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 "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

4) 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

5) 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

6) 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

7) 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

8) 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

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 "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

11) Product Images from "Sequential combinations of chemotherapeutic agents with BH3 mimetics to treat rhabdomyosarcoma and avoid resistance"

Article Title: Sequential combinations of chemotherapeutic agents with BH3 mimetics to treat rhabdomyosarcoma and avoid resistance

Journal: bioRxiv

doi: 10.1101/2020.01.24.918532

Vincristine induces resistance in RMS cells through BID inhibition by MCL-1. (A) Western blot results of the unbound fraction after MCL-1 immunoprecipitation. High efficiency of MCL-1 immunoprecipitation compared to Rabbit IgG control antibody. (B) Western blot results showing MCL-1 levels in CW9019 cell lysates (incubated with vincristine or DMSO for 36 hours) before performing the immunoprecipitation. (C) Left panel: Western blot results of the co-immunoprecipitation between MCL-1 and BID. Right panel: Quantification of the optical density of each protein and represented as binding ratio between BID and MCL-1. Results showed a significant increase in BID and MCL-1 binding after vincristine treatment, which is restored to control levels after the addition of S63845. Values indicate mean values ± SEM from at least three independent experiments, * p
Figure Legend Snippet: Vincristine induces resistance in RMS cells through BID inhibition by MCL-1. (A) Western blot results of the unbound fraction after MCL-1 immunoprecipitation. High efficiency of MCL-1 immunoprecipitation compared to Rabbit IgG control antibody. (B) Western blot results showing MCL-1 levels in CW9019 cell lysates (incubated with vincristine or DMSO for 36 hours) before performing the immunoprecipitation. (C) Left panel: Western blot results of the co-immunoprecipitation between MCL-1 and BID. Right panel: Quantification of the optical density of each protein and represented as binding ratio between BID and MCL-1. Results showed a significant increase in BID and MCL-1 binding after vincristine treatment, which is restored to control levels after the addition of S63845. Values indicate mean values ± SEM from at least three independent experiments, * p

Techniques Used: Inhibition, Western Blot, Immunoprecipitation, Incubation, Binding Assay

12) 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

13) 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

14) 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

15) 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

16) 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

17) 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

18) 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

19) 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

20) 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

21) 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

22) 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

23) 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

24) 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

25) 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

26) 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

27) 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

28) 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

29) 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

30) 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

31) 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

32) 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

33) 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

34) 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

35) 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

36) 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

37) 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

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 "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

40) 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

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Co-Immunoprecipitation Assay:

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Protease Inhibitor:

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Expressing:

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Mouse Assay:

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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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    Image Search Results


    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

    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

    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

    Mode of inhibition of TNFR1 signaling by zafirlukast and triclabendazole (A) Effect of zafirlukast and triclabendazole (200 μM) on TNFR1-LTα interaction was determined by co-immunoprecipitation with anti-FLAG–conjugated agarose beads. (B) Oligomeric states of PLAD were determined by analytical size-exclusion chromatography. Dimer peak is indicated. Open triangle indicates non-specific protein. (C) Effect of zafirlukast and triclabendazole on PLAD-PLAD interactions was determined by Native-PAGE. Open circle indicates nonspecific band. (D) Docking scores for zafirlukast and triclabendazole on TNFR1 chain B (PDB: 1NCF) interfacial residues. Boltzmann-weighted scores were calculated by multiplying the probability of contacting a given residue by the mean Boltzmann-weighted predicted free energy for each pose contacting that residue.

    Journal: SLAS discovery : advancing life sciences R & D

    Article Title: An innovative high-throughput screening approach for discovery of small molecules that inhibit TNF Receptors

    doi: 10.1177/2472555217706478

    Figure Lengend Snippet: Mode of inhibition of TNFR1 signaling by zafirlukast and triclabendazole (A) Effect of zafirlukast and triclabendazole (200 μM) on TNFR1-LTα interaction was determined by co-immunoprecipitation with anti-FLAG–conjugated agarose beads. (B) Oligomeric states of PLAD were determined by analytical size-exclusion chromatography. Dimer peak is indicated. Open triangle indicates non-specific protein. (C) Effect of zafirlukast and triclabendazole on PLAD-PLAD interactions was determined by Native-PAGE. Open circle indicates nonspecific band. (D) Docking scores for zafirlukast and triclabendazole on TNFR1 chain B (PDB: 1NCF) interfacial residues. Boltzmann-weighted scores were calculated by multiplying the probability of contacting a given residue by the mean Boltzmann-weighted predicted free energy for each pose contacting that residue.

    Article Snippet: HEK293 and transiently transfected cells were washed three times with ice-cold PBS before lysis with immunoprecipitation (IP) buffer (20 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, pH 7.5 and 0.5% NP-40) containing complete protease inhibitors cocktail (Roche).

    Techniques: Inhibition, Immunoprecipitation, Size-exclusion Chromatography, Clear Native PAGE