3 (middle panel), and actin (lower panel). The sample before immunoprecipitation was also loaded to confirm protein expression (right four lanes). (B) Direct recognition of mutant αB-crystallin by BAG3. Purified GST or GST-fused BAG3 beads were incubated with purified αB-crystallin wild type (WT) or mutant (R120G), and a pull-down assay was performed. Precipitated αB-crystallin was detected with anti-αB-crystallin antibody (upper panel). The same membrane stained with Ponceau is shown below. Mutated αB-crystallin preferentially binds to BAG3. Purified GST, GST-fused αB-crystallin wild type (WT) or mutant (R120G) was mixed with purified BAG3 for a pull-down assay. The detection of BAG3 was achieved with anti-BAG3 antibody after SDS-PAGE. The same membrane was stained with Ponceau (lower panel).

Techniques Used: Mutagenesis, SDS Page, Immunoprecipitation, Expressing, Purification, Incubation, Pull Down Assay, Staining

52) Product Images from "Cab45S inhibits the ER stress-induced IRE1-JNK pathway and apoptosis via GRP78/BiP"

Article Title: Cab45S inhibits the ER stress-induced IRE1-JNK pathway and apoptosis via GRP78/BiP

Journal: Cell Death & Disease

doi: 10.1038/cddis.2014.193

Cab45S interacts with the nucleotide-binding domain (NBD) of GRP78/BiP. ( a ) Immunoprecipitation assay (IP) with anti-Flag antibody in HEK293T cell lysates expressing 3 × Flag-Cab45S. Immunoprecipitates were subjected to SDS-PAGE and then MS analysis. ( b ) Extracts of HEK293T cells co-transfected with GRP78/BiP-EGFP and 3 × Flag, 3 × Flag-Cab45S, 3 × Flag-Cab45G or 3 × Flag-RCN1 were immunoprecipitated using anti-Flag antibody. The immunoprecipitates were immunoblotted with anti-Flag or anti-GFP antibody. ( c and d ) Mapping the domain at which GRP78/BiP interacted with Cab45S. Schematics of GRP78/BiP truncates ( c ). Extracts of HEK293T cells overexpressing GFP-tagged GRP78/BiP truncates and 3 × Flag-Cab45S were immunoprecipitated with anti-GFP antibody, and the immunoprecipitates were immunoblotted with anti-GFP or anti-Flag antibody ( d ). SBD, substrate-binding domain. ( e and f ) Mapping the domain of Cab45S, which interacted with GRP78/BiP. Schematics of Cab45S truncates ( e ). Extracts of HEK293T cells overexpressing 3 × Flag-tagged Cab45S truncates and GRP78/BiP-EGFP were immunoprecipitated with anti-Flag antibody, and the immunoprecipitates were immunoblotted with anti-GFP and anti-Flag antibodies ( f ). Asterisks indicate 3 × Flag-tagged Cab45S truncates immunoprecipitated by anti-Flag antibody. SP, signal peptide; EFh, EF-hand
Figure Legend Snippet: Cab45S interacts with the nucleotide-binding domain (NBD) of GRP78/BiP. ( a ) Immunoprecipitation assay (IP) with anti-Flag antibody in HEK293T cell lysates expressing 3 × Flag-Cab45S. Immunoprecipitates were subjected to SDS-PAGE and then MS analysis. ( b ) Extracts of HEK293T cells co-transfected with GRP78/BiP-EGFP and 3 × Flag, 3 × Flag-Cab45S, 3 × Flag-Cab45G or 3 × Flag-RCN1 were immunoprecipitated using anti-Flag antibody. The immunoprecipitates were immunoblotted with anti-Flag or anti-GFP antibody. ( c and d ) Mapping the domain at which GRP78/BiP interacted with Cab45S. Schematics of GRP78/BiP truncates ( c ). Extracts of HEK293T cells overexpressing GFP-tagged GRP78/BiP truncates and 3 × Flag-Cab45S were immunoprecipitated with anti-GFP antibody, and the immunoprecipitates were immunoblotted with anti-GFP or anti-Flag antibody ( d ). SBD, substrate-binding domain. ( e and f ) Mapping the domain of Cab45S, which interacted with GRP78/BiP. Schematics of Cab45S truncates ( e ). Extracts of HEK293T cells overexpressing 3 × Flag-tagged Cab45S truncates and GRP78/BiP-EGFP were immunoprecipitated with anti-Flag antibody, and the immunoprecipitates were immunoblotted with anti-GFP and anti-Flag antibodies ( f ). Asterisks indicate 3 × Flag-tagged Cab45S truncates immunoprecipitated by anti-Flag antibody. SP, signal peptide; EFh, EF-hand

Techniques Used: Binding Assay, Immunoprecipitation, Expressing, SDS Page, Mass Spectrometry, Transfection

53) Product Images from "Plant A20/AN1 protein serves as the important hub to mediate antiviral immunity"

Article Title: Plant A20/AN1 protein serves as the important hub to mediate antiviral immunity

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1007288

SA regulates Pha13 at post-translational level. (A) Total proteins were extracted from leaves of P . aphrodite without treatment (0 h), treated with H 2 O (Mock), or SA at different times (h), and used for in vivo immunoprecipitation (IP) assay using anti-Pha13 antibody. Samples after IP were analyzed by immunoblotting using anti-Pha13 or anti-ubiquitin antibody. (B) P . aphrodite was treated with H 2 O (Mock) or SA, and immediately followed by infiltration of DMSO (-) or MG132 (+). Total proteins were extracted from leaves of the treated samples at 3 h post-treatment, and were used for in vivo IP assay using anti-Pha13 antibodies. Samples after IP were analyzed by immunoblotting using anti-Pha13 antibody. (A and B) Extracted total proteins (input) stained by coomassie brilliant blue (CBB) served as a loading control.
Figure Legend Snippet: SA regulates Pha13 at post-translational level. (A) Total proteins were extracted from leaves of P . aphrodite without treatment (0 h), treated with H 2 O (Mock), or SA at different times (h), and used for in vivo immunoprecipitation (IP) assay using anti-Pha13 antibody. Samples after IP were analyzed by immunoblotting using anti-Pha13 or anti-ubiquitin antibody. (B) P . aphrodite was treated with H 2 O (Mock) or SA, and immediately followed by infiltration of DMSO (-) or MG132 (+). Total proteins were extracted from leaves of the treated samples at 3 h post-treatment, and were used for in vivo IP assay using anti-Pha13 antibodies. Samples after IP were analyzed by immunoblotting using anti-Pha13 antibody. (A and B) Extracted total proteins (input) stained by coomassie brilliant blue (CBB) served as a loading control.

Techniques Used: In Vivo, Immunoprecipitation, Staining

54) Product Images from "ccm2-like is required for cardiovascular development as a novel component of the Heg-CCM pathway"

Article Title: ccm2-like is required for cardiovascular development as a novel component of the Heg-CCM pathway

Journal: Developmental biology

doi: 10.1016/j.ydbio.2013.01.006

Biochemical interactions among proteins of the Heg-CCM pathway. (A) Schematic of all Ccm1 constructs used for co-immunoprecipitation experiments. (B) FLAG-ccm1 and FLAG-ccm1ΔFERM co-immunoprecipitate HA-ccm2 and HA-ccm2l, while the N-terminal Ccm1 deletion proteins do not bind HA-ccm2 and HA-ccm2l. In contrast, FLAG-ccm1ΔFERM does not co-immunoprecipitate Heg, while the N-terminal Ccm1 deletion proteins do. (C) HA-ccm2 binding to FLAG-ccm1 is severely weakened by mutation of Ccm1’s two NPXY motifs and further disrupted by mutations in the NPXF motif. The strength of the interaction between HA-ccm2l and FLAG-ccm1 is unaffected by mutation of Ccm1’s NPXY motifs but is severely diminished when all three NPXY/F motifs are mutated. Heg-Ccm1 interactions appear unaffected by mutation of either two or all three NPXY/F motifs in Ccm1. (D) The mutant Ccm1 proteins FLAG-ccm1ty219c and FLAG-ccm1m775 bind HA-ccm2 and HA-ccm2l as well as wildtype FLAG-ccm1 does, but they do not bind Heg.
Figure Legend Snippet: Biochemical interactions among proteins of the Heg-CCM pathway. (A) Schematic of all Ccm1 constructs used for co-immunoprecipitation experiments. (B) FLAG-ccm1 and FLAG-ccm1ΔFERM co-immunoprecipitate HA-ccm2 and HA-ccm2l, while the N-terminal Ccm1 deletion proteins do not bind HA-ccm2 and HA-ccm2l. In contrast, FLAG-ccm1ΔFERM does not co-immunoprecipitate Heg, while the N-terminal Ccm1 deletion proteins do. (C) HA-ccm2 binding to FLAG-ccm1 is severely weakened by mutation of Ccm1’s two NPXY motifs and further disrupted by mutations in the NPXF motif. The strength of the interaction between HA-ccm2l and FLAG-ccm1 is unaffected by mutation of Ccm1’s NPXY motifs but is severely diminished when all three NPXY/F motifs are mutated. Heg-Ccm1 interactions appear unaffected by mutation of either two or all three NPXY/F motifs in Ccm1. (D) The mutant Ccm1 proteins FLAG-ccm1ty219c and FLAG-ccm1m775 bind HA-ccm2 and HA-ccm2l as well as wildtype FLAG-ccm1 does, but they do not bind Heg.

Techniques Used: Construct, Immunoprecipitation, Binding Assay, Mutagenesis

55) Product Images from "Nuclear and Cytoplasmic Soluble Proteins Extraction from a Small Quantity of Drosophila’s Whole Larvae and Tissues"

Article Title: Nuclear and Cytoplasmic Soluble Proteins Extraction from a Small Quantity of Drosophila’s Whole Larvae and Tissues

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms160612360

In vitro and in vivo analysis of proteins extracted from Drosophila ’s small quantity of whole larva and tissues. Western Blot analysis of proteins extracted from WL to detect any contamination of cytoplasmic proteins in nuclear fractions and vice versa . The same amount (3 μg) of different protein fractions is used in an immunodetection assay with two different antibodies: anti-αH3 (1:4000) and anti-GAPDH (1:4000) ( A1 ). The same extracts are used in an immunodetection assay with anti-AGO2 (1:2000) ( A2 ); ( B1 ) Western Blot analysis of proteins extracted from different Drosophila ’s tissues like MT, SG and B. ( B2 ) 3 μg of nuclear and cytoplasmic protein fractions are used in an immunodetection assay with anti-AGO2 (1:2000), anti-αH3 (1:4000), and anti-GAPDH (1:4000); ( C ) Co-immunoprecipitation of Squid and Hrb87F hnRNPs from nuclear extracts. The same amount of nuclear protein fractions is used in an immunoprecipitation assay with anti-Squid antibody and IgG. For the immunodetection assay anti-Squid (1:200) and anti-Hrb87F (1:4000) were used. I = Input, U Squid = Unbound from anti-Squid IP, W Squid , wash from anti-Squid IP, U IgG = Unbound from IgG IP, W IgG , wash from anti-IgG IP, IP Squid = Immunoprecipitated material from anti-Squid, IP IP IgG = Immunoprecipitated material from IgG IP.
Figure Legend Snippet: In vitro and in vivo analysis of proteins extracted from Drosophila ’s small quantity of whole larva and tissues. Western Blot analysis of proteins extracted from WL to detect any contamination of cytoplasmic proteins in nuclear fractions and vice versa . The same amount (3 μg) of different protein fractions is used in an immunodetection assay with two different antibodies: anti-αH3 (1:4000) and anti-GAPDH (1:4000) ( A1 ). The same extracts are used in an immunodetection assay with anti-AGO2 (1:2000) ( A2 ); ( B1 ) Western Blot analysis of proteins extracted from different Drosophila ’s tissues like MT, SG and B. ( B2 ) 3 μg of nuclear and cytoplasmic protein fractions are used in an immunodetection assay with anti-AGO2 (1:2000), anti-αH3 (1:4000), and anti-GAPDH (1:4000); ( C ) Co-immunoprecipitation of Squid and Hrb87F hnRNPs from nuclear extracts. The same amount of nuclear protein fractions is used in an immunoprecipitation assay with anti-Squid antibody and IgG. For the immunodetection assay anti-Squid (1:200) and anti-Hrb87F (1:4000) were used. I = Input, U Squid = Unbound from anti-Squid IP, W Squid , wash from anti-Squid IP, U IgG = Unbound from IgG IP, W IgG , wash from anti-IgG IP, IP Squid = Immunoprecipitated material from anti-Squid, IP IP IgG = Immunoprecipitated material from IgG IP.

Techniques Used: In Vitro, In Vivo, Western Blot, Immunodetection, Immunoprecipitation

56) Product Images from "Dysregulation of the MiR-449b target TGFBI alters the TGFβ pathway to induce cisplatin resistance in nasopharyngeal carcinoma"

Article Title: Dysregulation of the MiR-449b target TGFBI alters the TGFβ pathway to induce cisplatin resistance in nasopharyngeal carcinoma

Journal: Oncogenesis

doi: 10.1038/s41389-018-0050-x

TGFBI regulates signaling pathways by binding ITGB3 and ITGB5. a ITGB3 immunoprecipitation (IP: αMyc) was performed on HEK293T cells expressing ITGB3 in the absence or the presence of TGFBI (via transfection). Interaction between ITGB3 and TGFBI was detected using anti-TGFBI (αTGFBI) antibody. b ITGB5 immunoprecipitation (IP: αGFP) was performed on HEK293T cells expressing ITGB5ΔC with or without presence of TGFBI (via transfection). Interaction between ITGB5 and TGFBI was detected using anti-TGFBI (αTGFBI) antibody. c Immunoprecipitation of pro-TGFβ1 (IP: αFlag) was performed on HEK293T cells expressing pro-TGFβ1 alone or with ITGB3. Interaction between ITGB3 and pro-TGFβ1 was identified using anti-Myc (αMyc) antibody. d Immunoprecipitation of ITGB5 (IP: αGFP) was performed on HEK293T cells expressing ITGB5ΔC alone or with pro-TGFβ1. Interaction between ITGB5 and pro-TGFβ1 was revealed using anti-Flag (αFlag) antibody. e Co-immunoprecipitations were performed on HEK293T cells expressing ITGB3 alone or with pro-TGFβ1 in the absence (TGFβ1-Flag) or presence of TGFBI (TGFβ1-Flag + TGFBI). These data show competition between TGFBI and pro-TGFβ1 for ITGB3 binding. f Co-immunoprecipitations were performed on HEK293T expressing ITGB5ΔC alone or with pro-TGFβ1 in the absence (TGFβ1-Flag) or presence of TGFBI (TGFβ1-Flag + TGFBI). These data show competition between TGFBI and pro-TGFβ1 for ITGB5 binding. For a–f Western blot of the whole-cell lysates (WCLs) before pull-down is shown as a control for specificity and expression. g Top: relative luciferase activity was assessed after transient transfection of pro-TGFβ1-Flag and TGFBI-Myc, as indicated, and the co-transfection of pSBE4-luciferase vector (150 ng) and Renilla plasmid (100 ng). Bottom: western blot of HEK293T cells transfected with pro-TGFβ1-Flag encoding plasmid (1 µg) and TGFBI-Myc encoding plasmid, as indicated. Anti-Myc (αMyc) antibody was used to assess the expression of TGFBI, and anti-Flag (αFlag) antibody was used to assess the expression of pro-TGFβ1 and active TGFβ1 forms. The expression of pro-TGFβ1 indicates similar transfection efficiency across the conditions. Anti-β-actin (αβ-actin) antibody was used as the loading control. h Top: relative luciferase activity was assessed after transient transfection of ITGB3-Myc (1 µg) or ITGB5ΔC-GFP (1 µg) and TGFBI (2 µg), and the co-transfection of pSBE4-luciferase vector (150 ng) and Renilla plasmid (100 ng). Bottom: western blot control of the plasmids expression: anti-TGFBI (αTGFBI) antibody was used to assess the expression of TGFBI, anti-Myc (αMyc), and anti-GFP (αGFP) for ITGB3 and ITGB5, respectively. Anti-β-actin (αβ-actin) antibody was used as the loading control. The data are expressed as the mean ± SEM of at least three independent experiments. * P
Figure Legend Snippet: TGFBI regulates signaling pathways by binding ITGB3 and ITGB5. a ITGB3 immunoprecipitation (IP: αMyc) was performed on HEK293T cells expressing ITGB3 in the absence or the presence of TGFBI (via transfection). Interaction between ITGB3 and TGFBI was detected using anti-TGFBI (αTGFBI) antibody. b ITGB5 immunoprecipitation (IP: αGFP) was performed on HEK293T cells expressing ITGB5ΔC with or without presence of TGFBI (via transfection). Interaction between ITGB5 and TGFBI was detected using anti-TGFBI (αTGFBI) antibody. c Immunoprecipitation of pro-TGFβ1 (IP: αFlag) was performed on HEK293T cells expressing pro-TGFβ1 alone or with ITGB3. Interaction between ITGB3 and pro-TGFβ1 was identified using anti-Myc (αMyc) antibody. d Immunoprecipitation of ITGB5 (IP: αGFP) was performed on HEK293T cells expressing ITGB5ΔC alone or with pro-TGFβ1. Interaction between ITGB5 and pro-TGFβ1 was revealed using anti-Flag (αFlag) antibody. e Co-immunoprecipitations were performed on HEK293T cells expressing ITGB3 alone or with pro-TGFβ1 in the absence (TGFβ1-Flag) or presence of TGFBI (TGFβ1-Flag + TGFBI). These data show competition between TGFBI and pro-TGFβ1 for ITGB3 binding. f Co-immunoprecipitations were performed on HEK293T expressing ITGB5ΔC alone or with pro-TGFβ1 in the absence (TGFβ1-Flag) or presence of TGFBI (TGFβ1-Flag + TGFBI). These data show competition between TGFBI and pro-TGFβ1 for ITGB5 binding. For a–f Western blot of the whole-cell lysates (WCLs) before pull-down is shown as a control for specificity and expression. g Top: relative luciferase activity was assessed after transient transfection of pro-TGFβ1-Flag and TGFBI-Myc, as indicated, and the co-transfection of pSBE4-luciferase vector (150 ng) and Renilla plasmid (100 ng). Bottom: western blot of HEK293T cells transfected with pro-TGFβ1-Flag encoding plasmid (1 µg) and TGFBI-Myc encoding plasmid, as indicated. Anti-Myc (αMyc) antibody was used to assess the expression of TGFBI, and anti-Flag (αFlag) antibody was used to assess the expression of pro-TGFβ1 and active TGFβ1 forms. The expression of pro-TGFβ1 indicates similar transfection efficiency across the conditions. Anti-β-actin (αβ-actin) antibody was used as the loading control. h Top: relative luciferase activity was assessed after transient transfection of ITGB3-Myc (1 µg) or ITGB5ΔC-GFP (1 µg) and TGFBI (2 µg), and the co-transfection of pSBE4-luciferase vector (150 ng) and Renilla plasmid (100 ng). Bottom: western blot control of the plasmids expression: anti-TGFBI (αTGFBI) antibody was used to assess the expression of TGFBI, anti-Myc (αMyc), and anti-GFP (αGFP) for ITGB3 and ITGB5, respectively. Anti-β-actin (αβ-actin) antibody was used as the loading control. The data are expressed as the mean ± SEM of at least three independent experiments. * P

Techniques Used: Binding Assay, Immunoprecipitation, Expressing, Transfection, Western Blot, Luciferase, Activity Assay, Cotransfection, Plasmid Preparation

57) Product Images from "A function-blocking CD47 antibody suppresses stem cell and EGF signaling in triple-negative breast cancer"

Article Title: A function-blocking CD47 antibody suppresses stem cell and EGF signaling in triple-negative breast cancer

Journal: Oncotarget

doi: 10.18632/oncotarget.7100

A. AEGFR-immunoprecipitation from MDA-MB-231 cell extracts followed by western blotting shows that B6H12 treatment for 15 min disrupts the association between EGFR and CD47 and inhibits EGFR-Y 1068 phosphorylation. B . CD47-immunoprecipitation showed that a fraction of EGFR co-immunoprecipitates with EGFR. B6H12 treatment for 15 min reduced interaction between CD47 and EGFR in MDA-MB-231 cells. ( C .- D .) MDA-MB-231 cells were pretreated with B6H12 for 15 minutes followed by EGF for 5 minutes, and IP-western blotting was performed using phospho-EGFR antibody. D . Quantification of three experiments was analyzed using the t-test (*P
Figure Legend Snippet: A. AEGFR-immunoprecipitation from MDA-MB-231 cell extracts followed by western blotting shows that B6H12 treatment for 15 min disrupts the association between EGFR and CD47 and inhibits EGFR-Y 1068 phosphorylation. B . CD47-immunoprecipitation showed that a fraction of EGFR co-immunoprecipitates with EGFR. B6H12 treatment for 15 min reduced interaction between CD47 and EGFR in MDA-MB-231 cells. ( C .- D .) MDA-MB-231 cells were pretreated with B6H12 for 15 minutes followed by EGF for 5 minutes, and IP-western blotting was performed using phospho-EGFR antibody. D . Quantification of three experiments was analyzed using the t-test (*P

Techniques Used: Immunoprecipitation, Multiple Displacement Amplification, Western Blot

58) Product Images from "Peptidylproline cis-trans-Isomerase Pin1 Interacts with Human T-Cell Leukemia Virus Type 1 Tax and Modulates Its Activation of NF-?B ▿"

Article Title: Peptidylproline cis-trans-Isomerase Pin1 Interacts with Human T-Cell Leukemia Virus Type 1 Tax and Modulates Its Activation of NF-?B ▿

Journal: Journal of Virology

doi: 10.1128/JVI.01824-08

Tax binds Pin1. (A) In vitro interaction between Tax and Pin1. GST pull-down assays were performed with control Jurkat and HTLV-1 (C8166-45 and HUT102) cell lysates using the indicated recombinant proteins, Pin1 fused to GST (GST-Pin1) or GST alone. The input lane shows approximately one-fifth the amount of protein material used in the pull-down assays. Immunoblotting for Tax is shown in the top panel, while the bottom panel shows Ponceau red staining of the blotted membrane. (B) Coimmunoprecipitation of Tax and Pin1. 293T cells were cotransfected with FLAG-Pin1 WT, FLAG-Pin1(W34A) (mutated in the phosphorylated S/T-P binding domain), or FLAG-Pin1(K63A) (mutated in the catalytic domain) without (lanes 1 to 4) or with (lanes 5 to 8) Tax. Pin1 was immunoprecipitated with anti-FLAG agarose beads, and the immunoprecipitates were immunoblotted with anti-Tax (upper panel) or anti-FLAG (lower panel). (C) 293T cells were transfected with FLAG-Pin1 vector (from lane 2 to lane 5) and WT Tax (lane 3) or different Tax point mutants as indicated (lanes 4 and 5). FLAG-Pin1 was immunoprecipitated using anti-FLAG agarose beads. GFP-tagged protein was detected using anti-GFP. Amounts of FLAG-Pin1 in the immunoprecipitation and GFP-Tax in cell extracts were verified using anti-FLAG or anti-GFP. (D) A schematic overview of the FLAG-tagged deletion mutants TD1, TD55, TD99, TD150, TD254, and TD319. (E) Mapping the interaction between Tax and Pin1. 293T cells were transfected with a Myc-Pin1 expression vector and different Tax deletion mutants (lanes 3 to 8). Myc-Pin1 was immunoprecipitated with anti-Myc agarose beads (Sigma). FLAG-bound protein was detected by anti-FLAG (upper panels). The amount of Myc-Pin1 in the immunoprecipitate and the amount of FLAG-Tax in the cell extracts were checked using anti-Myc and anti-FLAG (bottom panels). (F) Coimmunoprecipitation assay of Tax and the indicated Tax mutants with Pin1. 293 T cells were cotransfected with HA-Pin1 alone (lane 1) or with 2 μg of Tax WT (lane 2), 6 μg of Tax S116A (lane 3), or 6 μg of Tax S160A (lane 4). Pin1 was immunoprecipitated with anti-HA agarose beads, and the immunoprecipitates were resolved by SDS-PAGE and immunoblotted with anti-Tax (upper panel). The amount of Pin1 in the immunoprecipitates and the amount of Tax in the cell extracts were checked using anti-Pin1 and anti-Tax (bottom panels). IB, immunoblotting; IP, immunoprecipitation; α, anti.
Figure Legend Snippet: Tax binds Pin1. (A) In vitro interaction between Tax and Pin1. GST pull-down assays were performed with control Jurkat and HTLV-1 (C8166-45 and HUT102) cell lysates using the indicated recombinant proteins, Pin1 fused to GST (GST-Pin1) or GST alone. The input lane shows approximately one-fifth the amount of protein material used in the pull-down assays. Immunoblotting for Tax is shown in the top panel, while the bottom panel shows Ponceau red staining of the blotted membrane. (B) Coimmunoprecipitation of Tax and Pin1. 293T cells were cotransfected with FLAG-Pin1 WT, FLAG-Pin1(W34A) (mutated in the phosphorylated S/T-P binding domain), or FLAG-Pin1(K63A) (mutated in the catalytic domain) without (lanes 1 to 4) or with (lanes 5 to 8) Tax. Pin1 was immunoprecipitated with anti-FLAG agarose beads, and the immunoprecipitates were immunoblotted with anti-Tax (upper panel) or anti-FLAG (lower panel). (C) 293T cells were transfected with FLAG-Pin1 vector (from lane 2 to lane 5) and WT Tax (lane 3) or different Tax point mutants as indicated (lanes 4 and 5). FLAG-Pin1 was immunoprecipitated using anti-FLAG agarose beads. GFP-tagged protein was detected using anti-GFP. Amounts of FLAG-Pin1 in the immunoprecipitation and GFP-Tax in cell extracts were verified using anti-FLAG or anti-GFP. (D) A schematic overview of the FLAG-tagged deletion mutants TD1, TD55, TD99, TD150, TD254, and TD319. (E) Mapping the interaction between Tax and Pin1. 293T cells were transfected with a Myc-Pin1 expression vector and different Tax deletion mutants (lanes 3 to 8). Myc-Pin1 was immunoprecipitated with anti-Myc agarose beads (Sigma). FLAG-bound protein was detected by anti-FLAG (upper panels). The amount of Myc-Pin1 in the immunoprecipitate and the amount of FLAG-Tax in the cell extracts were checked using anti-Myc and anti-FLAG (bottom panels). (F) Coimmunoprecipitation assay of Tax and the indicated Tax mutants with Pin1. 293 T cells were cotransfected with HA-Pin1 alone (lane 1) or with 2 μg of Tax WT (lane 2), 6 μg of Tax S116A (lane 3), or 6 μg of Tax S160A (lane 4). Pin1 was immunoprecipitated with anti-HA agarose beads, and the immunoprecipitates were resolved by SDS-PAGE and immunoblotted with anti-Tax (upper panel). The amount of Pin1 in the immunoprecipitates and the amount of Tax in the cell extracts were checked using anti-Pin1 and anti-Tax (bottom panels). IB, immunoblotting; IP, immunoprecipitation; α, anti.

Techniques Used: In Vitro, Recombinant, Staining, Binding Assay, Immunoprecipitation, Transfection, Plasmid Preparation, Expressing, Co-Immunoprecipitation Assay, SDS Page

Pin1 contributes to Tax interaction with IKKγ. (A) 293T cells were cotransfected with 1.5 μg of control siRNA (lanes 1 and 2) or 1.5 μg of siRNA targeted against Pin1 (lanes 3 and 4) and FLAG-tagged Tax (lanes 2 and 4). FLAG-Tax was immunoprecipitated using anti-FLAG agarose beads. IKKγ was detected using anti-IKKγ (upper panel). Amounts of FLAG-Tax and IKKγ in the immunoprecipitate and Pin1 in the cell extract were verified using anti-FLAG, anti-IKKγ, and anti-Pin1, respectively. (B) WT MEFs (lanes 1 to 4) and Pin1 KO MEFs (lanes 5 to 8) were cotransfected with control plasmid (4 μg; lanes 1 and 5), with HA-Tax (3 μg; lanes 3, 4, 7, and 8), or with FLAG-Pin1-expressing vector (1 μg; lanes 2, 4, 6, and 8). HA-Tax was immunoprecipitated using anti-HA agarose beads. HA-Tax and IKKγ in the immunoprecipitate and Pin1 in the cell extract were detected using anti-HA, anti-IKKγ, and anti-Pin1, respectively. IB, immunoblotting; IP, immunoprecipitation; α, anti.
Figure Legend Snippet: Pin1 contributes to Tax interaction with IKKγ. (A) 293T cells were cotransfected with 1.5 μg of control siRNA (lanes 1 and 2) or 1.5 μg of siRNA targeted against Pin1 (lanes 3 and 4) and FLAG-tagged Tax (lanes 2 and 4). FLAG-Tax was immunoprecipitated using anti-FLAG agarose beads. IKKγ was detected using anti-IKKγ (upper panel). Amounts of FLAG-Tax and IKKγ in the immunoprecipitate and Pin1 in the cell extract were verified using anti-FLAG, anti-IKKγ, and anti-Pin1, respectively. (B) WT MEFs (lanes 1 to 4) and Pin1 KO MEFs (lanes 5 to 8) were cotransfected with control plasmid (4 μg; lanes 1 and 5), with HA-Tax (3 μg; lanes 3, 4, 7, and 8), or with FLAG-Pin1-expressing vector (1 μg; lanes 2, 4, 6, and 8). HA-Tax was immunoprecipitated using anti-HA agarose beads. HA-Tax and IKKγ in the immunoprecipitate and Pin1 in the cell extract were detected using anti-HA, anti-IKKγ, and anti-Pin1, respectively. IB, immunoblotting; IP, immunoprecipitation; α, anti.

Techniques Used: Immunoprecipitation, Plasmid Preparation, Expressing

59) Product Images from "Beclin1 restricts RNA virus infection in plants through suppression and degradation of the viral polymerase"

Article Title: Beclin1 restricts RNA virus infection in plants through suppression and degradation of the viral polymerase

Journal: Nature Communications

doi: 10.1038/s41467-018-03658-2

NbBeclin1 interacts with NIb. a Yeast-two hybrid (Y2H) assays for possible interactions between NbBeclin1 and each of the 11 TuMV proteins. NbBeclin1 and 11 viral proteins were fused with a GAL4 activation domain (AD-NbBeclin1) and a GAL4-binding domain (BD-P1, BD-HC-Pro, BD-P3, BD-P3N-PIPO, BD-6K1, BD-CI, BD-6K2, BD-NIa-VPg, BD-NIa-Pro, BD-NIb, BD-CP), respectively. Y2H Gold yeast cells co-transformed with the indicated plasmids were subjected to 10-fold serial dilutions and plated on synthetic dextrose (SD)/-Trp, -Leu, -His, -Ade or SD/-Trp, -Leu medium to screen for positive interactions at 3 days after transformation. Yeast co-transformed with AD-T7-T+BD-T7-53 serves as a positive control; yeast cells co-transformed with AD-NbBeclin1 and the empty BD or with the empty AD and BD-NIb are negative controls. b BiFC assays between NbBeclin1 and NIb in the leaves of H2B-RFP transgenic N . benthamiana . Confocal imaging was performed at 48 hpi. NbBeclin1 and NIb were fused to the N (YN) and C-terminal (YC) fragments of yellow fluorescent protein (YFP). The NbBeclin1-NIb interaction led to the reconstituted fluorescence-competent structure and restoration of yellow fluorescence (green). Nuclei of tobacco leaf epidermal cells are indicated by the expression of H2B-RFP transgene (red). Bars, 50 μm. c Co-localization of NIb-YFP with NbBeclin1-CFP in the leaf cells of H2B-RFP transgenic N . benthamiana by confocal microscopy at 48 hpi. Arrow indicates yellow fluorescence, which was produced from the overlapping of NIb-YFP (green) and NbBeclin1-CFP (red). Bars, 50 μm. d Co-immunoprecipitation (Co-IP) analysis of NbBeclin1-CFP and Myc-NIb in vivo. N . benthamiana leaves were co-infiltrated with A . tumefaciens cells harboring expression vectors to express NbBeclin1-CFP and Myc-NIb (Lane 1), NbBeclin1-CFP and Myc-P3N-PIPO (Lane 2), Myc-NIb and GFP (Lane 3), and GFP and Myc-P3N-PIPO (Lane 4). Leaf protein extracts were incubated with GFP-Trap®_MA magnetic agarose beads (ChromoTek). Samples before (Input) and after (IP) immunopurification were analyzed by immunoblotting using GFP or Myc antibody
Figure Legend Snippet: NbBeclin1 interacts with NIb. a Yeast-two hybrid (Y2H) assays for possible interactions between NbBeclin1 and each of the 11 TuMV proteins. NbBeclin1 and 11 viral proteins were fused with a GAL4 activation domain (AD-NbBeclin1) and a GAL4-binding domain (BD-P1, BD-HC-Pro, BD-P3, BD-P3N-PIPO, BD-6K1, BD-CI, BD-6K2, BD-NIa-VPg, BD-NIa-Pro, BD-NIb, BD-CP), respectively. Y2H Gold yeast cells co-transformed with the indicated plasmids were subjected to 10-fold serial dilutions and plated on synthetic dextrose (SD)/-Trp, -Leu, -His, -Ade or SD/-Trp, -Leu medium to screen for positive interactions at 3 days after transformation. Yeast co-transformed with AD-T7-T+BD-T7-53 serves as a positive control; yeast cells co-transformed with AD-NbBeclin1 and the empty BD or with the empty AD and BD-NIb are negative controls. b BiFC assays between NbBeclin1 and NIb in the leaves of H2B-RFP transgenic N . benthamiana . Confocal imaging was performed at 48 hpi. NbBeclin1 and NIb were fused to the N (YN) and C-terminal (YC) fragments of yellow fluorescent protein (YFP). The NbBeclin1-NIb interaction led to the reconstituted fluorescence-competent structure and restoration of yellow fluorescence (green). Nuclei of tobacco leaf epidermal cells are indicated by the expression of H2B-RFP transgene (red). Bars, 50 μm. c Co-localization of NIb-YFP with NbBeclin1-CFP in the leaf cells of H2B-RFP transgenic N . benthamiana by confocal microscopy at 48 hpi. Arrow indicates yellow fluorescence, which was produced from the overlapping of NIb-YFP (green) and NbBeclin1-CFP (red). Bars, 50 μm. d Co-immunoprecipitation (Co-IP) analysis of NbBeclin1-CFP and Myc-NIb in vivo. N . benthamiana leaves were co-infiltrated with A . tumefaciens cells harboring expression vectors to express NbBeclin1-CFP and Myc-NIb (Lane 1), NbBeclin1-CFP and Myc-P3N-PIPO (Lane 2), Myc-NIb and GFP (Lane 3), and GFP and Myc-P3N-PIPO (Lane 4). Leaf protein extracts were incubated with GFP-Trap®_MA magnetic agarose beads (ChromoTek). Samples before (Input) and after (IP) immunopurification were analyzed by immunoblotting using GFP or Myc antibody

Techniques Used: Activation Assay, Binding Assay, Transformation Assay, Positive Control, Bimolecular Fluorescence Complementation Assay, Transgenic Assay, Imaging, Fluorescence, Expressing, Confocal Microscopy, Produced, Immunoprecipitation, Co-Immunoprecipitation Assay, In Vivo, Incubation, Immu-Puri

60) Product Images from "CEACAM1 regulates TIM–3–mediated tolerance and exhaustion"

Article Title: CEACAM1 regulates TIM–3–mediated tolerance and exhaustion

Journal: Nature

doi: 10.1038/nature13848

Biochemical characterization of interactions between CEACAM1 and TIM-3 a , hTIM-3 does not co-immunoprecipitate (co-IP) with ITGA5 despite interactions with hCEACAM1. HEK293T cells transfected with Flag–ITGA5 and HA–TIM-3 (ITGA5Tw) or Flag–CEACAM1 and HA–TIM-3 (CwTw). Immunoprecipitation with anti-HA antibody and immunoblotted (IB) with anti-Flag antibody are shown. Input represents anti-Flag immunoblot of lysates. b, Co-immunoprecipitation of human TIM-3 and CEACAM1 from activated primary human T cells after N -glycanase treatment of lystates followed by immunoprecipitation with anti-human TIM-3 antibodies (2E2, 2E12 or 3F9) or IgG as control and immunoblotted with anti-human CEACAM1 antibody (5F4). Protein lystates from HeLa-CEACAM1 transfectants treated with N -glycanase followed by immunoprecipitation with 5F4 and the immune complex used as positive control (pos). c , mTIM-3 interacts with mCEACAM1 in mouse T cells. Splenocytes from Ceacam1 4S Tg Ceacam1 −/− and Ceacam1-4L Tg Ceacam1 −/− mice cultured with anti-CD3 (1 μg ml −1 ) or anti-CD3 (1 μg ml −1 ) and anti-CD28 (1 μg ml −1 ) or medium for 96 h. Cell lysates immunoprecipitated with anti-mCEACAM1 antibody (cc1) or with mIgG and IB with 5D12 (anti-mTIM-3 antibody) are shown. Locations of mTIM-3 protein variants are indicated. CHO, carbohydrate. d , Immunoprecipitation and immunoblot as in a with tunicamycin treated, wild-type HA–hTIM-3 and Flag–hCEACAM1 co-transfected HEK293T cells. Arrowhead denotes core CEACAM1 protein. e , Potential hCEACAM1-interacting residues on hTIM-3 highlighted in blue. f , HEK293 T cells transiently co-transfected with Flag–hCEACAM1 and HA–hTIM-3 mutants. Immunoblotting of anti-HA were used to analyse hTIM-3 expression in HEK293T transfectants. Except for Pro50Ala mutation displaying enhanced overall protein expression, all other mutations in the IgV domain of hTIM-3 are equally detected by anti-HA antibody. g , Quantification of association of hTIM-3 mutants associated with wild-type hCEACAM1 shown in summing all experiments performed. Association between wild-type hCEACAM1 and hTIM-3 core protein are depicted as reference (set as 1, n = 3, mean ± s.e.m. shown, unpaired Student’s t -test). h , Immunoprecipitation with anti-Flag (hCEACAM1) and immunoblot with anti-HA (hTIM-3) or anti-Flag of wild-type hCEACAM1 and mutant hTIM-3 proteins are shown. i , Quantification of h as performed in g. j , HEK293T cells co-transfected with Flag–hCEACAM1 wild-type and HA– hTIM-3 mutants and immunoprecipitation/immunblot as in h revealing no effects of Cys52Ala or Cys63Ala mutations in hTIM-3 in affecting association with hCEACAM1 in contrast to Cys109Ala mutation of hTIM-3 that disrupts interactions with hCEACAM1. k , Potential hTIM-3-interacting-residues around the FG–CC′ cleft of hCEACAM1 highlighted in red. l , HEK293T cells transiently co-transfected with Flag–hCEACAM1 mutants and wild-type HA–hTIM-3. Immunoblot with anti-Flag antibody was used to analyse hCEACAM1 expression in HEK293T co-transfectants. All hCEACAM1 mutations in IgV domain equally detected. m . n–p , Analysis of Gly47Ala mutation of hCEACAM1 in hTIM-3 co-transfected HEK293T cells by immunoprecipitation with anti-HA (hTIM-3) and immunoblot with anti-Flag (hCEACAM1) to detect association ( n ), IB with anti-Flag to confirm similarity of hCEACAM1 transfection ( o ) and quantification of associated hCEACAM1 of n as shown in m. q–s , Analysis of hCEACAM1 mutants Asn42Ala and Arg43Ala association with hTIM-3 ( q ), similarity of transfections ( r ) and quantification of q as in n–p . Representative of four ( d, h ), three ( f, g, i, l–s ), two ( a–c ) and one ( j ) independent experiments. * P
Figure Legend Snippet: Biochemical characterization of interactions between CEACAM1 and TIM-3 a , hTIM-3 does not co-immunoprecipitate (co-IP) with ITGA5 despite interactions with hCEACAM1. HEK293T cells transfected with Flag–ITGA5 and HA–TIM-3 (ITGA5Tw) or Flag–CEACAM1 and HA–TIM-3 (CwTw). Immunoprecipitation with anti-HA antibody and immunoblotted (IB) with anti-Flag antibody are shown. Input represents anti-Flag immunoblot of lysates. b, Co-immunoprecipitation of human TIM-3 and CEACAM1 from activated primary human T cells after N -glycanase treatment of lystates followed by immunoprecipitation with anti-human TIM-3 antibodies (2E2, 2E12 or 3F9) or IgG as control and immunoblotted with anti-human CEACAM1 antibody (5F4). Protein lystates from HeLa-CEACAM1 transfectants treated with N -glycanase followed by immunoprecipitation with 5F4 and the immune complex used as positive control (pos). c , mTIM-3 interacts with mCEACAM1 in mouse T cells. Splenocytes from Ceacam1 4S Tg Ceacam1 −/− and Ceacam1-4L Tg Ceacam1 −/− mice cultured with anti-CD3 (1 μg ml −1 ) or anti-CD3 (1 μg ml −1 ) and anti-CD28 (1 μg ml −1 ) or medium for 96 h. Cell lysates immunoprecipitated with anti-mCEACAM1 antibody (cc1) or with mIgG and IB with 5D12 (anti-mTIM-3 antibody) are shown. Locations of mTIM-3 protein variants are indicated. CHO, carbohydrate. d , Immunoprecipitation and immunoblot as in a with tunicamycin treated, wild-type HA–hTIM-3 and Flag–hCEACAM1 co-transfected HEK293T cells. Arrowhead denotes core CEACAM1 protein. e , Potential hCEACAM1-interacting residues on hTIM-3 highlighted in blue. f , HEK293 T cells transiently co-transfected with Flag–hCEACAM1 and HA–hTIM-3 mutants. Immunoblotting of anti-HA were used to analyse hTIM-3 expression in HEK293T transfectants. Except for Pro50Ala mutation displaying enhanced overall protein expression, all other mutations in the IgV domain of hTIM-3 are equally detected by anti-HA antibody. g , Quantification of association of hTIM-3 mutants associated with wild-type hCEACAM1 shown in summing all experiments performed. Association between wild-type hCEACAM1 and hTIM-3 core protein are depicted as reference (set as 1, n = 3, mean ± s.e.m. shown, unpaired Student’s t -test). h , Immunoprecipitation with anti-Flag (hCEACAM1) and immunoblot with anti-HA (hTIM-3) or anti-Flag of wild-type hCEACAM1 and mutant hTIM-3 proteins are shown. i , Quantification of h as performed in g. j , HEK293T cells co-transfected with Flag–hCEACAM1 wild-type and HA– hTIM-3 mutants and immunoprecipitation/immunblot as in h revealing no effects of Cys52Ala or Cys63Ala mutations in hTIM-3 in affecting association with hCEACAM1 in contrast to Cys109Ala mutation of hTIM-3 that disrupts interactions with hCEACAM1. k , Potential hTIM-3-interacting-residues around the FG–CC′ cleft of hCEACAM1 highlighted in red. l , HEK293T cells transiently co-transfected with Flag–hCEACAM1 mutants and wild-type HA–hTIM-3. Immunoblot with anti-Flag antibody was used to analyse hCEACAM1 expression in HEK293T co-transfectants. All hCEACAM1 mutations in IgV domain equally detected. m . n–p , Analysis of Gly47Ala mutation of hCEACAM1 in hTIM-3 co-transfected HEK293T cells by immunoprecipitation with anti-HA (hTIM-3) and immunoblot with anti-Flag (hCEACAM1) to detect association ( n ), IB with anti-Flag to confirm similarity of hCEACAM1 transfection ( o ) and quantification of associated hCEACAM1 of n as shown in m. q–s , Analysis of hCEACAM1 mutants Asn42Ala and Arg43Ala association with hTIM-3 ( q ), similarity of transfections ( r ) and quantification of q as in n–p . Representative of four ( d, h ), three ( f, g, i, l–s ), two ( a–c ) and one ( j ) independent experiments. * P

Techniques Used: Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Positive Control, Mouse Assay, Cell Culture, Expressing, Mutagenesis

CEACAM1 and TIM-3 heterodimerize and serve as heterophilic ligands a, b , Co-immunoprecipitation (IP) and immunoblot (IB) of wild-type hCEACAM1 and hTIM-3 in co-transfected HEK293T cells, c, d , Co-immunoprecipitation and immunoblot of wild-type hCEACAM1 and hTIM-3 mutants ( c ) or wild-type hTIM-3 and hCEACAM1 mutants ( d ) as in a and b. e , Human CEACAM1 (IgV)-TIM-3 (IgV) heterodimer structure, f, g , 2 F o — F c maps contoured at 0.9σ showing electron densities, h, i , Autoradiogram of anti-haemagglutinin (HA) (hTIM-3) immunoprecipitate from metabolic-labelled ( h ) and pulse-chase metabolic-labelled ( i ) co-transfected HEK293T cells. CHO, carbohydrate; core T, non-glycosylated hTIM-3; Cw, wild-type hCEACAM1; EndoH, endoglycosidaseH; H2-MA, HA-tagged influenza virus A M2 protein; T, hTIM-3 (Thr101Ile); Tw, wild-type hTIM-3. hTIM-3 isoforms noted. j , Quantification of densities in i ( n = 3 per group). k , Immunoblot for mTIM-3 from PBS-treated (−) or SEB-treated (+) CD4 + T cells. Labelling as in h and i. 1 , mTIM-3 expression after SEB tolerance induction, m , Column-bound glutathione S -transferase (GST)-hTIM-3 IgV-domain pull-down of hCEACAM1 detected by immunoblot. GST 2 , GST-hTIM-3 dimer. Ft, flow through, n , Suppression of mouse CD4 + T-cell proliferation by mCEACAM1 N-terminal domain-Fc fusion protein (NFc). o , Immunoprecipitation of mTIM-3 and immunoblot for BAT3 or mTIM-3 from lysates of CD4 + T cells. p, q , Proliferation of CD4 + T cells from wild-type ( p ) and CeaCAM1 −/− ( q ) mice transduced with wild-type mTIM-3 (Tw), mTIM-3 Δ252–281 (Tmut) or vector exposed to anti-CD3 and either NFc or IgG1-Fc (IgG1). Data are mean ± s.e.m. and represent five ( a, b ), four ( c, d ), three ( h-j, l, n, p, q ) and two ( k, m, o ) independent experiments. NS, not significant; * P
Figure Legend Snippet: CEACAM1 and TIM-3 heterodimerize and serve as heterophilic ligands a, b , Co-immunoprecipitation (IP) and immunoblot (IB) of wild-type hCEACAM1 and hTIM-3 in co-transfected HEK293T cells, c, d , Co-immunoprecipitation and immunoblot of wild-type hCEACAM1 and hTIM-3 mutants ( c ) or wild-type hTIM-3 and hCEACAM1 mutants ( d ) as in a and b. e , Human CEACAM1 (IgV)-TIM-3 (IgV) heterodimer structure, f, g , 2 F o — F c maps contoured at 0.9σ showing electron densities, h, i , Autoradiogram of anti-haemagglutinin (HA) (hTIM-3) immunoprecipitate from metabolic-labelled ( h ) and pulse-chase metabolic-labelled ( i ) co-transfected HEK293T cells. CHO, carbohydrate; core T, non-glycosylated hTIM-3; Cw, wild-type hCEACAM1; EndoH, endoglycosidaseH; H2-MA, HA-tagged influenza virus A M2 protein; T, hTIM-3 (Thr101Ile); Tw, wild-type hTIM-3. hTIM-3 isoforms noted. j , Quantification of densities in i ( n = 3 per group). k , Immunoblot for mTIM-3 from PBS-treated (−) or SEB-treated (+) CD4 + T cells. Labelling as in h and i. 1 , mTIM-3 expression after SEB tolerance induction, m , Column-bound glutathione S -transferase (GST)-hTIM-3 IgV-domain pull-down of hCEACAM1 detected by immunoblot. GST 2 , GST-hTIM-3 dimer. Ft, flow through, n , Suppression of mouse CD4 + T-cell proliferation by mCEACAM1 N-terminal domain-Fc fusion protein (NFc). o , Immunoprecipitation of mTIM-3 and immunoblot for BAT3 or mTIM-3 from lysates of CD4 + T cells. p, q , Proliferation of CD4 + T cells from wild-type ( p ) and CeaCAM1 −/− ( q ) mice transduced with wild-type mTIM-3 (Tw), mTIM-3 Δ252–281 (Tmut) or vector exposed to anti-CD3 and either NFc or IgG1-Fc (IgG1). Data are mean ± s.e.m. and represent five ( a, b ), four ( c, d ), three ( h-j, l, n, p, q ) and two ( k, m, o ) independent experiments. NS, not significant; * P

Techniques Used: Immunoprecipitation, Transfection, Pulse Chase, Expressing, Flow Cytometry, Mouse Assay, Transduction, Plasmid Preparation

61) Product Images from "RCN1 suppresses ER stress-induced apoptosis via calcium homeostasis and PERK–CHOP signaling"

Article Title: RCN1 suppresses ER stress-induced apoptosis via calcium homeostasis and PERK–CHOP signaling

Journal: Oncogenesis

doi: 10.1038/oncsis.2017.6

RCN1 interacts with IP 3 R1 and inhibits ER calcium release. ( a ) Representative Ca 2+ traces of cytosolic Ca 2+ after treatment with ATP (20 μ m ) in negative control (shNC) or RCN1 shRNA-transfected (shRCN1) HEK293T cells. ( b and c ) Representative Ca 2+ traces of cytosolic Ca 2+ after treatment with ATP (100 μ m ) ( b ) or histamine (50 μ m ) ( c ) in negative control or RCN1-knockdown HepG2 cells. ( d ) HEK293T cells co-transfected with 3 × Flag-RCN1 and GFP or IP 3 R1-TM (transmembrane domain)-GFP were subjected to immunoprecipitation (IP) using anti-GFP antibody. The immunoprecipitates were immunoblotted with anti-Flag or anti-GFP antibody. ( e ) HEK293T cells co-transfected with IP 3 R1-TM-GFP and 3 × Flag or 3 × Flag-RCN1 were subjected to immunoprecipitation using anti-Flag antibody. The immunoprecipitates were immunoblotted with anti-Flag or anti-GFP antibody. ( f ) HepG2 cells were subjected to immunoprecipitation using anti-IP 3 R1 antibody. The immunoprecipitates were immunoblotted with anti-RCN1 or anti-IP 3 R1 antibody. ( g ) Schematic of IP 3 R1 structure. ( h ) Mapping of the domains of IP 3 R1 required for interaction with RCN1. HEK293T cells co-overexpressing GFP-tagged IP 3 R1 truncations (IP 3 R1-L1-GFP, IP 3 R1-L2-GFP, IP 3 R1-L3-GFP) and 3 × Flag-RCN1 were subjected to immunoprecipitation with anti-GFP antibody. The immunoprecipitates were immunoblotted with anti-Flag or anti-GFP antibody. ( i ) Schematic of RCN1 truncation constructs. SP, signal peptide; EFh, EF-hand. ( j ) HEK293T cells co-transfected with IP 3 R1-TM-GFP and 3 × Flag, 3 × Flag-RCN1, 3 × Flag-RCN1-EFh1+2, or 3 × Flag-RCN1-EFh1 were subjected to immunoprecipitation using anti-Flag antibody. The immunoprecipitates were immunoblotted with anti-Flag or anti-GFP antibody. ( k ) Basal Ca 2+ level tracked by fluo-4 in ATP-treated HEK293T cells transfected with control, 3 × Flag-RCN1 or 3 × Flag-EFh1+2. Representative Ca 2+ traces of cytosolic Ca 2+ after treatment with ATP (100 μ m ) in control (3 × Flag)-, 3 × Flag-EFh1+2- or 3 × Flag-RCN1-transfected HEK293T cells.
Figure Legend Snippet: RCN1 interacts with IP 3 R1 and inhibits ER calcium release. ( a ) Representative Ca 2+ traces of cytosolic Ca 2+ after treatment with ATP (20 μ m ) in negative control (shNC) or RCN1 shRNA-transfected (shRCN1) HEK293T cells. ( b and c ) Representative Ca 2+ traces of cytosolic Ca 2+ after treatment with ATP (100 μ m ) ( b ) or histamine (50 μ m ) ( c ) in negative control or RCN1-knockdown HepG2 cells. ( d ) HEK293T cells co-transfected with 3 × Flag-RCN1 and GFP or IP 3 R1-TM (transmembrane domain)-GFP were subjected to immunoprecipitation (IP) using anti-GFP antibody. The immunoprecipitates were immunoblotted with anti-Flag or anti-GFP antibody. ( e ) HEK293T cells co-transfected with IP 3 R1-TM-GFP and 3 × Flag or 3 × Flag-RCN1 were subjected to immunoprecipitation using anti-Flag antibody. The immunoprecipitates were immunoblotted with anti-Flag or anti-GFP antibody. ( f ) HepG2 cells were subjected to immunoprecipitation using anti-IP 3 R1 antibody. The immunoprecipitates were immunoblotted with anti-RCN1 or anti-IP 3 R1 antibody. ( g ) Schematic of IP 3 R1 structure. ( h ) Mapping of the domains of IP 3 R1 required for interaction with RCN1. HEK293T cells co-overexpressing GFP-tagged IP 3 R1 truncations (IP 3 R1-L1-GFP, IP 3 R1-L2-GFP, IP 3 R1-L3-GFP) and 3 × Flag-RCN1 were subjected to immunoprecipitation with anti-GFP antibody. The immunoprecipitates were immunoblotted with anti-Flag or anti-GFP antibody. ( i ) Schematic of RCN1 truncation constructs. SP, signal peptide; EFh, EF-hand. ( j ) HEK293T cells co-transfected with IP 3 R1-TM-GFP and 3 × Flag, 3 × Flag-RCN1, 3 × Flag-RCN1-EFh1+2, or 3 × Flag-RCN1-EFh1 were subjected to immunoprecipitation using anti-Flag antibody. The immunoprecipitates were immunoblotted with anti-Flag or anti-GFP antibody. ( k ) Basal Ca 2+ level tracked by fluo-4 in ATP-treated HEK293T cells transfected with control, 3 × Flag-RCN1 or 3 × Flag-EFh1+2. Representative Ca 2+ traces of cytosolic Ca 2+ after treatment with ATP (100 μ m ) in control (3 × Flag)-, 3 × Flag-EFh1+2- or 3 × Flag-RCN1-transfected HEK293T cells.

Techniques Used: Negative Control, shRNA, Transfection, Immunoprecipitation, Construct

62) Product Images from "The Arkadia-ESRP2 axis suppresses tumor progression: analyses in clear-cell renal cell carcinoma"

Article Title: The Arkadia-ESRP2 axis suppresses tumor progression: analyses in clear-cell renal cell carcinoma

Journal: Oncogene

doi: 10.1038/onc.2015.412

Modulation of the splicing function of ESRP2 by Arkadia through ubiquitination. ( a and b ) Immunoprecipitation assay using HEK293T cells transiently transfected with ESRP1 ( a ) or ESRP2 ( b ), Arkadia wild-type or CA mutant, and ubiquitin. IP, immunoprecipitation; IB, immunoblotting; Ub, ubiquitin; WT, wild-type. ( c and d ) Immunoprecipitation assay using HEK293T cells transiently transfected with ESRP1 ( c ) or ESRP2 ( d ), WT Arkadia, and ubiquitin WT or its mutants. 7KR: all seven lysine residues are substituted by arginine residues; 6, 11, 27, 29, 33, 48 and 63K: one lysine residue is intact, but the others are substituted by arginine residues. KR, lysine-to-arginine mutation. ( e ) Schematic representation of ESRP2-KR mutants. ( f ) Immunoblot analysis to examine the protein expression of WT or ESRP2-KR mutants overexpressed in HEK293T cells. ( g ) qRT–PCR analysis of ENAH exon 11a expression in HEK293T cells transiently transfected with WT or ESRP2-KR mutants upon Arkadia knockdown. Data were normalized to total ENAH. Error bars indicate s.d. siNC, negative control siRNA. Experiments were repeated, and a representative set of data are shown in ( g ). ( h ) Schematic representation of the regulation of ESRP2 function by Arkadia through ubiquitination.
Figure Legend Snippet: Modulation of the splicing function of ESRP2 by Arkadia through ubiquitination. ( a and b ) Immunoprecipitation assay using HEK293T cells transiently transfected with ESRP1 ( a ) or ESRP2 ( b ), Arkadia wild-type or CA mutant, and ubiquitin. IP, immunoprecipitation; IB, immunoblotting; Ub, ubiquitin; WT, wild-type. ( c and d ) Immunoprecipitation assay using HEK293T cells transiently transfected with ESRP1 ( c ) or ESRP2 ( d ), WT Arkadia, and ubiquitin WT or its mutants. 7KR: all seven lysine residues are substituted by arginine residues; 6, 11, 27, 29, 33, 48 and 63K: one lysine residue is intact, but the others are substituted by arginine residues. KR, lysine-to-arginine mutation. ( e ) Schematic representation of ESRP2-KR mutants. ( f ) Immunoblot analysis to examine the protein expression of WT or ESRP2-KR mutants overexpressed in HEK293T cells. ( g ) qRT–PCR analysis of ENAH exon 11a expression in HEK293T cells transiently transfected with WT or ESRP2-KR mutants upon Arkadia knockdown. Data were normalized to total ENAH. Error bars indicate s.d. siNC, negative control siRNA. Experiments were repeated, and a representative set of data are shown in ( g ). ( h ) Schematic representation of the regulation of ESRP2 function by Arkadia through ubiquitination.

Techniques Used: Immunoprecipitation, Transfection, Mutagenesis, Expressing, Quantitative RT-PCR, Negative Control

63) Product Images from "Sequence-dependent cargo recognition by SNX-BARs mediates retromer-independent transport of CI-MPR"

Article Title: Sequence-dependent cargo recognition by SNX-BARs mediates retromer-independent transport of CI-MPR

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201703015

SNX1 is less efficiently recruited to CI-MPR tubules expressing a chimera harboring the WLM-AAA mutation. (A) Scheme of CI-MPR chimera constructs used. SP, signal peptide; TM, transmembrane domain. (B, top) The GFP–CI-MPR chimera WT and the GFP–CI-MPR chimera WLM-AAA mutant have comparable expression levels. HeLa cells were transfected with CI-MPR chimeras. 48 h after transfection, GFP levels were analyzed by Western blotting. (B, bottom) n = 3 independent experiments. (C) The GFP–CI-MPR chimera WLM-AAA mutant has a reduced ability to bind to the SNX1/2–SNX5/6 complex. GFP trap of GFP-tagged GFP–CI-MPR chimeras, each transiently transfected in HEK293T cells. Molecular masses are given in kilodaltons. IP, immunoprecipitation. (D, left) HeLa cells were transfected with WT or WLM-AAA mutant GFP–CI-MPR chimera constructs. Bars: (main images) 20 µm; (insets) 5 µm. (D, right) The percentages of cells with at least one GFP-positive tubule were blindly scored. n = 3 independent experiments; WT, 145 cells; WLM-AAA, 139 cells. (E) HeLa cells were transfected with WT or WLM-AAA mutant GFP–CI-MPR construct and immunostained for endogenous SNX1 and endogenous VPS35 after 48 h. Bars: (main images) 20 µm; (insets) 10 µm. (E, top right) Relative number of GFP-positive and SNX1-negative tubules per cell. n = 3 blindly scored independent experiments; WT, 40 cells; WLM-AAA, 38 cells. (E, bottom right) Relative number of GFP-positive and SNX1-positive tubules per cell. n = 3 blindly scored independent experiments; WT, 40 cells; WLM-AAA, 38 cells (means ± SEM; unpaired t test; *, P
Figure Legend Snippet: SNX1 is less efficiently recruited to CI-MPR tubules expressing a chimera harboring the WLM-AAA mutation. (A) Scheme of CI-MPR chimera constructs used. SP, signal peptide; TM, transmembrane domain. (B, top) The GFP–CI-MPR chimera WT and the GFP–CI-MPR chimera WLM-AAA mutant have comparable expression levels. HeLa cells were transfected with CI-MPR chimeras. 48 h after transfection, GFP levels were analyzed by Western blotting. (B, bottom) n = 3 independent experiments. (C) The GFP–CI-MPR chimera WLM-AAA mutant has a reduced ability to bind to the SNX1/2–SNX5/6 complex. GFP trap of GFP-tagged GFP–CI-MPR chimeras, each transiently transfected in HEK293T cells. Molecular masses are given in kilodaltons. IP, immunoprecipitation. (D, left) HeLa cells were transfected with WT or WLM-AAA mutant GFP–CI-MPR chimera constructs. Bars: (main images) 20 µm; (insets) 5 µm. (D, right) The percentages of cells with at least one GFP-positive tubule were blindly scored. n = 3 independent experiments; WT, 145 cells; WLM-AAA, 139 cells. (E) HeLa cells were transfected with WT or WLM-AAA mutant GFP–CI-MPR construct and immunostained for endogenous SNX1 and endogenous VPS35 after 48 h. Bars: (main images) 20 µm; (insets) 10 µm. (E, top right) Relative number of GFP-positive and SNX1-negative tubules per cell. n = 3 blindly scored independent experiments; WT, 40 cells; WLM-AAA, 38 cells. (E, bottom right) Relative number of GFP-positive and SNX1-positive tubules per cell. n = 3 blindly scored independent experiments; WT, 40 cells; WLM-AAA, 38 cells (means ± SEM; unpaired t test; *, P

Techniques Used: Expressing, Mutagenesis, Construct, Transfection, Western Blot, Immunoprecipitation

Interactome of the retromer-linked SNX-BAR complex. (A) Schematic representation of the SILAC methodology and the approach used to filter and merge SILAC datasets. (B) Venn diagram showing the interactors of SNX5, SNX6, and SNX32. (C) Venn diagram showing the interactors of SNX1 and SNX2 together with the shared interactors between SNX5, SNX6, and SNX32 (those within the red demarcated area in B). (D) STRING analysis of SNX-BAR interactors. Interactors were compiled and subjected to STRING analysis. Each connecting line represents an interaction indicated by experimental or database evidence. Color of the node indicates presence of the protein in a specific subset of the SNX-BAR interactome. (E) GFP trap of GFP-tagged retromer-linked SNX-BARs, retromer, and the retromer-independent SNX4 and SNX8, each transiently transfected in HEK293T cells. Molecular masses are given in kilodaltons. IP, immunoprecipitation.
Figure Legend Snippet: Interactome of the retromer-linked SNX-BAR complex. (A) Schematic representation of the SILAC methodology and the approach used to filter and merge SILAC datasets. (B) Venn diagram showing the interactors of SNX5, SNX6, and SNX32. (C) Venn diagram showing the interactors of SNX1 and SNX2 together with the shared interactors between SNX5, SNX6, and SNX32 (those within the red demarcated area in B). (D) STRING analysis of SNX-BAR interactors. Interactors were compiled and subjected to STRING analysis. Each connecting line represents an interaction indicated by experimental or database evidence. Color of the node indicates presence of the protein in a specific subset of the SNX-BAR interactome. (E) GFP trap of GFP-tagged retromer-linked SNX-BARs, retromer, and the retromer-independent SNX4 and SNX8, each transiently transfected in HEK293T cells. Molecular masses are given in kilodaltons. IP, immunoprecipitation.

Techniques Used: Transfection, Immunoprecipitation

64) Product Images from "Ago1 Interacts with RNA Polymerase II and Binds to the Promoters of Actively Transcribed Genes in Human Cancer Cells"

Article Title: Ago1 Interacts with RNA Polymerase II and Binds to the Promoters of Actively Transcribed Genes in Human Cancer Cells

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1003821

Nuclear Ago1 interacts with RNAP II. ( A ) Immunoprecipitation (IP) assays were performed on nuclear extracts from PC-3 cells using Ago1 or Ago2 antibodies. IgG served as a negative IP control. Immunoprecipitates were analyzed by immunoblotting (IB) with the indicated antibodies. RNAP II and Tubulin were also detected by IB analysis to validate nuclear (Nuc) and cytoplasmic (Cyto) fractions. ( B ) Reciprocal IP analysis was performed on nuclear extracts from PC-3 cells using an antibody specific to RNAP II. IB detected pulldown of Ago1 and RNAP II but not Ago2. Input control represents 10% nuclear extract used for IP. * denotes a nonspecific band. ( C ) IP was performed on nuclear extracts from LNCaP cells as in (A). Nuclear (Nuc) and cytoplasmic (Cyto) fractions were confirmed by IB analysis.
Figure Legend Snippet: Nuclear Ago1 interacts with RNAP II. ( A ) Immunoprecipitation (IP) assays were performed on nuclear extracts from PC-3 cells using Ago1 or Ago2 antibodies. IgG served as a negative IP control. Immunoprecipitates were analyzed by immunoblotting (IB) with the indicated antibodies. RNAP II and Tubulin were also detected by IB analysis to validate nuclear (Nuc) and cytoplasmic (Cyto) fractions. ( B ) Reciprocal IP analysis was performed on nuclear extracts from PC-3 cells using an antibody specific to RNAP II. IB detected pulldown of Ago1 and RNAP II but not Ago2. Input control represents 10% nuclear extract used for IP. * denotes a nonspecific band. ( C ) IP was performed on nuclear extracts from LNCaP cells as in (A). Nuclear (Nuc) and cytoplasmic (Cyto) fractions were confirmed by IB analysis.

Techniques Used: Immunoprecipitation

65) Product Images from "GPR158/179 regulate G protein signaling by controlling localization and activity of the RGS7 complexes"

Article Title: GPR158/179 regulate G protein signaling by controlling localization and activity of the RGS7 complexes

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201202123

GPR179 is a paralogue of GPR158 required for subcellular targeting of the RGS7–Gβ5 complex in vivo. (A) GPR158 shares considerable sequence homology and conservation among species with GPR179 as revealed by phylogenic analysis. (B) GPR179 forms complexes with RGS7 in transfected cells. Forward and reverse immunoprecipitation experiments were carried using the indicated antibodies after cotransfection of GPR158 with RGS7 in HEK293T/17 cells. (C) Coimmunoprecipitation of GPR179 with members of the R7 RGS subfamily upon expression in HEK293T/17 cells. (D) Expression profile of GPR179 as determined by the Western blotting of total tissue lysates. (E) GPR179 colocalizes with RGS7 and RGS11 at the dendritic tips of the ON bipolar cells in the outer plexiform layer of the retina. Retina cross sections were immunolabeled for GPR179 and RGS7. (F) Loss of GPR179 in nob5 retinas does not affect the expression of RGS7 and RGS11 as revealed by Western blot analysis of RGS7 and RGS11 expression in total retina lysates from wild-type (WT) mice or mice lacking GPR179 (nob5). (G and H) Elimination of GPR179 prevents targeting of RGS7 and RGS11, but not TRPM1, to the dendritic tips. Retina cross sections were double immunostained for RGS7 and GPR179 in G or RGS7/RGS11 and TRPM1 in H. Cell nuclei are labeled with DAPI. Bars: (E and G) 10 µm; (H) 5 µm. IP, immunoprecipitation; KO, knockout.
Figure Legend Snippet: GPR179 is a paralogue of GPR158 required for subcellular targeting of the RGS7–Gβ5 complex in vivo. (A) GPR158 shares considerable sequence homology and conservation among species with GPR179 as revealed by phylogenic analysis. (B) GPR179 forms complexes with RGS7 in transfected cells. Forward and reverse immunoprecipitation experiments were carried using the indicated antibodies after cotransfection of GPR158 with RGS7 in HEK293T/17 cells. (C) Coimmunoprecipitation of GPR179 with members of the R7 RGS subfamily upon expression in HEK293T/17 cells. (D) Expression profile of GPR179 as determined by the Western blotting of total tissue lysates. (E) GPR179 colocalizes with RGS7 and RGS11 at the dendritic tips of the ON bipolar cells in the outer plexiform layer of the retina. Retina cross sections were immunolabeled for GPR179 and RGS7. (F) Loss of GPR179 in nob5 retinas does not affect the expression of RGS7 and RGS11 as revealed by Western blot analysis of RGS7 and RGS11 expression in total retina lysates from wild-type (WT) mice or mice lacking GPR179 (nob5). (G and H) Elimination of GPR179 prevents targeting of RGS7 and RGS11, but not TRPM1, to the dendritic tips. Retina cross sections were double immunostained for RGS7 and GPR179 in G or RGS7/RGS11 and TRPM1 in H. Cell nuclei are labeled with DAPI. Bars: (E and G) 10 µm; (H) 5 µm. IP, immunoprecipitation; KO, knockout.

Techniques Used: In Vivo, Sequencing, Transfection, Immunoprecipitation, Cotransfection, Expressing, Western Blot, Immunolabeling, Mouse Assay, Labeling, Knock-Out

GPR158 is a novel binding partner of RGS7. (A) Summary of the mass spectrometric analysis of identified proteins. Positive identification criteria were set to 95% confidence. Only hits > 95% confidence threshold (yellow) with the number of unique peptides similar to RGS7 were considered (green). Red indicates those that did not meet the identification criteria. (B) Bioinformatics analysis of GPR158 organization. Key residues important for the G protein activation in class C GPCRs are marked. term, terminus. (C) Tissue specificity of GPR158 expression as indicated by Western blotting analysis. (D) RGS7 and GPR158 coimmunoprecipitate from native brain lysates when specific antibodies are used. (E) RGS7 and GPR158 coimmunoprecipitate from transfected HEK293T/17 cells. (F) Coimmunoprecipitation of GPR158 with RGS6 but not with RGS9 or RGS11 after expression in HEK293T/17 cells. (G) GPR158 does not coimmunoprecipitate with R7BP in the presence of the RGS7–Gβ5 complex in transfected HEK293T/17 cells. (H) GPR158 and R7BP compete for binding to RGS7. Transfection of increasing amounts of R7BP reduced coimmunoprecipitation of GPR158 with RGS7, and conversely, increasing concentrations of GPR158 reduced binding of RGS7 to R7BP. Error bars indicate SEM. IP, immunoprecipitation; WB, Western blot; wt, wild type.
Figure Legend Snippet: GPR158 is a novel binding partner of RGS7. (A) Summary of the mass spectrometric analysis of identified proteins. Positive identification criteria were set to 95% confidence. Only hits > 95% confidence threshold (yellow) with the number of unique peptides similar to RGS7 were considered (green). Red indicates those that did not meet the identification criteria. (B) Bioinformatics analysis of GPR158 organization. Key residues important for the G protein activation in class C GPCRs are marked. term, terminus. (C) Tissue specificity of GPR158 expression as indicated by Western blotting analysis. (D) RGS7 and GPR158 coimmunoprecipitate from native brain lysates when specific antibodies are used. (E) RGS7 and GPR158 coimmunoprecipitate from transfected HEK293T/17 cells. (F) Coimmunoprecipitation of GPR158 with RGS6 but not with RGS9 or RGS11 after expression in HEK293T/17 cells. (G) GPR158 does not coimmunoprecipitate with R7BP in the presence of the RGS7–Gβ5 complex in transfected HEK293T/17 cells. (H) GPR158 and R7BP compete for binding to RGS7. Transfection of increasing amounts of R7BP reduced coimmunoprecipitation of GPR158 with RGS7, and conversely, increasing concentrations of GPR158 reduced binding of RGS7 to R7BP. Error bars indicate SEM. IP, immunoprecipitation; WB, Western blot; wt, wild type.

Techniques Used: Binding Assay, Activation Assay, Expressing, Western Blot, Transfection, Immunoprecipitation

66) Product Images from "Absence of the Fragile X Mental Retardation Protein results in defects of RNA editing of neuronal mRNAs in mouse"

Article Title: Absence of the Fragile X Mental Retardation Protein results in defects of RNA editing of neuronal mRNAs in mouse

Journal: RNA Biology

doi: 10.1080/15476286.2017.1338232

ADAR2-FMRP interaction determined by co-immunoprecipitation experiments. (A) FMRP western blot on frontal cortex total cell lysate prior and after immunoprecipitation with ADAR2 and FMRP antibodies. Both WT and fmr1 KO murine FC were analyzed. (B) ADAR-FMRP interaction is RNA independent. FMRP western blot of frontal cortex total cell lysate from WT mice prior and after immunoprecipitation with ADAR2 and rabbit IgG antibodies treated or not with RNase A.
Figure Legend Snippet: ADAR2-FMRP interaction determined by co-immunoprecipitation experiments. (A) FMRP western blot on frontal cortex total cell lysate prior and after immunoprecipitation with ADAR2 and FMRP antibodies. Both WT and fmr1 KO murine FC were analyzed. (B) ADAR-FMRP interaction is RNA independent. FMRP western blot of frontal cortex total cell lysate from WT mice prior and after immunoprecipitation with ADAR2 and rabbit IgG antibodies treated or not with RNase A.

Techniques Used: Immunoprecipitation, Western Blot, Mouse Assay

67) Product Images from "The RNA helicase MOV10L1 binds piRNA precursors to initiate piRNA processing"

Article Title: The RNA helicase MOV10L1 binds piRNA precursors to initiate piRNA processing

Journal: Genes & Development

doi: 10.1101/gad.254631.114

G4 antigenicity of piRNA precursor transcripts increases in Mov10l1 mutant mice. ( A ) RNA immunoprecipitation of piRNA precursor transcripts using the BG4 antibody that specifically recognizes RNA G4s using testis lysates from wild-type (WT) and Mov10l1
Figure Legend Snippet: G4 antigenicity of piRNA precursor transcripts increases in Mov10l1 mutant mice. ( A ) RNA immunoprecipitation of piRNA precursor transcripts using the BG4 antibody that specifically recognizes RNA G4s using testis lysates from wild-type (WT) and Mov10l1

Techniques Used: Mutagenesis, Mouse Assay, Immunoprecipitation

68) Product Images from "Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity"

Article Title: Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.2548-14.2015

NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.
Figure Legend Snippet: NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.

Techniques Used: Western Blot, Immunoprecipitation, Molecular Weight, Recombinant, Transfection, Immunofluorescence, Confocal Microscopy, Isolation, Negative Control, Marker, Cell Culture

69) Product Images from "Anthrax Lethal Toxin Triggers the Formation of a Membrane-Associated Inflammasome Complex in Murine Macrophages ▿"

Article Title: Anthrax Lethal Toxin Triggers the Formation of a Membrane-Associated Inflammasome Complex in Murine Macrophages ▿

Journal: Infection and Immunity

doi: 10.1128/IAI.01032-08

Caspase-1 is able to pull down caspase-11 and α-enolase in LT-treated J774A.1 macrophages. (A) Pull-down assays were performed using lysates from untreated, LPS (2 μg/ml)-treated, and LT-treated J774A.1 macrophages. Lysates were immunoprecipitated using an anti-caspase-1 antibody, and following elution, samples were subjected to SDS-PAGE and immunoblotting using anti-caspase-11 and anti-α-enolase antibodies. Levels of expression of α-enolase and actin in J774A.1 lysates are shown as controls. IP, immunoprecipitation; WB, Western blotting. (B) LT-treated J774A.1 murine macrophages were subjected to immunostaining with anti-α-enolase (fluorescein isothiocyanate [FITC] conjugated, green) and anti-caspase-1 (Alexa Fluor 555 labeled, red) antibodies 90 min after LT exposure. Nuclei were stained with Hoechst (blue). (C) LT-treated J774A.1 cells were incubated with anti-MHC-I antibodies (FITC conjugated, green) for 20 min at 37°C to allow endocytosis of MHC-I. Subsequently, cells were immunostained using anti-caspase-1 antibodies (red). Colocalization of caspase-1 and α-enolase or MHC-1 is shown in yellow. As a negative control for the MHC-I immunostaining, cells were incubated with the secondary antibody alone (data not shown).
Figure Legend Snippet: Caspase-1 is able to pull down caspase-11 and α-enolase in LT-treated J774A.1 macrophages. (A) Pull-down assays were performed using lysates from untreated, LPS (2 μg/ml)-treated, and LT-treated J774A.1 macrophages. Lysates were immunoprecipitated using an anti-caspase-1 antibody, and following elution, samples were subjected to SDS-PAGE and immunoblotting using anti-caspase-11 and anti-α-enolase antibodies. Levels of expression of α-enolase and actin in J774A.1 lysates are shown as controls. IP, immunoprecipitation; WB, Western blotting. (B) LT-treated J774A.1 murine macrophages were subjected to immunostaining with anti-α-enolase (fluorescein isothiocyanate [FITC] conjugated, green) and anti-caspase-1 (Alexa Fluor 555 labeled, red) antibodies 90 min after LT exposure. Nuclei were stained with Hoechst (blue). (C) LT-treated J774A.1 cells were incubated with anti-MHC-I antibodies (FITC conjugated, green) for 20 min at 37°C to allow endocytosis of MHC-I. Subsequently, cells were immunostained using anti-caspase-1 antibodies (red). Colocalization of caspase-1 and α-enolase or MHC-1 is shown in yellow. As a negative control for the MHC-I immunostaining, cells were incubated with the secondary antibody alone (data not shown).

Techniques Used: Immunoprecipitation, SDS Page, Expressing, Western Blot, Immunostaining, Labeling, Staining, Incubation, Negative Control

Nalp1b interacts directly with caspase-1. Coimmunoprecipitation and pull-down assays were performed using lysates from 293T cells that had been transiently transfected with plasmids encoding murine caspase-1 (Casp-1) and V5-His-tagged Nalp1b, as indicated. Lysates were immunoprecipitated using an anti-caspase-1 antibody, and following elution, samples were subjected to SDS-PAGE and immunoblotting using anti-V5 antibodies. Lysates were pulled down with a nickel column, and the eluates were subjected to immunoblotting using anti-caspase-1 antibodies. A truncated version of Nalp1b is indicated with an asterisk. IP, immunoprecipitation; WB, Western blotting.
Figure Legend Snippet: Nalp1b interacts directly with caspase-1. Coimmunoprecipitation and pull-down assays were performed using lysates from 293T cells that had been transiently transfected with plasmids encoding murine caspase-1 (Casp-1) and V5-His-tagged Nalp1b, as indicated. Lysates were immunoprecipitated using an anti-caspase-1 antibody, and following elution, samples were subjected to SDS-PAGE and immunoblotting using anti-V5 antibodies. Lysates were pulled down with a nickel column, and the eluates were subjected to immunoblotting using anti-caspase-1 antibodies. A truncated version of Nalp1b is indicated with an asterisk. IP, immunoprecipitation; WB, Western blotting.

Techniques Used: Transfection, Immunoprecipitation, SDS Page, Nickel Column, Western Blot

The peak of caspase-1 activation is concurrent with membrane impairment in LT-treated murine macrophages. (A) Caspase-1 was immunoprecipitated from LT-treated murine J774A.1 macrophages. Triton X-100-soluble PNP fractions of J774A.1 macrophages were immunoprecipitated using anti-caspase-1 antibodies, and proteins were analyzed by SDS-PAGE and immunoblotting. (Bottom) Immunoblotting of whole-cell lysates from untreated and LT-treated macrophages was done using anti-IL-18 antibodies. IP, immunoprecipitation; WB, Western blotting. (B) Membrane integrity of murine macrophages in response to LT treatment, as determined by PI (red) exclusion. Nuclei were stained with the membrane-permeable Hoechst stain (blue). (C) PI uptake in J774A.1 macrophages following treatment with LT (250 and 500 ng/ml of LF and PA, respectively) for the indicated times. PI uptake was determined using a plate reader (Victor 3V; PerkinElmer). As a control, macrophages were treated with PA and LF only (250 and 500 ng/ml of LF and PA, respectively) for 120 min. Error bars indicate standard deviations. (D) Changes in the J774A.1 cytoskeleton 90 min after LT treatment. The changes in the cytoskeleton were determined by F-actin staining with phalloidin (green), and the nuclei were stained with Hoechst stain (blue). An LT-treated cell lacking its cytoskeleton is marked with a white arrowhead.
Figure Legend Snippet: The peak of caspase-1 activation is concurrent with membrane impairment in LT-treated murine macrophages. (A) Caspase-1 was immunoprecipitated from LT-treated murine J774A.1 macrophages. Triton X-100-soluble PNP fractions of J774A.1 macrophages were immunoprecipitated using anti-caspase-1 antibodies, and proteins were analyzed by SDS-PAGE and immunoblotting. (Bottom) Immunoblotting of whole-cell lysates from untreated and LT-treated macrophages was done using anti-IL-18 antibodies. IP, immunoprecipitation; WB, Western blotting. (B) Membrane integrity of murine macrophages in response to LT treatment, as determined by PI (red) exclusion. Nuclei were stained with the membrane-permeable Hoechst stain (blue). (C) PI uptake in J774A.1 macrophages following treatment with LT (250 and 500 ng/ml of LF and PA, respectively) for the indicated times. PI uptake was determined using a plate reader (Victor 3V; PerkinElmer). As a control, macrophages were treated with PA and LF only (250 and 500 ng/ml of LF and PA, respectively) for 120 min. Error bars indicate standard deviations. (D) Changes in the J774A.1 cytoskeleton 90 min after LT treatment. The changes in the cytoskeleton were determined by F-actin staining with phalloidin (green), and the nuclei were stained with Hoechst stain (blue). An LT-treated cell lacking its cytoskeleton is marked with a white arrowhead.

Techniques Used: Activation Assay, Immunoprecipitation, SDS Page, Western Blot, Staining

70) Product Images from "Measles virus entry inhibitors: A structural proposal for mechanism of action and the development of resistance"

Article Title: Measles virus entry inhibitors: A structural proposal for mechanism of action and the development of resistance

Journal: Biochemistry

doi: 10.1021/bi801513p

Co-immunoprecipitation experiments with AS-48 and two antibodies directed at epitopes in the Val94 microdomain. (A) Ab-359, directed against residues 88–104, and Ab-361, directed against residues 240–259 have opposing effects on the amount
Figure Legend Snippet: Co-immunoprecipitation experiments with AS-48 and two antibodies directed at epitopes in the Val94 microdomain. (A) Ab-359, directed against residues 88–104, and Ab-361, directed against residues 240–259 have opposing effects on the amount

Techniques Used: Immunoprecipitation

71) Product Images from "A CD317/tetherin-RICH2 complex plays a critical role in the organization of the subapical actin cytoskeleton in polarized epithelial cells"

Article Title: A CD317/tetherin-RICH2 complex plays a critical role in the organization of the subapical actin cytoskeleton in polarized epithelial cells

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200804154

CD317 interacts with RICH2. (A) Immunoblot probed with an anti-RICH2 polyclonal antibody showing the results of a pull-down assay between a GST-RICH2 fusion protein and a synthetic peptide corresponding to the entire cytosolic N terminus of CD317. Lysate = 10% of total E. coli lysate used in the pull-down; Biotin eluate = GST-RICH2 eluted from biotin-coated beads; CD317 eluate = GST-RICH2 eluted from CD317 peptide-coated beads. (B, top) Immunoblot (using an anti-RICH2 antibody) of immunoprecipitates (using the indicated antibodies) from lysate of COS cells expressing CD317-GFP and RICH2-RFP (R26.4C antibody detects ZO-1 and was used as a control). (bottom) Immunoblot (using an anti-RICH2 antibody) of immunoprecipitates (generated using an anti-CD317 antibody) of lysate from polarized control Caco-2 cells or polarized CD317 knockdown Caco-2 cells as indicated. (C) Surface plasmon resonance data from an experiment in which the indicated concentrations of GST-RICH2 were flowed over a streptavidin-coated surface (flow channel) to which biotinylated CD317 N-terminal peptide had been attached. GST-RICH2 at the indicated concentrations was added at 160 s and stopped being added at 320 s. Data represent the binding of GST-RICH2 minus binding to a biotin-blocked control flow channel linked in series to the test channel. (D) Cartoon of the domain organization of RICH2. The region of RICH2 indicated as binding to CD317 was identified because this was the region detected in the initial bacteriophage display screen. The C terminus of RICH2 is a PDZ domain–binding motif, ESTAL. (E) Immunoblot (using an anti-EBP50 antibody) of material immunoprecipitated from lysates of polarized control Caco-2 cells and polarized CD317 knockdown Caco-2 cells (CD317 siRNA) using an anti-CD317 antibody. The top band present in both lanes corresponds to the heavy chain of the anti-CD317 antibody used in the immunoprecipitation. (F) Immunoblot analysis of membrane and cytosol fractions from Caco-2 cells using antibodies to RICH2 and the integral membrane protein CD99 as indicated. (A, B, E, and F) Molecular mass is indicated in kilodaltons.
Figure Legend Snippet: CD317 interacts with RICH2. (A) Immunoblot probed with an anti-RICH2 polyclonal antibody showing the results of a pull-down assay between a GST-RICH2 fusion protein and a synthetic peptide corresponding to the entire cytosolic N terminus of CD317. Lysate = 10% of total E. coli lysate used in the pull-down; Biotin eluate = GST-RICH2 eluted from biotin-coated beads; CD317 eluate = GST-RICH2 eluted from CD317 peptide-coated beads. (B, top) Immunoblot (using an anti-RICH2 antibody) of immunoprecipitates (using the indicated antibodies) from lysate of COS cells expressing CD317-GFP and RICH2-RFP (R26.4C antibody detects ZO-1 and was used as a control). (bottom) Immunoblot (using an anti-RICH2 antibody) of immunoprecipitates (generated using an anti-CD317 antibody) of lysate from polarized control Caco-2 cells or polarized CD317 knockdown Caco-2 cells as indicated. (C) Surface plasmon resonance data from an experiment in which the indicated concentrations of GST-RICH2 were flowed over a streptavidin-coated surface (flow channel) to which biotinylated CD317 N-terminal peptide had been attached. GST-RICH2 at the indicated concentrations was added at 160 s and stopped being added at 320 s. Data represent the binding of GST-RICH2 minus binding to a biotin-blocked control flow channel linked in series to the test channel. (D) Cartoon of the domain organization of RICH2. The region of RICH2 indicated as binding to CD317 was identified because this was the region detected in the initial bacteriophage display screen. The C terminus of RICH2 is a PDZ domain–binding motif, ESTAL. (E) Immunoblot (using an anti-EBP50 antibody) of material immunoprecipitated from lysates of polarized control Caco-2 cells and polarized CD317 knockdown Caco-2 cells (CD317 siRNA) using an anti-CD317 antibody. The top band present in both lanes corresponds to the heavy chain of the anti-CD317 antibody used in the immunoprecipitation. (F) Immunoblot analysis of membrane and cytosol fractions from Caco-2 cells using antibodies to RICH2 and the integral membrane protein CD99 as indicated. (A, B, E, and F) Molecular mass is indicated in kilodaltons.

Techniques Used: Pull Down Assay, Expressing, Generated, SPR Assay, Flow Cytometry, Binding Assay, Immunoprecipitation

72) Product Images from "NP1 Regulates Neuronal Activity-Dependent Accumulation of BAX in Mitochondria and Mitochondrial Dynamics"

Article Title: NP1 Regulates Neuronal Activity-Dependent Accumulation of BAX in Mitochondria and Mitochondrial Dynamics

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.4604-11.2012

BAX immunoprecipitates NP1 and NP1 colocalizes with active BAX in mitochondria. A–C , Representative Western blots of total protein lysate (TL), the eluate of BAX immunoprecipitation (IP; Bax), and the eluate of IgG immunoprecipitation controls (IgG). A , Immunoprecipitation of BAX from total brain lysates. Rat brains were homogenized, and protein lysates prepared as described in Materials and Methods. B , Immunoprecipitation of BAX from CGN lysates. CGNs were treated with low K + for 2 h. C , Immunoprecipitation of BAX from HEK 293T cell lysates transfected with recombinant rat Bax and NP1 cDNAs. D , Confocal microscopy studies were performed in mature CGNs in culture after 4 h of K + deprivation. Orthogonal sectioning shows colocalization between NP1 and active BAX in mitochondria. Colocalization between NP1 and MtDsRed is seen as yellow, between NP1 and BAX as cyan, and between BAX and MtDsRed as magenta, and colocalization between all three proteins is shown in white. Scale bar, 5 μm.
Figure Legend Snippet: BAX immunoprecipitates NP1 and NP1 colocalizes with active BAX in mitochondria. A–C , Representative Western blots of total protein lysate (TL), the eluate of BAX immunoprecipitation (IP; Bax), and the eluate of IgG immunoprecipitation controls (IgG). A , Immunoprecipitation of BAX from total brain lysates. Rat brains were homogenized, and protein lysates prepared as described in Materials and Methods. B , Immunoprecipitation of BAX from CGN lysates. CGNs were treated with low K + for 2 h. C , Immunoprecipitation of BAX from HEK 293T cell lysates transfected with recombinant rat Bax and NP1 cDNAs. D , Confocal microscopy studies were performed in mature CGNs in culture after 4 h of K + deprivation. Orthogonal sectioning shows colocalization between NP1 and active BAX in mitochondria. Colocalization between NP1 and MtDsRed is seen as yellow, between NP1 and BAX as cyan, and between BAX and MtDsRed as magenta, and colocalization between all three proteins is shown in white. Scale bar, 5 μm.

Techniques Used: Western Blot, Immunoprecipitation, Transfection, Recombinant, Confocal Microscopy

73) Product Images from "Sequence-dependent cargo recognition by SNX-BARs mediates retromer-independent transport of CI-MPR"

Article Title: Sequence-dependent cargo recognition by SNX-BARs mediates retromer-independent transport of CI-MPR

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201703015

SNX1 is less efficiently recruited to CI-MPR tubules expressing a chimera harboring the WLM-AAA mutation. (A) Scheme of CI-MPR chimera constructs used. SP, signal peptide; TM, transmembrane domain. (B, top) The GFP–CI-MPR chimera WT and the GFP–CI-MPR chimera WLM-AAA mutant have comparable expression levels. HeLa cells were transfected with CI-MPR chimeras. 48 h after transfection, GFP levels were analyzed by Western blotting. (B, bottom) n = 3 independent experiments. (C) The GFP–CI-MPR chimera WLM-AAA mutant has a reduced ability to bind to the SNX1/2–SNX5/6 complex. GFP trap of GFP-tagged GFP–CI-MPR chimeras, each transiently transfected in HEK293T cells. Molecular masses are given in kilodaltons. IP, immunoprecipitation. (D, left) HeLa cells were transfected with WT or WLM-AAA mutant GFP–CI-MPR chimera constructs. Bars: (main images) 20 µm; (insets) 5 µm. (D, right) The percentages of cells with at least one GFP-positive tubule were blindly scored. n = 3 independent experiments; WT, 145 cells; WLM-AAA, 139 cells. (E) HeLa cells were transfected with WT or WLM-AAA mutant GFP–CI-MPR construct and immunostained for endogenous SNX1 and endogenous VPS35 after 48 h. Bars: (main images) 20 µm; (insets) 10 µm. (E, top right) Relative number of GFP-positive and SNX1-negative tubules per cell. n = 3 blindly scored independent experiments; WT, 40 cells; WLM-AAA, 38 cells. (E, bottom right) Relative number of GFP-positive and SNX1-positive tubules per cell. n = 3 blindly scored independent experiments; WT, 40 cells; WLM-AAA, 38 cells (means ± SEM; unpaired t test; *, P
Figure Legend Snippet: SNX1 is less efficiently recruited to CI-MPR tubules expressing a chimera harboring the WLM-AAA mutation. (A) Scheme of CI-MPR chimera constructs used. SP, signal peptide; TM, transmembrane domain. (B, top) The GFP–CI-MPR chimera WT and the GFP–CI-MPR chimera WLM-AAA mutant have comparable expression levels. HeLa cells were transfected with CI-MPR chimeras. 48 h after transfection, GFP levels were analyzed by Western blotting. (B, bottom) n = 3 independent experiments. (C) The GFP–CI-MPR chimera WLM-AAA mutant has a reduced ability to bind to the SNX1/2–SNX5/6 complex. GFP trap of GFP-tagged GFP–CI-MPR chimeras, each transiently transfected in HEK293T cells. Molecular masses are given in kilodaltons. IP, immunoprecipitation. (D, left) HeLa cells were transfected with WT or WLM-AAA mutant GFP–CI-MPR chimera constructs. Bars: (main images) 20 µm; (insets) 5 µm. (D, right) The percentages of cells with at least one GFP-positive tubule were blindly scored. n = 3 independent experiments; WT, 145 cells; WLM-AAA, 139 cells. (E) HeLa cells were transfected with WT or WLM-AAA mutant GFP–CI-MPR construct and immunostained for endogenous SNX1 and endogenous VPS35 after 48 h. Bars: (main images) 20 µm; (insets) 10 µm. (E, top right) Relative number of GFP-positive and SNX1-negative tubules per cell. n = 3 blindly scored independent experiments; WT, 40 cells; WLM-AAA, 38 cells. (E, bottom right) Relative number of GFP-positive and SNX1-positive tubules per cell. n = 3 blindly scored independent experiments; WT, 40 cells; WLM-AAA, 38 cells (means ± SEM; unpaired t test; *, P

Techniques Used: Expressing, Mutagenesis, Construct, Transfection, Western Blot, Immunoprecipitation

Interactome of the retromer-linked SNX-BAR complex. (A) Schematic representation of the SILAC methodology and the approach used to filter and merge SILAC datasets. (B) Venn diagram showing the interactors of SNX5, SNX6, and SNX32. (C) Venn diagram showing the interactors of SNX1 and SNX2 together with the shared interactors between SNX5, SNX6, and SNX32 (those within the red demarcated area in B). (D) STRING analysis of SNX-BAR interactors. Interactors were compiled and subjected to STRING analysis. Each connecting line represents an interaction indicated by experimental or database evidence. Color of the node indicates presence of the protein in a specific subset of the SNX-BAR interactome. (E) GFP trap of GFP-tagged retromer-linked SNX-BARs, retromer, and the retromer-independent SNX4 and SNX8, each transiently transfected in HEK293T cells. Molecular masses are given in kilodaltons. IP, immunoprecipitation.
Figure Legend Snippet: Interactome of the retromer-linked SNX-BAR complex. (A) Schematic representation of the SILAC methodology and the approach used to filter and merge SILAC datasets. (B) Venn diagram showing the interactors of SNX5, SNX6, and SNX32. (C) Venn diagram showing the interactors of SNX1 and SNX2 together with the shared interactors between SNX5, SNX6, and SNX32 (those within the red demarcated area in B). (D) STRING analysis of SNX-BAR interactors. Interactors were compiled and subjected to STRING analysis. Each connecting line represents an interaction indicated by experimental or database evidence. Color of the node indicates presence of the protein in a specific subset of the SNX-BAR interactome. (E) GFP trap of GFP-tagged retromer-linked SNX-BARs, retromer, and the retromer-independent SNX4 and SNX8, each transiently transfected in HEK293T cells. Molecular masses are given in kilodaltons. IP, immunoprecipitation.

Techniques Used: Transfection, Immunoprecipitation

74) Product Images from "Lipopolysaccharide regulation of intestinal tight junction permeability is mediated by TLR-4 signal transduction pathway activation of FAK and MyD88"

Article Title: Lipopolysaccharide regulation of intestinal tight junction permeability is mediated by TLR-4 signal transduction pathway activation of FAK and MyD88

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1402598

Co-immunoprecipitation of TLR-4, FAK and MyD88 using Dynabeads Protein G. After LPS treatment (5 d), Caco-2 monolayers were lysed, and immunoprecipitated with TLR-4 antibody, then phospho-FAK, FAK and MyD88 were detected by Western blot analysis. n=4. Densitometry analysis showed a significant increase in phospho-FAK compared to control and no change in total FAK and MyD88 expression. *, p
Figure Legend Snippet: Co-immunoprecipitation of TLR-4, FAK and MyD88 using Dynabeads Protein G. After LPS treatment (5 d), Caco-2 monolayers were lysed, and immunoprecipitated with TLR-4 antibody, then phospho-FAK, FAK and MyD88 were detected by Western blot analysis. n=4. Densitometry analysis showed a significant increase in phospho-FAK compared to control and no change in total FAK and MyD88 expression. *, p

Techniques Used: Immunoprecipitation, Western Blot, Expressing

75) Product Images from "Intracellular Distribution of Capsid-Associated pUL77 of Human Cytomegalovirus and Interactions with Packaging Proteins and pUL93"

Article Title: Intracellular Distribution of Capsid-Associated pUL77 of Human Cytomegalovirus and Interactions with Packaging Proteins and pUL93

Journal: Journal of Virology

doi: 10.1128/JVI.00351-16

Interaction of pUL77 with pUL93. (A) RPE-1 cells infected with HCMV AD169 were transfected at 48 h p.i. with pGEX_UL93 or pGEX5x-1. Cells were lysed at 24 h posttransfection, and immunoprecipitation (IP) was performed with antibodies to pUL77. Cell extracts and coimmunoprecipitated (Co-IP) proteins were subjected to SDS-PAGE prior to Western blot analyses with antibodies to pUL77 and GST. (B) HEK293T cells were cotransfected with pGEX_UL93 and pcDNA_UL77 or mutant variants of pcDNA_UL77. Immunoprecipitation was performed with antibodies to GST. Cell extracts and coimmunoprecipitated proteins were subjected to SDS-PAGE prior to Western blot analyses with antibodies to pUL77 and GST. Molecular mass markers (M) are indicated on the left of each panel.
Figure Legend Snippet: Interaction of pUL77 with pUL93. (A) RPE-1 cells infected with HCMV AD169 were transfected at 48 h p.i. with pGEX_UL93 or pGEX5x-1. Cells were lysed at 24 h posttransfection, and immunoprecipitation (IP) was performed with antibodies to pUL77. Cell extracts and coimmunoprecipitated (Co-IP) proteins were subjected to SDS-PAGE prior to Western blot analyses with antibodies to pUL77 and GST. (B) HEK293T cells were cotransfected with pGEX_UL93 and pcDNA_UL77 or mutant variants of pcDNA_UL77. Immunoprecipitation was performed with antibodies to GST. Cell extracts and coimmunoprecipitated proteins were subjected to SDS-PAGE prior to Western blot analyses with antibodies to pUL77 and GST. Molecular mass markers (M) are indicated on the left of each panel.

Techniques Used: Infection, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, SDS Page, Western Blot, Mutagenesis

Interaction of pUL77 with terminase subunits pUL56 and pUL89. (A) HFFs were infected with HCMV AD169 prior to coimmunoprecipitation (Co-IP) with an antibody specific to pUL77. Precipitated proteins were subjected to Western blot analyses with antibodies to pUL77, IE1, pUL56, and pUL89. (B) HEK293T cells were cotransfected with pEYFP_UL56 and pcDNA_UL77 or two mutant variants of pcDNA_UL77. Immunoprecipitation (IP) was performed with antibodies to the His tag. Cell extracts were subjected to Western blot analyses with antibodies to GFP. (C) HEK293T cells were cotransfected with pGEX_UL89 and pcDNA_UL77 or mutant variants of pcDNA_UL77. Immunoprecipitation was performed with antibodies to the His tag. Cell extracts were subjected to Western blot analyses with antibodies to GST and the His tag. Molecular mass markers (M) are indicated on the left.
Figure Legend Snippet: Interaction of pUL77 with terminase subunits pUL56 and pUL89. (A) HFFs were infected with HCMV AD169 prior to coimmunoprecipitation (Co-IP) with an antibody specific to pUL77. Precipitated proteins were subjected to Western blot analyses with antibodies to pUL77, IE1, pUL56, and pUL89. (B) HEK293T cells were cotransfected with pEYFP_UL56 and pcDNA_UL77 or two mutant variants of pcDNA_UL77. Immunoprecipitation (IP) was performed with antibodies to the His tag. Cell extracts were subjected to Western blot analyses with antibodies to GFP. (C) HEK293T cells were cotransfected with pGEX_UL89 and pcDNA_UL77 or mutant variants of pcDNA_UL77. Immunoprecipitation was performed with antibodies to the His tag. Cell extracts were subjected to Western blot analyses with antibodies to GST and the His tag. Molecular mass markers (M) are indicated on the left.

Techniques Used: Infection, Co-Immunoprecipitation Assay, Western Blot, Mutagenesis, Immunoprecipitation

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Article Snippet: Paragraph title: Extracts, immunoprecipitations, and Western blotting. ... Cell extracts were prepared in 0.5 ml of immunoprecipitation buffer (50 mM Tris [pH 8], 120 mM NaCl, 0.5% NP-40, 5 mM β-glycerophosphate, 0.1 mM Na3 VO4 , 10 mM NaF) supplemented with Roche protease inhibitors.

Article Title: Modular Structure of PACT: Distinct Domains for Binding and Activating PKR
Article Snippet: At 24 h after transfection, cells were lysed in immunoprecipitation buffer (20 mM Tris-HCl [pH 7.5], 1 mM DTT, 100 mM NaCl, 2 mM MgCl2 , complete protease inhibitors [Roche], 20% glycerol) on ice. .. The immunoprecipitates were analyzed by Western blotting with anti-PKR (Santa Cruz) and anti-FLAG polyclonal antibodies (Santa Cruz) ( ).

Article Title: Regulation of Sufu activity by p66β and Mycbp provides new insight into vertebrate Hedgehog signaling
Article Snippet: Cells were collected at 48 h post-transfection and lysed in immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl, 50mM Tris-Cl at pH 7.5, 1 mM EDTA, protease inhibitor cocktail [Roche], PhosSTOP [Roche]). .. Immunoprecipitates were analyzed by Western blotting using rabbit anti-Flag (1:2000; Sigma) and rabbit anti-Myc (1:2000; Sigma).

Article Title: Mechanism of suppressors of cytokine signaling 1 inhibition of epithelial-mesenchymal transition signaling through ROS regulation in colon cancer cells: suppression of Src leading to thioredoxin up-regulation
Article Snippet: Paragraph title: Western blot analysis and co-immunoprecipitation assay ... For immunoprecipitation, cell extracts were prepared in immunoprecipitation buffer (10 mM HEPES [pH 7.6], 15 mM KCl, 2 mM MgCl2, 0.1% Nonidet P-40, 1 mM PMSF) and complete protease inhibitor (Roche).

Transformation Assay:

Article Title: Trithorax Group Protein Oryza sativa Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] [OPEN]
Article Snippet: Each DNA combination of all vectors was transformed into the protoplasts isolated from rice mesophyll or Oc cells. .. Briefly, the protoplasts were resuspended in Immunoprecipitation buffer (50 m m Tris-HCl, pH 7.5, 1 m m EDTA, 150 m m sodium chloride, 1% [v/v] Triton X-100, 1 m m dithiothreitol, 2 m m NaF, 50 μ m MG132, and an adequate amount of Protease inhibitor cocktail [Roche].

Transfection:

Article Title: MyoD Stimulates RB Promoter Activity via the CREB/p300 Nuclear Transduction Pathway †
Article Snippet: For immunoprecipitation experiments, 3 × 105 C3H10T[1/2] cells were transfected with expression vectors for MyoD (pcDNA3-FLAG-MyoD) and/or CREB (RC/RSV-CREB341) at 2.5 μg each. .. Cell extracts were prepared in 0.5 ml of immunoprecipitation buffer (50 mM Tris [pH 8], 120 mM NaCl, 0.5% NP-40, 5 mM β-glycerophosphate, 0.1 mM Na3 VO4 , 10 mM NaF) supplemented with Roche protease inhibitors.

Article Title: Palmitoylation mediates membrane association of hepatitis E virus ORF3 protein and is required for infectious particle secretion
Article Snippet: To detect palmitoylation, U-2 OS cells were transfected with pCMVORF3, pCMVORF3C1-4 or pCMVORF3C5-8 plasmids and Hep293TT were electroporated with full-length HEV p6 or 83–2 RNAs prior to incubation with radiolabelled palmitate 24 h or 6 days post-transfection, respectively. .. After three washes with cold PBS, cells were lysed in immunoprecipitation buffer (50 mM Tris-HCl pH 7.4; 1 mM EDTA; 150 mM NaCl; 1% Triton X-100) supplemented with 1X cOmplete protease inhibitor cocktail (Roche, Basel, Switzerland).

Article Title: Modular Structure of PACT: Distinct Domains for Binding and Activating PKR
Article Snippet: .. At 24 h after transfection, cells were lysed in immunoprecipitation buffer (20 mM Tris-HCl [pH 7.5], 1 mM DTT, 100 mM NaCl, 2 mM MgCl2 , complete protease inhibitors [Roche], 20% glycerol) on ice. .. The cell extract was used to immunoprecipitate FLAG-PACT with anti-FLAG (M2) agarose as described above for the in vitro interaction assay.

Article Title: Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity
Article Snippet: HEK 293T cells were transfected using a standard calcium phosphate transfection protocol. .. For total rat brain extraction, the whole rat brain was homogenized in 10 volumes of immunoprecipitation buffer (50 m m Tris-HCl, pH 7.5, 100 m m NaCl, 2 m m CaCl2 , 1% Triton X-100) containing the mini-EDTA-free protease inhibitor cocktail (Roche).

Article Title: Regulation of Sufu activity by p66β and Mycbp provides new insight into vertebrate Hedgehog signaling
Article Snippet: Different combinations of Myc-Mycbp, Myc-p66β, Flag-Sufu, Flag-Gli1, and Flag-Gli2 were transfected into HEK293T cells by Lipofectamine 2000 (Life Technologies). .. Cells were collected at 48 h post-transfection and lysed in immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl, 50mM Tris-Cl at pH 7.5, 1 mM EDTA, protease inhibitor cocktail [Roche], PhosSTOP [Roche]).

Protease Inhibitor:

Article Title: Homodimerization of RBPMS2 through a new RRM-interaction motif is necessary to control smooth muscle plasticity
Article Snippet: .. For immunoprecipitation assays, 50 μg of total protein lysates were incubated in immunoprecipitation buffer (50-mM Tris pH8, 150-mM NaCl, 0.4% NP40, cOmplete, EDTA-free Protease Inhibitor Cocktail (Roche)) with rabbit anti-Myc antibodies (Ozyme) pre-adsorbed to protein A-Sepharose CL-4B (GE Healthcare) at 4°C for 1 h and washed extensively. .. For glutaraldehyde crosslinking, 4 μg of GFP-, Myc-RBPMS2 and Myc-RBPMS2-L410E-expressing DF1 total protein extracts were incubated at 4°C in 36 μl of 0,1% PBS-buffered glutaraldehyde solution (Sigma-Aldrich) during 10 or 30 s and reaction was stopped with 4 μl of Tris 1M pH8.

Article Title: Palmitoylation mediates membrane association of hepatitis E virus ORF3 protein and is required for infectious particle secretion
Article Snippet: .. After three washes with cold PBS, cells were lysed in immunoprecipitation buffer (50 mM Tris-HCl pH 7.4; 1 mM EDTA; 150 mM NaCl; 1% Triton X-100) supplemented with 1X cOmplete protease inhibitor cocktail (Roche, Basel, Switzerland). .. Immunoprecipitation was carried out by the incubation of each protein lysates overnight at 4°C with 50 μL of Dynabeads Protein G (Thermo Fischer Scientific) pre-adsorbed with 1 μl of rabbit anti-ORF3 pAb, gift from Suzanne Emerson (NIH, Bethesda, MD), or unrelated rabbit serum as control, following manufacturers’s recommendations.

Article Title: The Capsicum annuum class IV chitinase ChitIV interacts with receptor-like cytoplasmic protein kinase PIK1 to accelerate PIK1-triggered cell death and defence responses
Article Snippet: .. Immunoblotting For Co-IP, total proteins were extracted from leaves using immunoprecipitation buffer [50mM HEPES (pH 7.5), 50mM NaCl, 10mM EDTA, 0.2% Triton X-100, and protease inhibitor cocktail (Roche, Mannheim, Germany)]; insoluble debris was pelleted by centrifuging leaf extracts at 15 000 g for 30min at 4 °C. .. The soluble protein extracts were incubated with monoclonal anti-cMyc or anti-HA agarose conjugates (Sigma-Aldrich, St Louis, MO, USA) overnight.

Article Title: Sorting nexin-21 is a scaffold for the endosomal recruitment of huntingtin
Article Snippet: .. Cells were lysed in immunoprecipitation buffer [50 mM Tris-HCl (pH 7.4), 0.5% NP40, 1 mM PMSF, 200 µM Na3 VO4 and a Roche mini complete protease inhibitor tablet] and the GFP tags were precipitated with GFP-nanotrap beads (Chromotek) for 1 h at 4°C then combined prior to three washes in wash buffer (50 mM Tris-HCl, pH7.4, 0.2% NP40). .. Proteins were isolated in sample buffer, separated using NuPAGE (4-12%) pre cast gels (Invitrogen), visualised using All Blue protein stain (Invitrogen) and analysed by LC-MS-MS on an Orbitrap Velos (Thermo) spectrophotometer ( ; ; , ; ).

Article Title: Trithorax Group Protein Oryza sativa Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] [OPEN]
Article Snippet: .. Briefly, the protoplasts were resuspended in Immunoprecipitation buffer (50 m m Tris-HCl, pH 7.5, 1 m m EDTA, 150 m m sodium chloride, 1% [v/v] Triton X-100, 1 m m dithiothreitol, 2 m m NaF, 50 μ m MG132, and an adequate amount of Protease inhibitor cocktail [Roche]. .. After brief vortexing, the samples were centrifuged at 12,000 rpm for 10 min. We added 10 μ L each of protein A and G conjugated to agarose beads (Millipore) to the supernatant for 1 h of preclearing to prevent nonspecific binding.

Article Title: Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity
Article Snippet: .. For total rat brain extraction, the whole rat brain was homogenized in 10 volumes of immunoprecipitation buffer (50 m m Tris-HCl, pH 7.5, 100 m m NaCl, 2 m m CaCl2 , 1% Triton X-100) containing the mini-EDTA-free protease inhibitor cocktail (Roche). .. The immunoprecipitation of the supernatants, both 293T and total brain, was performed with Dynabeads (Invitrogen) following the manufacturer's instructions.

Article Title: CNTNAP2 stabilizes interneuron dendritic arbors through CASK
Article Snippet: .. Expression Time Course Cortices from CD1 WT mice aged P0, P14, P28, 4 months, and 6 months (sex not determined at P0, P14; males for P28, 4 months, 6 months) were homogenized in immunoprecipitation buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.5% Triton X-100 with Roche protease inhibitor cocktail) and solubilized for 1 hour at 4°C. ..

Article Title: Regulation of Sufu activity by p66β and Mycbp provides new insight into vertebrate Hedgehog signaling
Article Snippet: .. Cells were collected at 48 h post-transfection and lysed in immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl, 50mM Tris-Cl at pH 7.5, 1 mM EDTA, protease inhibitor cocktail [Roche], PhosSTOP [Roche]). .. The supernatant was removed and bound to 20 μL of anti-Flag M2 beads (Sigma) or anti-Myc beads (Santa Cruz Biotechnology) overnight at 4°C with constant nutation.

Article Title: Mechanism of suppressors of cytokine signaling 1 inhibition of epithelial-mesenchymal transition signaling through ROS regulation in colon cancer cells: suppression of Src leading to thioredoxin up-regulation
Article Snippet: .. For immunoprecipitation, cell extracts were prepared in immunoprecipitation buffer (10 mM HEPES [pH 7.6], 15 mM KCl, 2 mM MgCl2, 0.1% Nonidet P-40, 1 mM PMSF) and complete protease inhibitor (Roche). .. The extracts (600 μg proteins) were incubated with rabbit monoclonal anti-Src Ab(Abcam) or rabbit IgG for 12 h at 4°C.

Cell Culture:

Article Title: MyoD Stimulates RB Promoter Activity via the CREB/p300 Nuclear Transduction Pathway †
Article Snippet: Whole-cell extracts of C2.7, CH3/MyoD, or C3H10T1/2 cells, cultured in GM or DM, were prepared in lysis buffer (50 mM Tris [pH 7.5], 250 mM NaCl, 1% Nonidet P-40 [NP-40], 5 mM ATP, 5 mM MgCl2 , 5 mM EDTA, 5 mM β-glycerophosphate, 0.1 mM Na3 VO4 , 10 mM NaF) supplemented with a mixture of protease inhibitors (Roche). .. Cell extracts were prepared in 0.5 ml of immunoprecipitation buffer (50 mM Tris [pH 8], 120 mM NaCl, 0.5% NP-40, 5 mM β-glycerophosphate, 0.1 mM Na3 VO4 , 10 mM NaF) supplemented with Roche protease inhibitors.

Article Title: Sorting nexin-21 is a scaffold for the endosomal recruitment of huntingtin
Article Snippet: RPE1 cells virally expressing selected plasmids were seeded in six-well plates in SILAC labelling medium supplemented with dialysed FCS and cultured over a minimum of six passages to achieve full labelling with respective isotopes and a minimum of two confluent 20 cm dishes for generation of lysates. .. Cells were lysed in immunoprecipitation buffer [50 mM Tris-HCl (pH 7.4), 0.5% NP40, 1 mM PMSF, 200 µM Na3 VO4 and a Roche mini complete protease inhibitor tablet] and the GFP tags were precipitated with GFP-nanotrap beads (Chromotek) for 1 h at 4°C then combined prior to three washes in wash buffer (50 mM Tris-HCl, pH7.4, 0.2% NP40).

Article Title: CNTNAP2 stabilizes interneuron dendritic arbors through CASK
Article Snippet: Expression Time Course Cortices from CD1 WT mice aged P0, P14, P28, 4 months, and 6 months (sex not determined at P0, P14; males for P28, 4 months, 6 months) were homogenized in immunoprecipitation buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.5% Triton X-100 with Roche protease inhibitor cocktail) and solubilized for 1 hour at 4°C. .. For in vitro studies, the same procedure was performed on cultured WT rat neurons aged 7, 14, 21, and 28 DIV.

Binding Assay:

Article Title: Trithorax Group Protein Oryza sativa Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] [OPEN]
Article Snippet: Briefly, the protoplasts were resuspended in Immunoprecipitation buffer (50 m m Tris-HCl, pH 7.5, 1 m m EDTA, 150 m m sodium chloride, 1% [v/v] Triton X-100, 1 m m dithiothreitol, 2 m m NaF, 50 μ m MG132, and an adequate amount of Protease inhibitor cocktail [Roche]. .. After brief vortexing, the samples were centrifuged at 12,000 rpm for 10 min. We added 10 μ L each of protein A and G conjugated to agarose beads (Millipore) to the supernatant for 1 h of preclearing to prevent nonspecific binding.

Radioactivity:

Article Title: Palmitoylation mediates membrane association of hepatitis E virus ORF3 protein and is required for infectious particle secretion
Article Snippet: Paragraph title: Radiolabeling and immunoprecipitation ... After three washes with cold PBS, cells were lysed in immunoprecipitation buffer (50 mM Tris-HCl pH 7.4; 1 mM EDTA; 150 mM NaCl; 1% Triton X-100) supplemented with 1X cOmplete protease inhibitor cocktail (Roche, Basel, Switzerland).

Mutagenesis:

Article Title: Modular Structure of PACT: Distinct Domains for Binding and Activating PKR
Article Snippet: HT1080 cells were transfected in 100-mm culture dishes with 10 μg of total DNA (5 μg of CMV-PKR [K296R] and 5 μg of FLAG-PACT mutant DNA) using the Lipofectamine reagent (Gibco-BRL). .. At 24 h after transfection, cells were lysed in immunoprecipitation buffer (20 mM Tris-HCl [pH 7.5], 1 mM DTT, 100 mM NaCl, 2 mM MgCl2 , complete protease inhibitors [Roche], 20% glycerol) on ice.

Isolation:

Article Title: Sorting nexin-21 is a scaffold for the endosomal recruitment of huntingtin
Article Snippet: Cells were lysed in immunoprecipitation buffer [50 mM Tris-HCl (pH 7.4), 0.5% NP40, 1 mM PMSF, 200 µM Na3 VO4 and a Roche mini complete protease inhibitor tablet] and the GFP tags were precipitated with GFP-nanotrap beads (Chromotek) for 1 h at 4°C then combined prior to three washes in wash buffer (50 mM Tris-HCl, pH7.4, 0.2% NP40). .. Proteins were isolated in sample buffer, separated using NuPAGE (4-12%) pre cast gels (Invitrogen), visualised using All Blue protein stain (Invitrogen) and analysed by LC-MS-MS on an Orbitrap Velos (Thermo) spectrophotometer ( ; ; , ; ).

Article Title: Trithorax Group Protein Oryza sativa Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] [OPEN]
Article Snippet: Each DNA combination of all vectors was transformed into the protoplasts isolated from rice mesophyll or Oc cells. .. Briefly, the protoplasts were resuspended in Immunoprecipitation buffer (50 m m Tris-HCl, pH 7.5, 1 m m EDTA, 150 m m sodium chloride, 1% [v/v] Triton X-100, 1 m m dithiothreitol, 2 m m NaF, 50 μ m MG132, and an adequate amount of Protease inhibitor cocktail [Roche].

Article Title: Mechanism of suppressors of cytokine signaling 1 inhibition of epithelial-mesenchymal transition signaling through ROS regulation in colon cancer cells: suppression of Src leading to thioredoxin up-regulation
Article Snippet: Western blot analysis and co-immunoprecipitation assay Whole cell, cytosolic, or nuclear lysates were isolated as previously described [ ]. .. For immunoprecipitation, cell extracts were prepared in immunoprecipitation buffer (10 mM HEPES [pH 7.6], 15 mM KCl, 2 mM MgCl2, 0.1% Nonidet P-40, 1 mM PMSF) and complete protease inhibitor (Roche).

Labeling:

Article Title: Identification of a Novel Recycling Sequence in the C-tail of FPR2/ALX Receptor
Article Snippet: Biotin Protection Degradation Assay HEK293 cells stably expressing N-terminal FLAG-tagged FPR2/ALX, N333-stop, P342-stop, or T346-stop were grown to 100% confluency and labeled with 3 μg/ml disulfide-cleavable biotin (Pierce) for 30 min at 4 °C ( ). .. Cells were then placed in 5 ml of medium stimulated for 30, 90, or 180 min. All plates (except the 100%) were then washed in PBS, stripped (50 mm glutathione, 0.3 mm NaCl, 75 mm NaOH, 1% FBS) at 4 °C for 30 min (to remove remaining cell surface-biotinylated receptors), quenched (PBS containing 1 mm iodoacetamide, 0.1% BSA), and then lysed in immunoprecipitation buffer (containing 0.1% Triton X-100, 150 mm NaCl, 25 mm KCl, 10 mm Tris-HCl (pH 7.4), and protease inhibitors (Roche Applied Science)).

Mouse Assay:

Article Title: CNTNAP2 stabilizes interneuron dendritic arbors through CASK
Article Snippet: .. Expression Time Course Cortices from CD1 WT mice aged P0, P14, P28, 4 months, and 6 months (sex not determined at P0, P14; males for P28, 4 months, 6 months) were homogenized in immunoprecipitation buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.5% Triton X-100 with Roche protease inhibitor cocktail) and solubilized for 1 hour at 4°C. ..

Degradation Assay:

Article Title: Identification of a Novel Recycling Sequence in the C-tail of FPR2/ALX Receptor
Article Snippet: Paragraph title: Biotin Protection Degradation Assay ... Cells were then placed in 5 ml of medium stimulated for 30, 90, or 180 min. All plates (except the 100%) were then washed in PBS, stripped (50 mm glutathione, 0.3 mm NaCl, 75 mm NaOH, 1% FBS) at 4 °C for 30 min (to remove remaining cell surface-biotinylated receptors), quenched (PBS containing 1 mm iodoacetamide, 0.1% BSA), and then lysed in immunoprecipitation buffer (containing 0.1% Triton X-100, 150 mm NaCl, 25 mm KCl, 10 mm Tris-HCl (pH 7.4), and protease inhibitors (Roche Applied Science)).

Lysis:

Article Title: Homodimerization of RBPMS2 through a new RRM-interaction motif is necessary to control smooth muscle plasticity
Article Snippet: Immunoprecipitation and glutaraldehyde crosslinking DF-1 cells were lysed in lysis buffer (20-mM Tris pH8, 50-mM NaCl, 1% NP40, cOmplete ethylenediaminetetraacetic acid (EDTA)-free Protease Inhibitor Cocktail (Roche)). .. For immunoprecipitation assays, 50 μg of total protein lysates were incubated in immunoprecipitation buffer (50-mM Tris pH8, 150-mM NaCl, 0.4% NP40, cOmplete, EDTA-free Protease Inhibitor Cocktail (Roche)) with rabbit anti-Myc antibodies (Ozyme) pre-adsorbed to protein A-Sepharose CL-4B (GE Healthcare) at 4°C for 1 h and washed extensively.

Article Title: MyoD Stimulates RB Promoter Activity via the CREB/p300 Nuclear Transduction Pathway †
Article Snippet: Whole-cell extracts of C2.7, CH3/MyoD, or C3H10T1/2 cells, cultured in GM or DM, were prepared in lysis buffer (50 mM Tris [pH 7.5], 250 mM NaCl, 1% Nonidet P-40 [NP-40], 5 mM ATP, 5 mM MgCl2 , 5 mM EDTA, 5 mM β-glycerophosphate, 0.1 mM Na3 VO4 , 10 mM NaF) supplemented with a mixture of protease inhibitors (Roche). .. Cell extracts were prepared in 0.5 ml of immunoprecipitation buffer (50 mM Tris [pH 8], 120 mM NaCl, 0.5% NP-40, 5 mM β-glycerophosphate, 0.1 mM Na3 VO4 , 10 mM NaF) supplemented with Roche protease inhibitors.

SDS Page:

Article Title: Palmitoylation mediates membrane association of hepatitis E virus ORF3 protein and is required for infectious particle secretion
Article Snippet: After three washes with cold PBS, cells were lysed in immunoprecipitation buffer (50 mM Tris-HCl pH 7.4; 1 mM EDTA; 150 mM NaCl; 1% Triton X-100) supplemented with 1X cOmplete protease inhibitor cocktail (Roche, Basel, Switzerland). .. Samples were then incubated for 5 min at 90°C in Laemmli buffer and separated onto a 17% SDS-PAGE.

Article Title: Identification of a Novel Recycling Sequence in the C-tail of FPR2/ALX Receptor
Article Snippet: Cells were then placed in 5 ml of medium stimulated for 30, 90, or 180 min. All plates (except the 100%) were then washed in PBS, stripped (50 mm glutathione, 0.3 mm NaCl, 75 mm NaOH, 1% FBS) at 4 °C for 30 min (to remove remaining cell surface-biotinylated receptors), quenched (PBS containing 1 mm iodoacetamide, 0.1% BSA), and then lysed in immunoprecipitation buffer (containing 0.1% Triton X-100, 150 mm NaCl, 25 mm KCl, 10 mm Tris-HCl (pH 7.4), and protease inhibitors (Roche Applied Science)). .. Samples were resolved by SDS-PAGE and visualized with streptavidin overlay (VECTASTAIN ABC immunoperoxidase reagent, Vector Laboratories).

Article Title: Mechanism of suppressors of cytokine signaling 1 inhibition of epithelial-mesenchymal transition signaling through ROS regulation in colon cancer cells: suppression of Src leading to thioredoxin up-regulation
Article Snippet: The lysates (60 μg each) were separated on SDS-PAGE and transferred to polyvinylidene difluoride membranes. .. For immunoprecipitation, cell extracts were prepared in immunoprecipitation buffer (10 mM HEPES [pH 7.6], 15 mM KCl, 2 mM MgCl2, 0.1% Nonidet P-40, 1 mM PMSF) and complete protease inhibitor (Roche).

Plasmid Preparation:

Article Title: Identification of a Novel Recycling Sequence in the C-tail of FPR2/ALX Receptor
Article Snippet: Cells were then placed in 5 ml of medium stimulated for 30, 90, or 180 min. All plates (except the 100%) were then washed in PBS, stripped (50 mm glutathione, 0.3 mm NaCl, 75 mm NaOH, 1% FBS) at 4 °C for 30 min (to remove remaining cell surface-biotinylated receptors), quenched (PBS containing 1 mm iodoacetamide, 0.1% BSA), and then lysed in immunoprecipitation buffer (containing 0.1% Triton X-100, 150 mm NaCl, 25 mm KCl, 10 mm Tris-HCl (pH 7.4), and protease inhibitors (Roche Applied Science)). .. Samples were resolved by SDS-PAGE and visualized with streptavidin overlay (VECTASTAIN ABC immunoperoxidase reagent, Vector Laboratories).

Article Title: Trithorax Group Protein Oryza sativa Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] [OPEN]
Article Snippet: An Myc-tagged full-length Ehd3 vector was constructed using the Hpa I site of the pGA3697 vector that carried the maize Ubiquitin1 promoter and the 4x Myc coding region. .. Briefly, the protoplasts were resuspended in Immunoprecipitation buffer (50 m m Tris-HCl, pH 7.5, 1 m m EDTA, 150 m m sodium chloride, 1% [v/v] Triton X-100, 1 m m dithiothreitol, 2 m m NaF, 50 μ m MG132, and an adequate amount of Protease inhibitor cocktail [Roche].

Co-Immunoprecipitation Assay:

Article Title: Modular Structure of PACT: Distinct Domains for Binding and Activating PKR
Article Snippet: Paragraph title: Expression in mammalian cells and coimmunoprecipitation assay. ... At 24 h after transfection, cells were lysed in immunoprecipitation buffer (20 mM Tris-HCl [pH 7.5], 1 mM DTT, 100 mM NaCl, 2 mM MgCl2 , complete protease inhibitors [Roche], 20% glycerol) on ice.

Article Title: The Capsicum annuum class IV chitinase ChitIV interacts with receptor-like cytoplasmic protein kinase PIK1 to accelerate PIK1-triggered cell death and defence responses
Article Snippet: .. Immunoblotting For Co-IP, total proteins were extracted from leaves using immunoprecipitation buffer [50mM HEPES (pH 7.5), 50mM NaCl, 10mM EDTA, 0.2% Triton X-100, and protease inhibitor cocktail (Roche, Mannheim, Germany)]; insoluble debris was pelleted by centrifuging leaf extracts at 15 000 g for 30min at 4 °C. .. The soluble protein extracts were incubated with monoclonal anti-cMyc or anti-HA agarose conjugates (Sigma-Aldrich, St Louis, MO, USA) overnight.

Article Title: Mechanism of suppressors of cytokine signaling 1 inhibition of epithelial-mesenchymal transition signaling through ROS regulation in colon cancer cells: suppression of Src leading to thioredoxin up-regulation
Article Snippet: Paragraph title: Western blot analysis and co-immunoprecipitation assay ... For immunoprecipitation, cell extracts were prepared in immunoprecipitation buffer (10 mM HEPES [pH 7.6], 15 mM KCl, 2 mM MgCl2, 0.1% Nonidet P-40, 1 mM PMSF) and complete protease inhibitor (Roche).

Recombinant:

Article Title: Identification of a Novel Recycling Sequence in the C-tail of FPR2/ALX Receptor
Article Snippet: Cells were then placed in 5 ml of medium stimulated for 30, 90, or 180 min. All plates (except the 100%) were then washed in PBS, stripped (50 mm glutathione, 0.3 mm NaCl, 75 mm NaOH, 1% FBS) at 4 °C for 30 min (to remove remaining cell surface-biotinylated receptors), quenched (PBS containing 1 mm iodoacetamide, 0.1% BSA), and then lysed in immunoprecipitation buffer (containing 0.1% Triton X-100, 150 mm NaCl, 25 mm KCl, 10 mm Tris-HCl (pH 7.4), and protease inhibitors (Roche Applied Science)). .. Lysates were immunoprecipitated (anti-FLAG M2) overnight and incubated for 2 h with recombinant protein G-Sepharose (Life Technologies) and deglycosylated.

In Vitro:

Article Title: Modular Structure of PACT: Distinct Domains for Binding and Activating PKR
Article Snippet: At 24 h after transfection, cells were lysed in immunoprecipitation buffer (20 mM Tris-HCl [pH 7.5], 1 mM DTT, 100 mM NaCl, 2 mM MgCl2 , complete protease inhibitors [Roche], 20% glycerol) on ice. .. The cell extract was used to immunoprecipitate FLAG-PACT with anti-FLAG (M2) agarose as described above for the in vitro interaction assay.

Article Title: CNTNAP2 stabilizes interneuron dendritic arbors through CASK
Article Snippet: Expression Time Course Cortices from CD1 WT mice aged P0, P14, P28, 4 months, and 6 months (sex not determined at P0, P14; males for P28, 4 months, 6 months) were homogenized in immunoprecipitation buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.5% Triton X-100 with Roche protease inhibitor cocktail) and solubilized for 1 hour at 4°C. .. For in vitro studies, the same procedure was performed on cultured WT rat neurons aged 7, 14, 21, and 28 DIV.

Spectrophotometry:

Article Title: Sorting nexin-21 is a scaffold for the endosomal recruitment of huntingtin
Article Snippet: Cells were lysed in immunoprecipitation buffer [50 mM Tris-HCl (pH 7.4), 0.5% NP40, 1 mM PMSF, 200 µM Na3 VO4 and a Roche mini complete protease inhibitor tablet] and the GFP tags were precipitated with GFP-nanotrap beads (Chromotek) for 1 h at 4°C then combined prior to three washes in wash buffer (50 mM Tris-HCl, pH7.4, 0.2% NP40). .. Proteins were isolated in sample buffer, separated using NuPAGE (4-12%) pre cast gels (Invitrogen), visualised using All Blue protein stain (Invitrogen) and analysed by LC-MS-MS on an Orbitrap Velos (Thermo) spectrophotometer ( ; ; , ; ).

Produced:

Article Title: Trithorax Group Protein Oryza sativa Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] [OPEN]
Article Snippet: HA-tagged vectors were produced with the Hpa I and Kpn I sites of the pGA3698 vector, which contained the maize Ubiquitin1 promoter and the 3x HA coding region. .. Briefly, the protoplasts were resuspended in Immunoprecipitation buffer (50 m m Tris-HCl, pH 7.5, 1 m m EDTA, 150 m m sodium chloride, 1% [v/v] Triton X-100, 1 m m dithiothreitol, 2 m m NaF, 50 μ m MG132, and an adequate amount of Protease inhibitor cocktail [Roche].

Immunoprecipitation:

Article Title: Homodimerization of RBPMS2 through a new RRM-interaction motif is necessary to control smooth muscle plasticity
Article Snippet: .. For immunoprecipitation assays, 50 μg of total protein lysates were incubated in immunoprecipitation buffer (50-mM Tris pH8, 150-mM NaCl, 0.4% NP40, cOmplete, EDTA-free Protease Inhibitor Cocktail (Roche)) with rabbit anti-Myc antibodies (Ozyme) pre-adsorbed to protein A-Sepharose CL-4B (GE Healthcare) at 4°C for 1 h and washed extensively. .. For glutaraldehyde crosslinking, 4 μg of GFP-, Myc-RBPMS2 and Myc-RBPMS2-L410E-expressing DF1 total protein extracts were incubated at 4°C in 36 μl of 0,1% PBS-buffered glutaraldehyde solution (Sigma-Aldrich) during 10 or 30 s and reaction was stopped with 4 μl of Tris 1M pH8.

Article Title: MyoD Stimulates RB Promoter Activity via the CREB/p300 Nuclear Transduction Pathway †
Article Snippet: .. Cell extracts were prepared in 0.5 ml of immunoprecipitation buffer (50 mM Tris [pH 8], 120 mM NaCl, 0.5% NP-40, 5 mM β-glycerophosphate, 0.1 mM Na3 VO4 , 10 mM NaF) supplemented with Roche protease inhibitors. ..

Article Title: Palmitoylation mediates membrane association of hepatitis E virus ORF3 protein and is required for infectious particle secretion
Article Snippet: .. After three washes with cold PBS, cells were lysed in immunoprecipitation buffer (50 mM Tris-HCl pH 7.4; 1 mM EDTA; 150 mM NaCl; 1% Triton X-100) supplemented with 1X cOmplete protease inhibitor cocktail (Roche, Basel, Switzerland). .. Immunoprecipitation was carried out by the incubation of each protein lysates overnight at 4°C with 50 μL of Dynabeads Protein G (Thermo Fischer Scientific) pre-adsorbed with 1 μl of rabbit anti-ORF3 pAb, gift from Suzanne Emerson (NIH, Bethesda, MD), or unrelated rabbit serum as control, following manufacturers’s recommendations.

Article Title: Modular Structure of PACT: Distinct Domains for Binding and Activating PKR
Article Snippet: .. At 24 h after transfection, cells were lysed in immunoprecipitation buffer (20 mM Tris-HCl [pH 7.5], 1 mM DTT, 100 mM NaCl, 2 mM MgCl2 , complete protease inhibitors [Roche], 20% glycerol) on ice. .. The cell extract was used to immunoprecipitate FLAG-PACT with anti-FLAG (M2) agarose as described above for the in vitro interaction assay.

Article Title: Identification of a Novel Recycling Sequence in the C-tail of FPR2/ALX Receptor
Article Snippet: .. Cells were then placed in 5 ml of medium stimulated for 30, 90, or 180 min. All plates (except the 100%) were then washed in PBS, stripped (50 mm glutathione, 0.3 mm NaCl, 75 mm NaOH, 1% FBS) at 4 °C for 30 min (to remove remaining cell surface-biotinylated receptors), quenched (PBS containing 1 mm iodoacetamide, 0.1% BSA), and then lysed in immunoprecipitation buffer (containing 0.1% Triton X-100, 150 mm NaCl, 25 mm KCl, 10 mm Tris-HCl (pH 7.4), and protease inhibitors (Roche Applied Science)). .. Lysates were immunoprecipitated (anti-FLAG M2) overnight and incubated for 2 h with recombinant protein G-Sepharose (Life Technologies) and deglycosylated.

Article Title: The Capsicum annuum class IV chitinase ChitIV interacts with receptor-like cytoplasmic protein kinase PIK1 to accelerate PIK1-triggered cell death and defence responses
Article Snippet: .. Immunoblotting For Co-IP, total proteins were extracted from leaves using immunoprecipitation buffer [50mM HEPES (pH 7.5), 50mM NaCl, 10mM EDTA, 0.2% Triton X-100, and protease inhibitor cocktail (Roche, Mannheim, Germany)]; insoluble debris was pelleted by centrifuging leaf extracts at 15 000 g for 30min at 4 °C. .. The soluble protein extracts were incubated with monoclonal anti-cMyc or anti-HA agarose conjugates (Sigma-Aldrich, St Louis, MO, USA) overnight.

Article Title: Sorting nexin-21 is a scaffold for the endosomal recruitment of huntingtin
Article Snippet: .. Cells were lysed in immunoprecipitation buffer [50 mM Tris-HCl (pH 7.4), 0.5% NP40, 1 mM PMSF, 200 µM Na3 VO4 and a Roche mini complete protease inhibitor tablet] and the GFP tags were precipitated with GFP-nanotrap beads (Chromotek) for 1 h at 4°C then combined prior to three washes in wash buffer (50 mM Tris-HCl, pH7.4, 0.2% NP40). .. Proteins were isolated in sample buffer, separated using NuPAGE (4-12%) pre cast gels (Invitrogen), visualised using All Blue protein stain (Invitrogen) and analysed by LC-MS-MS on an Orbitrap Velos (Thermo) spectrophotometer ( ; ; , ; ).

Article Title: Trithorax Group Protein Oryza sativa Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] [OPEN]
Article Snippet: .. Briefly, the protoplasts were resuspended in Immunoprecipitation buffer (50 m m Tris-HCl, pH 7.5, 1 m m EDTA, 150 m m sodium chloride, 1% [v/v] Triton X-100, 1 m m dithiothreitol, 2 m m NaF, 50 μ m MG132, and an adequate amount of Protease inhibitor cocktail [Roche]. .. After brief vortexing, the samples were centrifuged at 12,000 rpm for 10 min. We added 10 μ L each of protein A and G conjugated to agarose beads (Millipore) to the supernatant for 1 h of preclearing to prevent nonspecific binding.

Article Title: Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity
Article Snippet: .. For total rat brain extraction, the whole rat brain was homogenized in 10 volumes of immunoprecipitation buffer (50 m m Tris-HCl, pH 7.5, 100 m m NaCl, 2 m m CaCl2 , 1% Triton X-100) containing the mini-EDTA-free protease inhibitor cocktail (Roche). .. The immunoprecipitation of the supernatants, both 293T and total brain, was performed with Dynabeads (Invitrogen) following the manufacturer's instructions.

Article Title: CNTNAP2 stabilizes interneuron dendritic arbors through CASK
Article Snippet: .. Expression Time Course Cortices from CD1 WT mice aged P0, P14, P28, 4 months, and 6 months (sex not determined at P0, P14; males for P28, 4 months, 6 months) were homogenized in immunoprecipitation buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.5% Triton X-100 with Roche protease inhibitor cocktail) and solubilized for 1 hour at 4°C. ..

Article Title: Regulation of Sufu activity by p66β and Mycbp provides new insight into vertebrate Hedgehog signaling
Article Snippet: .. Cells were collected at 48 h post-transfection and lysed in immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl, 50mM Tris-Cl at pH 7.5, 1 mM EDTA, protease inhibitor cocktail [Roche], PhosSTOP [Roche]). .. The supernatant was removed and bound to 20 μL of anti-Flag M2 beads (Sigma) or anti-Myc beads (Santa Cruz Biotechnology) overnight at 4°C with constant nutation.

Article Title: Mechanism of suppressors of cytokine signaling 1 inhibition of epithelial-mesenchymal transition signaling through ROS regulation in colon cancer cells: suppression of Src leading to thioredoxin up-regulation
Article Snippet: .. For immunoprecipitation, cell extracts were prepared in immunoprecipitation buffer (10 mM HEPES [pH 7.6], 15 mM KCl, 2 mM MgCl2, 0.1% Nonidet P-40, 1 mM PMSF) and complete protease inhibitor (Roche). .. The extracts (600 μg proteins) were incubated with rabbit monoclonal anti-Src Ab(Abcam) or rabbit IgG for 12 h at 4°C.

Staining:

Article Title: Sorting nexin-21 is a scaffold for the endosomal recruitment of huntingtin
Article Snippet: Cells were lysed in immunoprecipitation buffer [50 mM Tris-HCl (pH 7.4), 0.5% NP40, 1 mM PMSF, 200 µM Na3 VO4 and a Roche mini complete protease inhibitor tablet] and the GFP tags were precipitated with GFP-nanotrap beads (Chromotek) for 1 h at 4°C then combined prior to three washes in wash buffer (50 mM Tris-HCl, pH7.4, 0.2% NP40). .. Proteins were isolated in sample buffer, separated using NuPAGE (4-12%) pre cast gels (Invitrogen), visualised using All Blue protein stain (Invitrogen) and analysed by LC-MS-MS on an Orbitrap Velos (Thermo) spectrophotometer ( ; ; , ; ).

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    Roche immunoprecipitation buffer
    CNTNAP2 interacts with CASK at the plasma membrane in cortical GABAergic interneurons (a) Only yeast cells co-expressing CNTNAP2 bait and CASK prey constructs grow on high stringency yeast plates (QDO/X/A). (b–c) Cropped western blots of <t>co-immunoprecipitation</t> experiments with CASK and CNTNAP2 in mouse cortex. (d) Cropped western blots showing co-immunoprecipitation of various FLAG-CNTNAP2 truncation mutants ( Supplementary Figure 5a ; red lines) with untagged, full-length CASK in HEK293T cells. (e) Representative cropped western blots of membrane/cytosol fractions of HEK293T cells expressing pCS2-FLAG + CASK-mCherry (CASK), FLAG-CNTNAP2 + CASK-mCherry (CNTNAP2 + CASK), or FLAG-CNTNAP2 + CASKΔPDZ-mCherry (CNTNAP2 + CASKΔPDZ) and subsequent quantification of protein localization (CASK vs. CNTNAP2 + CASK vs. CNTNAP2 + CASKΔPDZ: 6 independent experiments; CASK alone vs. CASKΔPDZ alone: 3 independent experiments). Percentages were calculated by dividing the densitometry value of CASK/CNTNAP2 in either membrane or cytosol fraction by the summation of both. (f) Representative SIM image of endogenous CNTNAP2 and CASK co-localization (white) on a GFP-transfected interneuronal dendrite (scale bar = 5 μm). (g) Histogram showing distribution of CASK/CNTNAP2 co-localized puncta relative to the dendrite’s lateral edge from (f) (CASK colocalized with CNTNAP2: n = 79 puncta from 3 cultures; CNTNAP2 colocalized with CASK: n = 70 puncta from 3 cultures). (h) Representative confocal image showing PLA signal from endogenous CASK/CNTNAP2 staining, which occurs only when CASK and CNTNAP2 primary antibodies are both applied (scale bar = 1 μm). (i) Cropped immunoblots of subcellular fractionations from adult mouse forebrain probed with CNTNAP2, CASK, and β-tubulin. CNTNAP2 and CASK, but not β-tubulin, are found in the washed membrane fraction (S5; red box). (j) Cropped western blot of time course and (k) quantification of CASK expression in cultured cortical neurons (n = 3 independent experiments). Values are means ± SEM. * P≤0.05, ** P≤0.01, *** P≤0.001; one-way ANOVA with Bonferroni’s correction (k, top graph; e). Student’s t-test (middle and bottom graphs; e).
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    CNTNAP2 interacts with CASK at the plasma membrane in cortical GABAergic interneurons (a) Only yeast cells co-expressing CNTNAP2 bait and CASK prey constructs grow on high stringency yeast plates (QDO/X/A). (b–c) Cropped western blots of co-immunoprecipitation experiments with CASK and CNTNAP2 in mouse cortex. (d) Cropped western blots showing co-immunoprecipitation of various FLAG-CNTNAP2 truncation mutants ( Supplementary Figure 5a ; red lines) with untagged, full-length CASK in HEK293T cells. (e) Representative cropped western blots of membrane/cytosol fractions of HEK293T cells expressing pCS2-FLAG + CASK-mCherry (CASK), FLAG-CNTNAP2 + CASK-mCherry (CNTNAP2 + CASK), or FLAG-CNTNAP2 + CASKΔPDZ-mCherry (CNTNAP2 + CASKΔPDZ) and subsequent quantification of protein localization (CASK vs. CNTNAP2 + CASK vs. CNTNAP2 + CASKΔPDZ: 6 independent experiments; CASK alone vs. CASKΔPDZ alone: 3 independent experiments). Percentages were calculated by dividing the densitometry value of CASK/CNTNAP2 in either membrane or cytosol fraction by the summation of both. (f) Representative SIM image of endogenous CNTNAP2 and CASK co-localization (white) on a GFP-transfected interneuronal dendrite (scale bar = 5 μm). (g) Histogram showing distribution of CASK/CNTNAP2 co-localized puncta relative to the dendrite’s lateral edge from (f) (CASK colocalized with CNTNAP2: n = 79 puncta from 3 cultures; CNTNAP2 colocalized with CASK: n = 70 puncta from 3 cultures). (h) Representative confocal image showing PLA signal from endogenous CASK/CNTNAP2 staining, which occurs only when CASK and CNTNAP2 primary antibodies are both applied (scale bar = 1 μm). (i) Cropped immunoblots of subcellular fractionations from adult mouse forebrain probed with CNTNAP2, CASK, and β-tubulin. CNTNAP2 and CASK, but not β-tubulin, are found in the washed membrane fraction (S5; red box). (j) Cropped western blot of time course and (k) quantification of CASK expression in cultured cortical neurons (n = 3 independent experiments). Values are means ± SEM. * P≤0.05, ** P≤0.01, *** P≤0.001; one-way ANOVA with Bonferroni’s correction (k, top graph; e). Student’s t-test (middle and bottom graphs; e).

    Journal: Molecular psychiatry

    Article Title: CNTNAP2 stabilizes interneuron dendritic arbors through CASK

    doi: 10.1038/s41380-018-0027-3

    Figure Lengend Snippet: CNTNAP2 interacts with CASK at the plasma membrane in cortical GABAergic interneurons (a) Only yeast cells co-expressing CNTNAP2 bait and CASK prey constructs grow on high stringency yeast plates (QDO/X/A). (b–c) Cropped western blots of co-immunoprecipitation experiments with CASK and CNTNAP2 in mouse cortex. (d) Cropped western blots showing co-immunoprecipitation of various FLAG-CNTNAP2 truncation mutants ( Supplementary Figure 5a ; red lines) with untagged, full-length CASK in HEK293T cells. (e) Representative cropped western blots of membrane/cytosol fractions of HEK293T cells expressing pCS2-FLAG + CASK-mCherry (CASK), FLAG-CNTNAP2 + CASK-mCherry (CNTNAP2 + CASK), or FLAG-CNTNAP2 + CASKΔPDZ-mCherry (CNTNAP2 + CASKΔPDZ) and subsequent quantification of protein localization (CASK vs. CNTNAP2 + CASK vs. CNTNAP2 + CASKΔPDZ: 6 independent experiments; CASK alone vs. CASKΔPDZ alone: 3 independent experiments). Percentages were calculated by dividing the densitometry value of CASK/CNTNAP2 in either membrane or cytosol fraction by the summation of both. (f) Representative SIM image of endogenous CNTNAP2 and CASK co-localization (white) on a GFP-transfected interneuronal dendrite (scale bar = 5 μm). (g) Histogram showing distribution of CASK/CNTNAP2 co-localized puncta relative to the dendrite’s lateral edge from (f) (CASK colocalized with CNTNAP2: n = 79 puncta from 3 cultures; CNTNAP2 colocalized with CASK: n = 70 puncta from 3 cultures). (h) Representative confocal image showing PLA signal from endogenous CASK/CNTNAP2 staining, which occurs only when CASK and CNTNAP2 primary antibodies are both applied (scale bar = 1 μm). (i) Cropped immunoblots of subcellular fractionations from adult mouse forebrain probed with CNTNAP2, CASK, and β-tubulin. CNTNAP2 and CASK, but not β-tubulin, are found in the washed membrane fraction (S5; red box). (j) Cropped western blot of time course and (k) quantification of CASK expression in cultured cortical neurons (n = 3 independent experiments). Values are means ± SEM. * P≤0.05, ** P≤0.01, *** P≤0.001; one-way ANOVA with Bonferroni’s correction (k, top graph; e). Student’s t-test (middle and bottom graphs; e).

    Article Snippet: Expression Time Course Cortices from CD1 WT mice aged P0, P14, P28, 4 months, and 6 months (sex not determined at P0, P14; males for P28, 4 months, 6 months) were homogenized in immunoprecipitation buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.5% Triton X-100 with Roche protease inhibitor cocktail) and solubilized for 1 hour at 4°C.

    Techniques: Expressing, Construct, Western Blot, Immunoprecipitation, Transfection, Proximity Ligation Assay, Staining, Cell Culture

    Co-immunoprecipitation of Htt with SNX21 requires negatively charged residues in the SNX21 N-terminus, but does not require SNX21 to be endosomally localised. (A) Adaptation of SNX20 and SNX21 protein alignment previously generated by Clairfeuille and colleagues ( Clairfeuille et al., 2015 ) . Green boxed regions represent clusters of negatively charged amino acids not present in the SNX20 N-terminal extension. Red boxes denote conserved amino acids within SNX20 and SNX21 sequences and asterisks count every ten residues starting with the fist methionine of SNX20. (B) HEK293-T cells were transiently transfected to express GFP, GFP-tagged full-length SNX21 and two truncation mutants representing the two halves of the N-terminal region of SNX21. Precipitates from the GFP-nanotrap-isolated variants were analysed by western blotting and demonstrate the necessity for a full N-terminal extension in order to facilitate Htt binding. Data are representative of three biological replicates. (C) Site-directed mutagenesis was used to engineer a variety of charge swap mutants targeting the negatively charged clusters of amino acids, prior to probing for Htt binding as above. Two aspartic acid residues in the first N-terminal cluster are essential for precipitation of Htt with SNX21. (D) Both the point mutated GFP-SNX21 and truncation variants were expressed in HeLa cells prior to fixation and immunolabelling with anti-EEA1. Except for the N-terminal 1-129 construct, which lacks a PX domain, all mutants retained an endosomal localisation. Scale bars: 20 µm.

    Journal: Journal of Cell Science

    Article Title: Sorting nexin-21 is a scaffold for the endosomal recruitment of huntingtin

    doi: 10.1242/jcs.211672

    Figure Lengend Snippet: Co-immunoprecipitation of Htt with SNX21 requires negatively charged residues in the SNX21 N-terminus, but does not require SNX21 to be endosomally localised. (A) Adaptation of SNX20 and SNX21 protein alignment previously generated by Clairfeuille and colleagues ( Clairfeuille et al., 2015 ) . Green boxed regions represent clusters of negatively charged amino acids not present in the SNX20 N-terminal extension. Red boxes denote conserved amino acids within SNX20 and SNX21 sequences and asterisks count every ten residues starting with the fist methionine of SNX20. (B) HEK293-T cells were transiently transfected to express GFP, GFP-tagged full-length SNX21 and two truncation mutants representing the two halves of the N-terminal region of SNX21. Precipitates from the GFP-nanotrap-isolated variants were analysed by western blotting and demonstrate the necessity for a full N-terminal extension in order to facilitate Htt binding. Data are representative of three biological replicates. (C) Site-directed mutagenesis was used to engineer a variety of charge swap mutants targeting the negatively charged clusters of amino acids, prior to probing for Htt binding as above. Two aspartic acid residues in the first N-terminal cluster are essential for precipitation of Htt with SNX21. (D) Both the point mutated GFP-SNX21 and truncation variants were expressed in HeLa cells prior to fixation and immunolabelling with anti-EEA1. Except for the N-terminal 1-129 construct, which lacks a PX domain, all mutants retained an endosomal localisation. Scale bars: 20 µm.

    Article Snippet: Cells were lysed in immunoprecipitation buffer [50 mM Tris-HCl (pH 7.4), 0.5% NP40, 1 mM PMSF, 200 µM Na3 VO4 and a Roche mini complete protease inhibitor tablet] and the GFP tags were precipitated with GFP-nanotrap beads (Chromotek) for 1 h at 4°C then combined prior to three washes in wash buffer (50 mM Tris-HCl, pH7.4, 0.2% NP40).

    Techniques: Immunoprecipitation, Generated, Transfection, Isolation, Western Blot, Binding Assay, Mutagenesis, Construct

    Co-immunoprecipitation of septins with SNX21 requires a surface exposed leucine in the PXB domain. (A) HEK293-T cells were transiently transfected with constructs encoding GFP, GFP-SNX20, GFP-SNX21 and various SNX21 point mutants. After GFP-nanotrap immunoisolation, precipitates were analysed by SDS-PAGE and western blotting. GFP-SNX21 precipitates both septin 2 and septin 7, an interaction that occurs via the SNX21 PXB domain and appears to require the endosomal localisation of SNX21. Data are representative of three biological replicates. (B) Amino acid residues mutated in the current study mapped onto the published structure of the mouse SNX21 ( Clairfeuille et al., 2015 ). (C) Site-directed mutagenesis of the SNX21 PXB domain, targeting predicted surface exposed residues. Constructs encoding GFP-tag chimeras of the various SNX21 mutants were transiently expressed in HEK293-T cells prior to GFP-nanotrap, SDS-PAGE and western blotting. Mutation of an evolutionarily conserved leucine (L363A) and a neighbouring lysine (K364E) is sufficient to perturb association with both septin 2 and septin 7. Data are representative of three biological replicates. (D) GFP-SNX21 mutants were expressed in HeLa cells prior to fixation and immunolabelling with anti-EEA1. Each of the mutants analysed retained an endosomal localisation in accordance with the wild-type protein. Scale bars: 20 µm.

    Journal: Journal of Cell Science

    Article Title: Sorting nexin-21 is a scaffold for the endosomal recruitment of huntingtin

    doi: 10.1242/jcs.211672

    Figure Lengend Snippet: Co-immunoprecipitation of septins with SNX21 requires a surface exposed leucine in the PXB domain. (A) HEK293-T cells were transiently transfected with constructs encoding GFP, GFP-SNX20, GFP-SNX21 and various SNX21 point mutants. After GFP-nanotrap immunoisolation, precipitates were analysed by SDS-PAGE and western blotting. GFP-SNX21 precipitates both septin 2 and septin 7, an interaction that occurs via the SNX21 PXB domain and appears to require the endosomal localisation of SNX21. Data are representative of three biological replicates. (B) Amino acid residues mutated in the current study mapped onto the published structure of the mouse SNX21 ( Clairfeuille et al., 2015 ). (C) Site-directed mutagenesis of the SNX21 PXB domain, targeting predicted surface exposed residues. Constructs encoding GFP-tag chimeras of the various SNX21 mutants were transiently expressed in HEK293-T cells prior to GFP-nanotrap, SDS-PAGE and western blotting. Mutation of an evolutionarily conserved leucine (L363A) and a neighbouring lysine (K364E) is sufficient to perturb association with both septin 2 and septin 7. Data are representative of three biological replicates. (D) GFP-SNX21 mutants were expressed in HeLa cells prior to fixation and immunolabelling with anti-EEA1. Each of the mutants analysed retained an endosomal localisation in accordance with the wild-type protein. Scale bars: 20 µm.

    Article Snippet: Cells were lysed in immunoprecipitation buffer [50 mM Tris-HCl (pH 7.4), 0.5% NP40, 1 mM PMSF, 200 µM Na3 VO4 and a Roche mini complete protease inhibitor tablet] and the GFP tags were precipitated with GFP-nanotrap beads (Chromotek) for 1 h at 4°C then combined prior to three washes in wash buffer (50 mM Tris-HCl, pH7.4, 0.2% NP40).

    Techniques: Immunoprecipitation, Transfection, Construct, SDS Page, Western Blot, Mutagenesis

    AKIN10 interacts directly with EIN3. ( a ) Structure modeling of protein kinase domain of Snf1 in S . pombe and AKIN10 in A . thaliana . Individual protein structures were generated using SWISS-MODEL ( http://swissmodel.expasy.org ) and visualized in a superimposed image using PyMOL ( http://www.pymol.org ). ( b ) Binary protein-protein interaction of AKIN10 and EIN3 was analyzed using a yeast two-hybrid system. ( c ) Protein-protein interaction of AKIN10 and EIN3 was confirmed by co-immunoprecipitation using Arabidopsis protoplasts transfected with a combination of AKIN10 and EIN3 constructs. ( d ) Binary protein-protein interactions between AKIN10 with MYB2, MYC3, or MYC4 were analyzed using a yeast two-hybrid system. ( e ) AKIN10-dependent EIN3 phosphorylation in vitro was shown with GST-EIN3 fragments as substrate. Coomassie blue staining was used for protein substrate visualization. All experiments were repeated with consistent results.

    Journal: Scientific Reports

    Article Title: Regulatory Functions of Cellular Energy Sensor SNF1-Related Kinase1 for Leaf Senescence Delay through ETHYLENE- INSENSITIVE3 Repression

    doi: 10.1038/s41598-017-03506-1

    Figure Lengend Snippet: AKIN10 interacts directly with EIN3. ( a ) Structure modeling of protein kinase domain of Snf1 in S . pombe and AKIN10 in A . thaliana . Individual protein structures were generated using SWISS-MODEL ( http://swissmodel.expasy.org ) and visualized in a superimposed image using PyMOL ( http://www.pymol.org ). ( b ) Binary protein-protein interaction of AKIN10 and EIN3 was analyzed using a yeast two-hybrid system. ( c ) Protein-protein interaction of AKIN10 and EIN3 was confirmed by co-immunoprecipitation using Arabidopsis protoplasts transfected with a combination of AKIN10 and EIN3 constructs. ( d ) Binary protein-protein interactions between AKIN10 with MYB2, MYC3, or MYC4 were analyzed using a yeast two-hybrid system. ( e ) AKIN10-dependent EIN3 phosphorylation in vitro was shown with GST-EIN3 fragments as substrate. Coomassie blue staining was used for protein substrate visualization. All experiments were repeated with consistent results.

    Article Snippet: co-Immunoprecipitation and Protein immunoblotting Total protein was extracted from the transfected protoplasts using immunoprecipitation (IP) buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM DTT, 1% Triton-X100, and Complete™ protease inhibitors (Roche, Basel, Switzerland).

    Techniques: Generated, Immunoprecipitation, Transfection, Construct, In Vitro, Staining

    Hedgehog signaling leads to reduced nuclear Sufu protein levels and Sufu dissociation from Gli2 and Gli3 primarily in the nucleus. ( A ) Western blot analysis and quantification of Sufu protein levels in the nuclear and cytoplasmic fractions derived from MEFs treated with Shh-conditioned medium. Cytoplasmic tubulin and nuclear lamin A were used to assess the purity of cytoplasmic and nuclear fractions. Nuclear Sufu protein levels were reduced by 60% upon Hh pathway activation, while cytoplasmic Sufu levels were largely unaltered. ( B ) Western blot analysis and quantification of Sufu protein levels in the nuclear and cytoplasmic fractions in the presence of Shh-conditioned medium and proteasome inhibitor MG132. Nuclear Sufu protein levels were notably restored upon MG132 addition. ( C ) Western blot analysis and quantification of immunoprecipitated Sufu, Gli2, and Gli3 using lysates from the nuclear and cytoplasmic fractions derived from MEFs expressing Flag-tagged Sufu. The amount of coimmunoprecipitated Gli2 and Gli3 by Sufu was significantly reduced in the nuclear fraction but only marginally decreased in the cytoplasmic fraction at indicated time points after Hh stimulation. (In) Input; (IP) immunoprecipitation. (*) P

    Journal: Genes & Development

    Article Title: Regulation of Sufu activity by p66β and Mycbp provides new insight into vertebrate Hedgehog signaling

    doi: 10.1101/gad.249425.114

    Figure Lengend Snippet: Hedgehog signaling leads to reduced nuclear Sufu protein levels and Sufu dissociation from Gli2 and Gli3 primarily in the nucleus. ( A ) Western blot analysis and quantification of Sufu protein levels in the nuclear and cytoplasmic fractions derived from MEFs treated with Shh-conditioned medium. Cytoplasmic tubulin and nuclear lamin A were used to assess the purity of cytoplasmic and nuclear fractions. Nuclear Sufu protein levels were reduced by 60% upon Hh pathway activation, while cytoplasmic Sufu levels were largely unaltered. ( B ) Western blot analysis and quantification of Sufu protein levels in the nuclear and cytoplasmic fractions in the presence of Shh-conditioned medium and proteasome inhibitor MG132. Nuclear Sufu protein levels were notably restored upon MG132 addition. ( C ) Western blot analysis and quantification of immunoprecipitated Sufu, Gli2, and Gli3 using lysates from the nuclear and cytoplasmic fractions derived from MEFs expressing Flag-tagged Sufu. The amount of coimmunoprecipitated Gli2 and Gli3 by Sufu was significantly reduced in the nuclear fraction but only marginally decreased in the cytoplasmic fraction at indicated time points after Hh stimulation. (In) Input; (IP) immunoprecipitation. (*) P

    Article Snippet: Cells were collected at 48 h post-transfection and lysed in immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl, 50mM Tris-Cl at pH 7.5, 1 mM EDTA, protease inhibitor cocktail [Roche], PhosSTOP [Roche]).

    Techniques: Western Blot, Derivative Assay, Activation Assay, Immunoprecipitation, Expressing

    A proteomic approach to identify Sufu-interacting proteins uncovers p66β and Mycbp. ( A ) Coomassie Blue-stained gel of control and Sufu immunoprecipitates treated with mock- or Shh-conditioned medium. Distinct bands were detected and were candidates for new Sufu-interacting proteins. Numbers at the right indicate locations of protein size standards. Large-scale immunoprecipitation (IP) and mass spectrometry were performed to identify new Sufu-interacting proteins and Sufu phosphorylation sites. Mass spectrometric analysis was performed directly on immunoprecipitates or specific bands cut out from SDS-PAGE gels. Immunoprecipitation and mass spectrometric analysis were repeated multiple times to eliminate nonspecific Sufu-binding proteins. ( B ) Western blot analysis of Sufu immunoprecipitates probed with anti-Sufu, anti-Gli2, and Gli3 antibodies. Endogenous Gli2 and Gli3 were detected in Sufu immunoprecipitates (but not in the control), suggesting that a physiologically relevant protein complex was pulled down. ( C ). ( D ) Western blot analysis of proteins pulled down by Sufu from lysates expressing Sufu and the indicated proteins, which were epitope-tagged. Both p66β and Mycbp physically interacted with Sufu by coimmunoprecipitation. Fu and Prc1 served as negative controls. ( E , top panels) Western blot analysis of Sufu immunoprecipitates using lysates derived from MEFs expressing Flag-tagged Sufu. Endogenous p66β and HDAC1 were coimmunoprecipitated. In contrast, HDAC2 and RBBP7/4 could not be detected in Sufu immunoprecipitates. ( Bottom panels) Western blot analysis of endogenous Sufu immunoprecipitated by an anti-Sufu antibody. p66β was coimmunoprecipitated by Sufu in wild-type MEFs but not in Sufu -deficient MEFs. p66β/Sufu interaction was not altered by Hh stimulation (Supplemental Fig. S6). ( F–H ) Immunofluorescence studies to assess the subcellular distribution of p66β and Mycbp. MEFs were transfected or transduced with p66β- and Mycbp-expressing constructs. p66β and Mycbp localized to the nucleus (marked by DAPI) of Hh-responsive cells. Cytoplasmic expression of Mycbp was also detected. Acetylated (Ac)-tubulin marks the primary cilium. Interestingly, Mycbp immunoreactivity can also be detected at the base of the cilium (white arrow).

    Journal: Genes & Development

    Article Title: Regulation of Sufu activity by p66β and Mycbp provides new insight into vertebrate Hedgehog signaling

    doi: 10.1101/gad.249425.114

    Figure Lengend Snippet: A proteomic approach to identify Sufu-interacting proteins uncovers p66β and Mycbp. ( A ) Coomassie Blue-stained gel of control and Sufu immunoprecipitates treated with mock- or Shh-conditioned medium. Distinct bands were detected and were candidates for new Sufu-interacting proteins. Numbers at the right indicate locations of protein size standards. Large-scale immunoprecipitation (IP) and mass spectrometry were performed to identify new Sufu-interacting proteins and Sufu phosphorylation sites. Mass spectrometric analysis was performed directly on immunoprecipitates or specific bands cut out from SDS-PAGE gels. Immunoprecipitation and mass spectrometric analysis were repeated multiple times to eliminate nonspecific Sufu-binding proteins. ( B ) Western blot analysis of Sufu immunoprecipitates probed with anti-Sufu, anti-Gli2, and Gli3 antibodies. Endogenous Gli2 and Gli3 were detected in Sufu immunoprecipitates (but not in the control), suggesting that a physiologically relevant protein complex was pulled down. ( C ). ( D ) Western blot analysis of proteins pulled down by Sufu from lysates expressing Sufu and the indicated proteins, which were epitope-tagged. Both p66β and Mycbp physically interacted with Sufu by coimmunoprecipitation. Fu and Prc1 served as negative controls. ( E , top panels) Western blot analysis of Sufu immunoprecipitates using lysates derived from MEFs expressing Flag-tagged Sufu. Endogenous p66β and HDAC1 were coimmunoprecipitated. In contrast, HDAC2 and RBBP7/4 could not be detected in Sufu immunoprecipitates. ( Bottom panels) Western blot analysis of endogenous Sufu immunoprecipitated by an anti-Sufu antibody. p66β was coimmunoprecipitated by Sufu in wild-type MEFs but not in Sufu -deficient MEFs. p66β/Sufu interaction was not altered by Hh stimulation (Supplemental Fig. S6). ( F–H ) Immunofluorescence studies to assess the subcellular distribution of p66β and Mycbp. MEFs were transfected or transduced with p66β- and Mycbp-expressing constructs. p66β and Mycbp localized to the nucleus (marked by DAPI) of Hh-responsive cells. Cytoplasmic expression of Mycbp was also detected. Acetylated (Ac)-tubulin marks the primary cilium. Interestingly, Mycbp immunoreactivity can also be detected at the base of the cilium (white arrow).

    Article Snippet: Cells were collected at 48 h post-transfection and lysed in immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl, 50mM Tris-Cl at pH 7.5, 1 mM EDTA, protease inhibitor cocktail [Roche], PhosSTOP [Roche]).

    Techniques: Staining, Immunoprecipitation, Mass Spectrometry, SDS Page, Binding Assay, Western Blot, Expressing, Derivative Assay, Immunofluorescence, Transfection, Transduction, Construct