immunoprecipitation cells  (Roche)


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    Structured Review

    Roche immunoprecipitation cells
    EGFR-MET interaction is modulated by EGFR mutations. (A) Western blot (WB) of total MET levels in H1975 derived cells. Tubulin levels are shown as loading control. Values beneath blots are relative levels of MET compared to the total levels in the H1975 L858R/T790M cell line from 2 independent experiments +/−SD (B) MET (7q31) (red signal) copy number analysis by FISH in the three H1975 cell lines using the Leica Kreatech C-MET (7q31)/SE7 FISH probe (KBI-10719). The green signal indicates the chromosome 7 centromere control probe. Scale bar 10 mm. Average copy number and ratio between MET and chromosome 7 centromere probe are also indicated (n = 30 cells). (C) Immunofluorescence of total EGFR (Alexa546 –red in the image) and MET (Cyanine 5 –green in the image) in H1975 derived cells. Hoescht dye was used to stain the nuclei of the cells. Merge panels are also shown. Bars, 20 μm. (D) <t>Co-immunoprecipitation</t> (IP) of EGFR in H1975 derived cell lines. The EGFR antibody was used to immunoprecipitate. EGFR and MET levels are shown in both bound and input fractions. The gels shown in the figure were run separately for the bound and input fractions, as indicated by the dotted line, under the same experimental conditions. (E) Fluorescence lifetime imaging was performed on cells plated to sub-confluence on cover-slips and time-resolved analysis in Tri2. Quantification of average FRET efficiency (*** p
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    Images

    1) Product Images from "MET-EGFR dimerization in lung adenocarcinoma is dependent on EGFR mtations and altered by MET kinase inhibition"

    Article Title: MET-EGFR dimerization in lung adenocarcinoma is dependent on EGFR mtations and altered by MET kinase inhibition

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0170798

    EGFR-MET interaction is modulated by EGFR mutations. (A) Western blot (WB) of total MET levels in H1975 derived cells. Tubulin levels are shown as loading control. Values beneath blots are relative levels of MET compared to the total levels in the H1975 L858R/T790M cell line from 2 independent experiments +/−SD (B) MET (7q31) (red signal) copy number analysis by FISH in the three H1975 cell lines using the Leica Kreatech C-MET (7q31)/SE7 FISH probe (KBI-10719). The green signal indicates the chromosome 7 centromere control probe. Scale bar 10 mm. Average copy number and ratio between MET and chromosome 7 centromere probe are also indicated (n = 30 cells). (C) Immunofluorescence of total EGFR (Alexa546 –red in the image) and MET (Cyanine 5 –green in the image) in H1975 derived cells. Hoescht dye was used to stain the nuclei of the cells. Merge panels are also shown. Bars, 20 μm. (D) Co-immunoprecipitation (IP) of EGFR in H1975 derived cell lines. The EGFR antibody was used to immunoprecipitate. EGFR and MET levels are shown in both bound and input fractions. The gels shown in the figure were run separately for the bound and input fractions, as indicated by the dotted line, under the same experimental conditions. (E) Fluorescence lifetime imaging was performed on cells plated to sub-confluence on cover-slips and time-resolved analysis in Tri2. Quantification of average FRET efficiency (*** p
    Figure Legend Snippet: EGFR-MET interaction is modulated by EGFR mutations. (A) Western blot (WB) of total MET levels in H1975 derived cells. Tubulin levels are shown as loading control. Values beneath blots are relative levels of MET compared to the total levels in the H1975 L858R/T790M cell line from 2 independent experiments +/−SD (B) MET (7q31) (red signal) copy number analysis by FISH in the three H1975 cell lines using the Leica Kreatech C-MET (7q31)/SE7 FISH probe (KBI-10719). The green signal indicates the chromosome 7 centromere control probe. Scale bar 10 mm. Average copy number and ratio between MET and chromosome 7 centromere probe are also indicated (n = 30 cells). (C) Immunofluorescence of total EGFR (Alexa546 –red in the image) and MET (Cyanine 5 –green in the image) in H1975 derived cells. Hoescht dye was used to stain the nuclei of the cells. Merge panels are also shown. Bars, 20 μm. (D) Co-immunoprecipitation (IP) of EGFR in H1975 derived cell lines. The EGFR antibody was used to immunoprecipitate. EGFR and MET levels are shown in both bound and input fractions. The gels shown in the figure were run separately for the bound and input fractions, as indicated by the dotted line, under the same experimental conditions. (E) Fluorescence lifetime imaging was performed on cells plated to sub-confluence on cover-slips and time-resolved analysis in Tri2. Quantification of average FRET efficiency (*** p

    Techniques Used: Western Blot, Derivative Assay, Fluorescence In Situ Hybridization, Immunofluorescence, Staining, Immunoprecipitation, Fluorescence, Imaging

    2) Product Images from "A Dual Role of Caspase-8 in Triggering and Sensing Proliferation-Associated DNA Damage, a Key Determinant of Liver Cancer Development"

    Article Title: A Dual Role of Caspase-8 in Triggering and Sensing Proliferation-Associated DNA Damage, a Key Determinant of Liver Cancer Development

    Journal: Cancer Cell

    doi: 10.1016/j.ccell.2017.08.010

    Caspase-8, RIPK1, FADD, and c-FLIP Are Crucial for Phosphorylation of H2AX in Hepatocytes upon Doxorubicin Treatment (A) IF for γH2AX in untreated wild-type mice and wild-type, Casp8 Δhep , and QVD-OPH-treated wild-type mice following doxorubicin treatment. Arrow heads illustrate γH2AX + foci in nuclei. Scale bar, 10 μm. (B) PFGE on livers of doxorubicin-treated mice. (C) γH2AX staining of doxorubicin-treated wild-type and caspase-8 D387-mutant mice. Scale bar, 50 μm. (D) γH2AX IF staining 12 hr post-doxorubicin-induced DNA damage in hepatocytes of Casp8 −/− /RIPK3 −/− mice (n = 5), RIPK3 −/− mice (n = 4), RIPK1 KD mice (n = 9), RIPK1 −/− RIPK3 −/− FADD −/− (labeled as R1 −/− R3 −/− FADD −/− , n = 2), RIPK1 +/− RIPK3 −/− FADD −/− (labeled as R1 +/− R3 −/− FADD −/− , n = 2), c-FLIP Δhep (n = 6), and TNFR1/2 −/− mice (n = 6). Arrowheads illustrate γH2AX + foci in nuclei. Scale bar, 10 μm. (E) Quantification of IF stainings (A and D). (F) Immunoprecipitation with anti-caspase-8 antibody (upper panel) and immunoblotting of lysates (lower panel), 0–24 hr after doxorubicin (5 μM) treatment. Red box: RIPK1, FADD, and caspase-8 interaction at 1 hr; blue boxes: low-level activation of apoptosis starting at 4 hr post-treatment. (The signal visible in the t = 0 column, cl.PARP lane, does not originate from cl.PARP, but from a lower unspecific band.) Control cells treated for 1 hr with CD95L/FasL (B, beads; L, lysates). (G) Immunoblotting of lysates, 0–24 hr after doxorubicin (5 μM) treatment looking at levels of total and cl.PARP, blue boxes (F and G): low-level activation of apoptosis starting at 4 hr post-treatment. (H) Levels of LUBAC (HOIP, HOIL-1, and SHARPIN), cIAP1, cIAP2, and XIAP in U2OS cells at 15 min (red box) post-doxorubicin stimulation (5 μM). (I) Subcellular fractionation of U2OS cells. (J) RIPK1 and γH2AX IF staining in U2OS cells after doxorubicin treatment. The arrowhead indicates colocalizing signals. Scale bar, 10 μm. Statistical significance was calculated using ANOVA with Bonferroni correction (E). ∗∗∗ p
    Figure Legend Snippet: Caspase-8, RIPK1, FADD, and c-FLIP Are Crucial for Phosphorylation of H2AX in Hepatocytes upon Doxorubicin Treatment (A) IF for γH2AX in untreated wild-type mice and wild-type, Casp8 Δhep , and QVD-OPH-treated wild-type mice following doxorubicin treatment. Arrow heads illustrate γH2AX + foci in nuclei. Scale bar, 10 μm. (B) PFGE on livers of doxorubicin-treated mice. (C) γH2AX staining of doxorubicin-treated wild-type and caspase-8 D387-mutant mice. Scale bar, 50 μm. (D) γH2AX IF staining 12 hr post-doxorubicin-induced DNA damage in hepatocytes of Casp8 −/− /RIPK3 −/− mice (n = 5), RIPK3 −/− mice (n = 4), RIPK1 KD mice (n = 9), RIPK1 −/− RIPK3 −/− FADD −/− (labeled as R1 −/− R3 −/− FADD −/− , n = 2), RIPK1 +/− RIPK3 −/− FADD −/− (labeled as R1 +/− R3 −/− FADD −/− , n = 2), c-FLIP Δhep (n = 6), and TNFR1/2 −/− mice (n = 6). Arrowheads illustrate γH2AX + foci in nuclei. Scale bar, 10 μm. (E) Quantification of IF stainings (A and D). (F) Immunoprecipitation with anti-caspase-8 antibody (upper panel) and immunoblotting of lysates (lower panel), 0–24 hr after doxorubicin (5 μM) treatment. Red box: RIPK1, FADD, and caspase-8 interaction at 1 hr; blue boxes: low-level activation of apoptosis starting at 4 hr post-treatment. (The signal visible in the t = 0 column, cl.PARP lane, does not originate from cl.PARP, but from a lower unspecific band.) Control cells treated for 1 hr with CD95L/FasL (B, beads; L, lysates). (G) Immunoblotting of lysates, 0–24 hr after doxorubicin (5 μM) treatment looking at levels of total and cl.PARP, blue boxes (F and G): low-level activation of apoptosis starting at 4 hr post-treatment. (H) Levels of LUBAC (HOIP, HOIL-1, and SHARPIN), cIAP1, cIAP2, and XIAP in U2OS cells at 15 min (red box) post-doxorubicin stimulation (5 μM). (I) Subcellular fractionation of U2OS cells. (J) RIPK1 and γH2AX IF staining in U2OS cells after doxorubicin treatment. The arrowhead indicates colocalizing signals. Scale bar, 10 μm. Statistical significance was calculated using ANOVA with Bonferroni correction (E). ∗∗∗ p

    Techniques Used: Mouse Assay, Staining, Mutagenesis, Labeling, Immunoprecipitation, Activation Assay, Fractionation

    3) Product Images from "p125A exists as part of the mammalian Sec13/Sec31 COPII subcomplex to facilitate ER-Golgi transport"

    Article Title: p125A exists as part of the mammalian Sec13/Sec31 COPII subcomplex to facilitate ER-Golgi transport

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201003005

    p125A exists as a ternary complex with the Sec13/Sec31A heterotetramer in the cytosol. (A) Characterization of p125A antibodies. Residues 500–758 of p125 were expressed as fusion protein with GST (GST-p125A) and used to raise rabbit antibodies. Affinity-purified antibodies were used for immunoblot analysis. 30 µg of HeLa cell (lanes 1, 3, and 5) and HEK293 cell lysate (lanes 2, 4, and 6) were loaded per lane. The lysates were resolved on SDS-PAGE, transferred to PVDF membrane, and then subjected to immunoblot analysis using anti-p125A antibodies. For the second and third panels, anti-p125A antibodies were preincubated with 500 µg of recombinant GST-Bet3 and GST-p125A, respectively. (B) p125A fractionated with Sec31A and Sec13 in gel filtration. HeLa cell cytosol was subjected to gel filtration in Superose 6 at a flow rate of 0.3 ml/min. Fractions (0.6 ml each) were collected and then TCA precipitated. The proteins were subjected to SDS-PAGE and transferred to PVDF and immunoblotted using antibodies against Sec31A, p125A, Sec23A, and Sec13 as indicated. The arrows indicate the fractions in which the molecular weight markers peaked. (C) Sec31A is co-immunodepleted with p125A. Cytosol was prepared from HEK293 cells transfected to express HA-p125A. The cytosol was subjected to three consecutive cycles of immunodepletion using anti-HA or control anti-GFP antibody-conjugated beads. One tenth of the cytosol was collected and TCA precipitated after each round of immunodepletion. The proteins (along with the cytosol before immunodepletion) were subjected to SDS-PAGE and transferred to PVDF and immunoblotted using antibodies against HA, Sec31A, p125A, Sec23A, and Rab8. Sec31A and total p125A were efficiently and proportionally depleted along with HA-p125A. (D) p125A exists in a multimeric form. myc-p125A and HA-p125A were exogenously expressed in transfected HEK293 cells either singly or in combination as indicated. Cell lysates were subjected to immunoprecipitation with anti-myc (lanes 4–6), anti-HA (lanes 7–9), and control mouse IgG (lanes 10–12). The immunoprecipitates, along with the starting materials, were resolved on SDS-PAGE and analyzed by immunoblot with antibodies against myc, HA, Sec31A, and Sec23A. Molecular size markers are in kD.
    Figure Legend Snippet: p125A exists as a ternary complex with the Sec13/Sec31A heterotetramer in the cytosol. (A) Characterization of p125A antibodies. Residues 500–758 of p125 were expressed as fusion protein with GST (GST-p125A) and used to raise rabbit antibodies. Affinity-purified antibodies were used for immunoblot analysis. 30 µg of HeLa cell (lanes 1, 3, and 5) and HEK293 cell lysate (lanes 2, 4, and 6) were loaded per lane. The lysates were resolved on SDS-PAGE, transferred to PVDF membrane, and then subjected to immunoblot analysis using anti-p125A antibodies. For the second and third panels, anti-p125A antibodies were preincubated with 500 µg of recombinant GST-Bet3 and GST-p125A, respectively. (B) p125A fractionated with Sec31A and Sec13 in gel filtration. HeLa cell cytosol was subjected to gel filtration in Superose 6 at a flow rate of 0.3 ml/min. Fractions (0.6 ml each) were collected and then TCA precipitated. The proteins were subjected to SDS-PAGE and transferred to PVDF and immunoblotted using antibodies against Sec31A, p125A, Sec23A, and Sec13 as indicated. The arrows indicate the fractions in which the molecular weight markers peaked. (C) Sec31A is co-immunodepleted with p125A. Cytosol was prepared from HEK293 cells transfected to express HA-p125A. The cytosol was subjected to three consecutive cycles of immunodepletion using anti-HA or control anti-GFP antibody-conjugated beads. One tenth of the cytosol was collected and TCA precipitated after each round of immunodepletion. The proteins (along with the cytosol before immunodepletion) were subjected to SDS-PAGE and transferred to PVDF and immunoblotted using antibodies against HA, Sec31A, p125A, Sec23A, and Rab8. Sec31A and total p125A were efficiently and proportionally depleted along with HA-p125A. (D) p125A exists in a multimeric form. myc-p125A and HA-p125A were exogenously expressed in transfected HEK293 cells either singly or in combination as indicated. Cell lysates were subjected to immunoprecipitation with anti-myc (lanes 4–6), anti-HA (lanes 7–9), and control mouse IgG (lanes 10–12). The immunoprecipitates, along with the starting materials, were resolved on SDS-PAGE and analyzed by immunoblot with antibodies against myc, HA, Sec31A, and Sec23A. Molecular size markers are in kD.

    Techniques Used: Affinity Purification, SDS Page, Recombinant, Filtration, Flow Cytometry, Molecular Weight, Transfection, Immunoprecipitation

    p125A binds to Sec31A. (A) GST-Sec31A and GST immobilized onto glutathione beads were incubated with rat liver cytosol and proteins retained on the beads were eluted and resolved on a 7% SDS-PAGE gel. Distinct bands were cut out and subjected to protein identification by mass spectrometry. Arrow indicates mammalian p125A with the identified peptide sequences shown. (B) myc-p125A and GFP-Sec31A were exogenously expressed in HEK293 cells either singly or in combination as indicated. Cell lysates were prepared and subjected to immunoprecipitation by either anti-myc (9E10) antibodies or control mouse IgG. The samples (together with 5% starting materials as controls) were resolved by SDS-PAGE and transferred to PVDF and immunoblotted using antibodies against GFP and myc. Molecular size markers are in kD.
    Figure Legend Snippet: p125A binds to Sec31A. (A) GST-Sec31A and GST immobilized onto glutathione beads were incubated with rat liver cytosol and proteins retained on the beads were eluted and resolved on a 7% SDS-PAGE gel. Distinct bands were cut out and subjected to protein identification by mass spectrometry. Arrow indicates mammalian p125A with the identified peptide sequences shown. (B) myc-p125A and GFP-Sec31A were exogenously expressed in HEK293 cells either singly or in combination as indicated. Cell lysates were prepared and subjected to immunoprecipitation by either anti-myc (9E10) antibodies or control mouse IgG. The samples (together with 5% starting materials as controls) were resolved by SDS-PAGE and transferred to PVDF and immunoblotted using antibodies against GFP and myc. Molecular size markers are in kD.

    Techniques Used: Incubation, SDS Page, Mass Spectrometry, Immunoprecipitation

    4) Product Images from "An interdomain helix in IRE1α mediates the conformational change required for the sensor's activation"

    Article Title: An interdomain helix in IRE1α mediates the conformational change required for the sensor's activation

    Journal: The Journal of Biological Chemistry

    doi: 10.1016/j.jbc.2021.100781

    The L827P mutant inhibits WT IRE1α in HAP1 and Kms11 cells . A , IRE1GFP L827P inhibits XBP1 splicing in response to Tm in leukemic HAP1 cells. Parental HAP1 cells expressing IRE1GFP L827P in addition to the endogenous WT IRE1α were induced with the indicated concentrations of dox (μg/ml). The cells were then treated with 4 μg/ml Tm for 4 h, and XBP1 splicing was assessed by RT-PCR. B , L827P IRE1GFP inhibits ER stress-induced XBP1 splicing in multiple myeloma Kms11 cells. Kms11 cells expressing WT or L827P IRE1GFP were treated with 0.5 μM Tg for 4 h where indicated. RNA was extracted and XBP1 splicing was assessed by RT-PCR and quantified. C , L827P inhibits regulated IRE1-dependent decay activity in response to ER stress in Kms11 cells. The same samples as in B were used to perform a qPCR to detect BLOC1S1 expression levels as in Figure 1 C . D , expression of L827P decreases endogenous WT IRE1α phosphorylation in response to ER stress. Parental HAP1 cells, with constitutive expression of WT IRE1α and inducible expression of IRE1GFP L827P were induced with dox and treated with Tg (0.2 μM for 4 h). Cells were lysed and proteins were analyzed by Western blot. Arrow , endogenous phospho-S724 IRE1α; ∗, nonspecific bands. E. Quantification of phospho-IRE1α S724 and L827 mutant. The intensities of the Western blot bands from the experiment described in D were normalized to total protein contents of the samples, measured by Ponceau S staining, quantified, and plotted. F , L827P inhibits WT IRE1α clustering. Representative images of WT IRE1mCherry/L827P IRE1GFP-expressing cells treated with 4 μg/ml Tm for 3 h. Cells that coexpress mCherry and GFP typically exhibit reticular signal with no clusters. mCherry-expressing cells with low GFP expression form faint cluster-like foci termed dim clusters or the bright foci typical of WT IRE1α clusters. Arrowheads , two typical dim clusters; arrows , two typical bright clusters. G , L827P binds full-length WT IRE1α. HAP1KO cells were recomplemented with WT IRE1GFP containing only the GFP tag, WT IRE1HA, D123P IRE1HA, L827P IRE1HA containing only the HA tag or combinations of constructs. The cells were induced with dox and treated with 4 μg/ml Tm for 4 h where indicated. Cells were collected, lysed, and subjected to immunoprecipitation with GFP-Trap beads. Beads-bound proteins were analyzed by Western blot. Input: 5% of the lysates. Arrow , full-length IRE1GFP or IRE1HA; §, lower-molecular-weight bands that appear to be IRE1α specific and size sensitive to Tm treatment. ER, endoplasmic reticulum.
    Figure Legend Snippet: The L827P mutant inhibits WT IRE1α in HAP1 and Kms11 cells . A , IRE1GFP L827P inhibits XBP1 splicing in response to Tm in leukemic HAP1 cells. Parental HAP1 cells expressing IRE1GFP L827P in addition to the endogenous WT IRE1α were induced with the indicated concentrations of dox (μg/ml). The cells were then treated with 4 μg/ml Tm for 4 h, and XBP1 splicing was assessed by RT-PCR. B , L827P IRE1GFP inhibits ER stress-induced XBP1 splicing in multiple myeloma Kms11 cells. Kms11 cells expressing WT or L827P IRE1GFP were treated with 0.5 μM Tg for 4 h where indicated. RNA was extracted and XBP1 splicing was assessed by RT-PCR and quantified. C , L827P inhibits regulated IRE1-dependent decay activity in response to ER stress in Kms11 cells. The same samples as in B were used to perform a qPCR to detect BLOC1S1 expression levels as in Figure 1 C . D , expression of L827P decreases endogenous WT IRE1α phosphorylation in response to ER stress. Parental HAP1 cells, with constitutive expression of WT IRE1α and inducible expression of IRE1GFP L827P were induced with dox and treated with Tg (0.2 μM for 4 h). Cells were lysed and proteins were analyzed by Western blot. Arrow , endogenous phospho-S724 IRE1α; ∗, nonspecific bands. E. Quantification of phospho-IRE1α S724 and L827 mutant. The intensities of the Western blot bands from the experiment described in D were normalized to total protein contents of the samples, measured by Ponceau S staining, quantified, and plotted. F , L827P inhibits WT IRE1α clustering. Representative images of WT IRE1mCherry/L827P IRE1GFP-expressing cells treated with 4 μg/ml Tm for 3 h. Cells that coexpress mCherry and GFP typically exhibit reticular signal with no clusters. mCherry-expressing cells with low GFP expression form faint cluster-like foci termed dim clusters or the bright foci typical of WT IRE1α clusters. Arrowheads , two typical dim clusters; arrows , two typical bright clusters. G , L827P binds full-length WT IRE1α. HAP1KO cells were recomplemented with WT IRE1GFP containing only the GFP tag, WT IRE1HA, D123P IRE1HA, L827P IRE1HA containing only the HA tag or combinations of constructs. The cells were induced with dox and treated with 4 μg/ml Tm for 4 h where indicated. Cells were collected, lysed, and subjected to immunoprecipitation with GFP-Trap beads. Beads-bound proteins were analyzed by Western blot. Input: 5% of the lysates. Arrow , full-length IRE1GFP or IRE1HA; §, lower-molecular-weight bands that appear to be IRE1α specific and size sensitive to Tm treatment. ER, endoplasmic reticulum.

    Techniques Used: Mutagenesis, Expressing, Reverse Transcription Polymerase Chain Reaction, Activity Assay, Real-time Polymerase Chain Reaction, Western Blot, Staining, Construct, Immunoprecipitation, Molecular Weight

    5) Product Images from "CHMP6 and VPS4A mediate recycling of Ras to the plasma membrane to promote growth factor signaling"

    Article Title: CHMP6 and VPS4A mediate recycling of Ras to the plasma membrane to promote growth factor signaling

    Journal: Oncogene

    doi: 10.1038/onc.2011.607

    CHMP6 and VPS4A are novel H-Ras binding proteins. ( a ) HT1080 cells expressing Yn-H-Ras(12V) and either Yc-tagged CHMP6 or VPS4A, together with CFP-tagged GalT, Rab5A, Rab7A, or Rab11A, which mark Golgi, early (E.) endosomes, late (L.) endosomes/MVBs, or recycling (R.) endosomes, respectively, were analyzed by confocal microscopy. CFP and the reconstituted YFP signals were pseudo-colored red and green. Dotted lines mark the cell boundaries. Insets are scaled up images to better show co-localization between the CFP and YFP signals. ( b ) FLAG-tagged CHMP6 or VPS4A were co-expressed with indicated H-Ras proteins in 293 cells. Immunoprecipitation was performed using either a pan-reactive Ras antibody or an antibody against the FLAG tag. Immunoprecipitated proteins as well as the total lysate inputs were analyzed by Western blots using indicated antibodies. ( c ) YFP-VPS4A was coexpressed with CFP-tagged H-Ras(12V)-8RK, H-Ras(61L), or Ub-H-Ras in COS-1 cells. These cells were examined by laser confocal microscopy. Regions of interest are scaled up in the inset to better show co-localization between CFP and YFP signals, as well as FRET. A heat-map is included to show the relative FRET efficiency from low (violet) to high (red). The quantified FRET efficiencies are shown at the bottom. The number of cells (n) examined were: 15 (H-Ras(12V)-8RK), 11 (H-Ras(61L)), and 12 (Ub-H-Ras). **P
    Figure Legend Snippet: CHMP6 and VPS4A are novel H-Ras binding proteins. ( a ) HT1080 cells expressing Yn-H-Ras(12V) and either Yc-tagged CHMP6 or VPS4A, together with CFP-tagged GalT, Rab5A, Rab7A, or Rab11A, which mark Golgi, early (E.) endosomes, late (L.) endosomes/MVBs, or recycling (R.) endosomes, respectively, were analyzed by confocal microscopy. CFP and the reconstituted YFP signals were pseudo-colored red and green. Dotted lines mark the cell boundaries. Insets are scaled up images to better show co-localization between the CFP and YFP signals. ( b ) FLAG-tagged CHMP6 or VPS4A were co-expressed with indicated H-Ras proteins in 293 cells. Immunoprecipitation was performed using either a pan-reactive Ras antibody or an antibody against the FLAG tag. Immunoprecipitated proteins as well as the total lysate inputs were analyzed by Western blots using indicated antibodies. ( c ) YFP-VPS4A was coexpressed with CFP-tagged H-Ras(12V)-8RK, H-Ras(61L), or Ub-H-Ras in COS-1 cells. These cells were examined by laser confocal microscopy. Regions of interest are scaled up in the inset to better show co-localization between CFP and YFP signals, as well as FRET. A heat-map is included to show the relative FRET efficiency from low (violet) to high (red). The quantified FRET efficiencies are shown at the bottom. The number of cells (n) examined were: 15 (H-Ras(12V)-8RK), 11 (H-Ras(61L)), and 12 (Ub-H-Ras). **P

    Techniques Used: Binding Assay, Expressing, Confocal Microscopy, Immunoprecipitation, FLAG-tag, Western Blot

    6) Product Images from "Ubiquitination by the Membrane-associated RING-CH-8 (MARCH-8) Ligase Controls Steady-state Cell Surface Expression of Tumor Necrosis Factor-related Apoptosis Inducing Ligand (TRAIL) Receptor 1 *"

    Article Title: Ubiquitination by the Membrane-associated RING-CH-8 (MARCH-8) Ligase Controls Steady-state Cell Surface Expression of Tumor Necrosis Factor-related Apoptosis Inducing Ligand (TRAIL) Receptor 1 *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.448209

    Steady-state ubiquitination of TRAIL-R1 on lysine residue 273 by an endogenous machinery. A , MCF-7 Casp-3 cells were transfected to express FLAG-ubiquitin, together with mRFP only (−), or with mRFP-chimeras of WT TRAIL-R1 ( WT ) or its K273A lysine mutant ( K / A ). MARCH-8.HA cDNA (+) or an empty control vector (−) were additionally transfected as indicated. Cells were lysed in Nonidet P-40 buffer, TRAIL-R1 was isolated with anti(α)-mRFP antibody and immunoprecipitates ( IP ) were analyzed by immunoblotting ( IB ) with α-mRFP antibody to detect TRAIL-R1, α-FLAG antibody to detect ubiquitin and α-HA antibody to detect MARCH-8. Asterisk denotes the heavy chain of the antibody used for IP. Solid and open arrowheads indicate, respectively, TRAIL-R1.mRFP and mRFP only. Blot is representative of 4 independent experiments. B , MCF-7 Casp-3 cells were transfected to express FLAG-ubiquitin, together with either mRFP only (−), with mRFP chimeras of WT TRAIL-R1 ( WT ) or the K273A TRAIL-R1 mutant ( K / A ), or with a truncated TRAIL-R1 lacking the C-terminal 116 residues (ΔWT). TRAIL-R1 was isolated with α-mRFP antibody and immunoprecipitates were analyzed by immunoblotting with α-mRFP antibody to detect TRAIL-R1 and with α-FLAG antibody to detect ubiquitin. Data shown are representative of two independent experiments. C , MCF-7 Casp-3 cells were transfected to express FLAG-ubiquitin, together with mRFP only (−), or with mRFP-chimeras of WT TRAIL-R1 ( WT ) or its K273A lysine mutant ( K / A ). Cells were lysed by boiling in SDS, Nonidet P-40 buffer was added in excess and immunoprecipitation of TRAIL-R1 and analysis were performed as outlined for panel A. Asterisk denotes the heavy chain of the antibody used for IP. Solid and open arrowheads indicate, respectively, TRAIL-R1.mRFP and mRFP only. The blot is representative of 2 independent experiments. D , alignment of primary amino acid sequence of part of the transmembrane segment ( italic ) and the remaining 14 residues of the cytoplasmic tail of the truncated TRAIL-R1 mutants used in E . Relevant potential ubiquitination sites are shown in bold. E , MCF-7 Casp-3 cells were transfected to express FLAG-ubiquitin, together with mRFP-tagged TRAIL-R1 WT or mutants shown in D . TRAIL-R1 was isolated with α-mRFP antibody and immunoprecipitates were analyzed by immunoblotting with α-mRFP antibody to detect TRAIL-R1 and α-FLAG antibody to detect ubiquitin. The blot is representative of 2 independent experiments. Asterisk denotes the heavy chain of the antibody used for IP.
    Figure Legend Snippet: Steady-state ubiquitination of TRAIL-R1 on lysine residue 273 by an endogenous machinery. A , MCF-7 Casp-3 cells were transfected to express FLAG-ubiquitin, together with mRFP only (−), or with mRFP-chimeras of WT TRAIL-R1 ( WT ) or its K273A lysine mutant ( K / A ). MARCH-8.HA cDNA (+) or an empty control vector (−) were additionally transfected as indicated. Cells were lysed in Nonidet P-40 buffer, TRAIL-R1 was isolated with anti(α)-mRFP antibody and immunoprecipitates ( IP ) were analyzed by immunoblotting ( IB ) with α-mRFP antibody to detect TRAIL-R1, α-FLAG antibody to detect ubiquitin and α-HA antibody to detect MARCH-8. Asterisk denotes the heavy chain of the antibody used for IP. Solid and open arrowheads indicate, respectively, TRAIL-R1.mRFP and mRFP only. Blot is representative of 4 independent experiments. B , MCF-7 Casp-3 cells were transfected to express FLAG-ubiquitin, together with either mRFP only (−), with mRFP chimeras of WT TRAIL-R1 ( WT ) or the K273A TRAIL-R1 mutant ( K / A ), or with a truncated TRAIL-R1 lacking the C-terminal 116 residues (ΔWT). TRAIL-R1 was isolated with α-mRFP antibody and immunoprecipitates were analyzed by immunoblotting with α-mRFP antibody to detect TRAIL-R1 and with α-FLAG antibody to detect ubiquitin. Data shown are representative of two independent experiments. C , MCF-7 Casp-3 cells were transfected to express FLAG-ubiquitin, together with mRFP only (−), or with mRFP-chimeras of WT TRAIL-R1 ( WT ) or its K273A lysine mutant ( K / A ). Cells were lysed by boiling in SDS, Nonidet P-40 buffer was added in excess and immunoprecipitation of TRAIL-R1 and analysis were performed as outlined for panel A. Asterisk denotes the heavy chain of the antibody used for IP. Solid and open arrowheads indicate, respectively, TRAIL-R1.mRFP and mRFP only. The blot is representative of 2 independent experiments. D , alignment of primary amino acid sequence of part of the transmembrane segment ( italic ) and the remaining 14 residues of the cytoplasmic tail of the truncated TRAIL-R1 mutants used in E . Relevant potential ubiquitination sites are shown in bold. E , MCF-7 Casp-3 cells were transfected to express FLAG-ubiquitin, together with mRFP-tagged TRAIL-R1 WT or mutants shown in D . TRAIL-R1 was isolated with α-mRFP antibody and immunoprecipitates were analyzed by immunoblotting with α-mRFP antibody to detect TRAIL-R1 and α-FLAG antibody to detect ubiquitin. The blot is representative of 2 independent experiments. Asterisk denotes the heavy chain of the antibody used for IP.

    Techniques Used: Transfection, Mutagenesis, Plasmid Preparation, Isolation, Immunoprecipitation, Sequencing

    MARCH-1 and -8 interact with and down-regulate wild-type TRAIL-R1, but not the TRAIL-R1 K273A mutant. A , MCF-7 Casp-3 cells were transfected to express mRFP only (−), mRFP-tagged WT TRAIL-R1 or K273A ( K / A ) mutant, together with HA-tagged MARCH-1, MARCH-8, or empty vector (−), as indicated. Immunoprecipitation was performed with α-mRFP antibody and immunoprecipitates ( IP ) were analyzed by immunoblotting with α-mRFP and α-HA antibodies to detect TRAIL-R1 and MARCH-1/8, respectively. Panel I , mRFP detection in IP of TRAIL-R1.mRFP and control mRFP; panel II , MARCH-1 and -8 detection in IP of TRAIL-R1.mRFP and control mRFP; panel III , mRFP detection (TRAIL-R1.mRFP or RFP only) in total cell lysates ( TCL ); panel IV , MARCH-1 and -8 detection in TCL. B , quantification of TRAIL-R1 down-regulation in total cell lysates. Total protein levels of WT TRAIL-R1 and the K/A mutant in TCL of control cells (−), or those expressing HA-tagged MARCH-1 or -8 were quantified from Western blots as depicted in panel III of A and plotted as percentage of the WT TRAIL-R1.mRFP expression in control cells. Data represent mean ± S.D. of values from the experiment depicted in A and 2 additional experiments. C , impact of MARCH-1 or MARCH-8 on WT and K/A mutant TRAIL-R1 cell surface expression. Cells stably expressing WT or K273A TRAIL-R1.mRFP were transfected to express GFP (−), GFP-tagged MARCH-1 or MARCH-8, and stained with antibody to TRAIL-R1 as outlined for Fig. 2 . Quantification of 2–4 independent experiments assessing TRAIL-R1 MFI in GFP + cells as the percentage of TRAIL-R1 MFI in GFP − cells, whereby the values in control cells were set at 100%. Values represent mean ± S.D. Asterisks indicate statistically significant differences between TRAIL-R1 WT or K/A mutant (Student's t test; *, p
    Figure Legend Snippet: MARCH-1 and -8 interact with and down-regulate wild-type TRAIL-R1, but not the TRAIL-R1 K273A mutant. A , MCF-7 Casp-3 cells were transfected to express mRFP only (−), mRFP-tagged WT TRAIL-R1 or K273A ( K / A ) mutant, together with HA-tagged MARCH-1, MARCH-8, or empty vector (−), as indicated. Immunoprecipitation was performed with α-mRFP antibody and immunoprecipitates ( IP ) were analyzed by immunoblotting with α-mRFP and α-HA antibodies to detect TRAIL-R1 and MARCH-1/8, respectively. Panel I , mRFP detection in IP of TRAIL-R1.mRFP and control mRFP; panel II , MARCH-1 and -8 detection in IP of TRAIL-R1.mRFP and control mRFP; panel III , mRFP detection (TRAIL-R1.mRFP or RFP only) in total cell lysates ( TCL ); panel IV , MARCH-1 and -8 detection in TCL. B , quantification of TRAIL-R1 down-regulation in total cell lysates. Total protein levels of WT TRAIL-R1 and the K/A mutant in TCL of control cells (−), or those expressing HA-tagged MARCH-1 or -8 were quantified from Western blots as depicted in panel III of A and plotted as percentage of the WT TRAIL-R1.mRFP expression in control cells. Data represent mean ± S.D. of values from the experiment depicted in A and 2 additional experiments. C , impact of MARCH-1 or MARCH-8 on WT and K/A mutant TRAIL-R1 cell surface expression. Cells stably expressing WT or K273A TRAIL-R1.mRFP were transfected to express GFP (−), GFP-tagged MARCH-1 or MARCH-8, and stained with antibody to TRAIL-R1 as outlined for Fig. 2 . Quantification of 2–4 independent experiments assessing TRAIL-R1 MFI in GFP + cells as the percentage of TRAIL-R1 MFI in GFP − cells, whereby the values in control cells were set at 100%. Values represent mean ± S.D. Asterisks indicate statistically significant differences between TRAIL-R1 WT or K/A mutant (Student's t test; *, p

    Techniques Used: Mutagenesis, Transfection, Plasmid Preparation, Immunoprecipitation, Expressing, Western Blot, Stable Transfection, Staining

    TRAIL-R1 is a substrate of endogenous MARCH-8. A , endogenous MARCH-8 expression in MCF-7 Casp-3 (MCF-7) and Mel Juso (MJ) cells, as determined by RT-PCR on cDNA. Non-reverse transcribed RNA ( RNA ) was used as a control template to exclude amplification of genomic DNA. B , down-regulation of endogenous MARCH-8 by RNAi. MCF-7 Casp-3 cells were transfected with MARCH-8 shRNA, together with GFP. MARCH-8 shRNA expressing cells ( GFP + ) and nonexpressing cells ( GFP − ) cells were separated by flow cytometric sorting and analyzed for endogenous MARCH-8 transcript levels by quantitative RT-PCR. Signals that were corrected for GAPDH and MARCH-8 transcript levels in the GFP + population were normalized to the levels in the GFP − population. C , MCF-7 Casp-3 cells were transfected to express mRFP alone, WT TRAIL-R1.mRFP, or K273A TRAIL-R1.mRFP, together with FLAG-ubiquitin and either an empty vector, or the MARCH-8 targeting shRNA. TRAIL-R1 was isolated by immunoprecipitation with α-mRFP antibody and immunoprecipitates ( IP ) were analyzed by immunoblotting for TRAIL-R1 (α- mRFP ) or ubiquitin (α- FLAG ). Solid and open arrowheads indicate, respectively, TRAIL-R1.mRFP and mRFP only. Data shown are representative of 3 independent experiments.
    Figure Legend Snippet: TRAIL-R1 is a substrate of endogenous MARCH-8. A , endogenous MARCH-8 expression in MCF-7 Casp-3 (MCF-7) and Mel Juso (MJ) cells, as determined by RT-PCR on cDNA. Non-reverse transcribed RNA ( RNA ) was used as a control template to exclude amplification of genomic DNA. B , down-regulation of endogenous MARCH-8 by RNAi. MCF-7 Casp-3 cells were transfected with MARCH-8 shRNA, together with GFP. MARCH-8 shRNA expressing cells ( GFP + ) and nonexpressing cells ( GFP − ) cells were separated by flow cytometric sorting and analyzed for endogenous MARCH-8 transcript levels by quantitative RT-PCR. Signals that were corrected for GAPDH and MARCH-8 transcript levels in the GFP + population were normalized to the levels in the GFP − population. C , MCF-7 Casp-3 cells were transfected to express mRFP alone, WT TRAIL-R1.mRFP, or K273A TRAIL-R1.mRFP, together with FLAG-ubiquitin and either an empty vector, or the MARCH-8 targeting shRNA. TRAIL-R1 was isolated by immunoprecipitation with α-mRFP antibody and immunoprecipitates ( IP ) were analyzed by immunoblotting for TRAIL-R1 (α- mRFP ) or ubiquitin (α- FLAG ). Solid and open arrowheads indicate, respectively, TRAIL-R1.mRFP and mRFP only. Data shown are representative of 3 independent experiments.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Transfection, shRNA, Flow Cytometry, Quantitative RT-PCR, Plasmid Preparation, Isolation, Immunoprecipitation

    7) Product Images from "DDR1 promotes E-cadherin stability via inhibition of integrin-β1-Src activation-mediated E-cadherin endocytosis"

    Article Title: DDR1 promotes E-cadherin stability via inhibition of integrin-β1-Src activation-mediated E-cadherin endocytosis

    Journal: Scientific Reports

    doi: 10.1038/srep36336

    Clathrin-mediated endocytosis is involved in the knockdown of DDR1-induced E-cadherin endocytosis. ( a ) The interaction between E-cadherin and clathrin in Mock or Sh-DDR1 cells was assessed using immunoprecipitation of E-cadherin, followed by immunoblotting with anti-clathrin heavy chain or anti-E-cadherin antibodies. TCL represents total cell lysate; “-” represents immunoprecipitation with the control antibody. ( b ) Quantification results of the ratio of clathrin/E-cadherin intensity. Each bar represents mean ± SE of three independent experiments. *Indicates P
    Figure Legend Snippet: Clathrin-mediated endocytosis is involved in the knockdown of DDR1-induced E-cadherin endocytosis. ( a ) The interaction between E-cadherin and clathrin in Mock or Sh-DDR1 cells was assessed using immunoprecipitation of E-cadherin, followed by immunoblotting with anti-clathrin heavy chain or anti-E-cadherin antibodies. TCL represents total cell lysate; “-” represents immunoprecipitation with the control antibody. ( b ) Quantification results of the ratio of clathrin/E-cadherin intensity. Each bar represents mean ± SE of three independent experiments. *Indicates P

    Techniques Used: Immunoprecipitation

    8) Product Images from "MicroRNAs Distinguish Cytogenetic Subgroups in Pediatric AML and Contribute to Complex Regulatory Networks in AML-Relevant Pathways"

    Article Title: MicroRNAs Distinguish Cytogenetic Subgroups in Pediatric AML and Contribute to Complex Regulatory Networks in AML-Relevant Pathways

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0056334

    Ago-associated miRNAs and - mRNAs using the PAR-CLIP-Array method. (A) Western Blot analysis of immunoprecipitates of human Ago1-4 from AML cell lines, KASUMI-1 with t(8;21) and NB4 carrying t(15;17). The immunoprecipitates show a specific band of the Argonaute protein (∼97 kDa; ←) in contrast to the isotype control antibody (rat IgG2a) and empty-bead control. A representative sample of the biological triplicate is shown. Please note that more material was loaded for Ago3 and Ago4 since these two Ago proteins are much lower expressed as was also validated by qRT-PCR (not shown). Antibodies were tested for specificity for detection of native and denatured protein prior to this experiment with cell lines overexpressing tagged Ago protein (not shown) (B) Validation of miRNA- and mRNA-enrichment in immunoprecipitation experiments. Argonaute proteins (black bar) are compared to the isotype (white bar) and empty bead controls (grey bar) using TaqMan qRT-PCR assays for microRNA- (upper panel) and SYBR Green qRT-PCR assays for mRNA-quantification (lower panel). Shown are the measured levels (2 −C T) of six and five miRNAs of KASUMI-1 cells (upper left panel) and NB4 cells (upper right panel), respectively. Immunoprecipitation experiments as well as cDNA synthesis were each done in triplicates and the mean value of the nine values as well as one standard deviation is depicted. miRNAs differentially expressed in patient samples between the t(8;21) and t(15;17) were selected together with ubiquitously expressed miR-16. Please note that calculation of ΔC T -values is not possible due to the lack of a housekeeping gene bound to Argonaute proteins. Six Ago-associated mRNAs in the KASUMI-1 cells (lower left panel) and NB4 cells (lower right panel), covering the whole range from low to high enrichment over isotype control according to microarray data, were selected for qRT-PCR validation. Graphs are centered around a C T -value of 29.9 cycles (2 −C T = 0 −9 ).
    Figure Legend Snippet: Ago-associated miRNAs and - mRNAs using the PAR-CLIP-Array method. (A) Western Blot analysis of immunoprecipitates of human Ago1-4 from AML cell lines, KASUMI-1 with t(8;21) and NB4 carrying t(15;17). The immunoprecipitates show a specific band of the Argonaute protein (∼97 kDa; ←) in contrast to the isotype control antibody (rat IgG2a) and empty-bead control. A representative sample of the biological triplicate is shown. Please note that more material was loaded for Ago3 and Ago4 since these two Ago proteins are much lower expressed as was also validated by qRT-PCR (not shown). Antibodies were tested for specificity for detection of native and denatured protein prior to this experiment with cell lines overexpressing tagged Ago protein (not shown) (B) Validation of miRNA- and mRNA-enrichment in immunoprecipitation experiments. Argonaute proteins (black bar) are compared to the isotype (white bar) and empty bead controls (grey bar) using TaqMan qRT-PCR assays for microRNA- (upper panel) and SYBR Green qRT-PCR assays for mRNA-quantification (lower panel). Shown are the measured levels (2 −C T) of six and five miRNAs of KASUMI-1 cells (upper left panel) and NB4 cells (upper right panel), respectively. Immunoprecipitation experiments as well as cDNA synthesis were each done in triplicates and the mean value of the nine values as well as one standard deviation is depicted. miRNAs differentially expressed in patient samples between the t(8;21) and t(15;17) were selected together with ubiquitously expressed miR-16. Please note that calculation of ΔC T -values is not possible due to the lack of a housekeeping gene bound to Argonaute proteins. Six Ago-associated mRNAs in the KASUMI-1 cells (lower left panel) and NB4 cells (lower right panel), covering the whole range from low to high enrichment over isotype control according to microarray data, were selected for qRT-PCR validation. Graphs are centered around a C T -value of 29.9 cycles (2 −C T = 0 −9 ).

    Techniques Used: Cross-linking Immunoprecipitation, Western Blot, Quantitative RT-PCR, Immunoprecipitation, SYBR Green Assay, Standard Deviation, Microarray

    9) 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: Cell Death & Disease

    doi: 10.1038/s41419-020-02887-y

    Vincristine induces resistance in RMS cells through BID and BAK inhibition by MCL-1. a Left panel: Western blot results of the unbound fraction after MCL-1 immunoprecipitation. Right panel: MCL-1 levels in the initial cell lysates. High efficiency of MCL-1 immunoprecipitation compared to Rabbit IgG control antibody. b Western blot results of the co-immunoprecipitation between MCL-1 and tBID in control conditions and after 1 nM vincristine treatment for 36 h. Results showed a significant increase in tBID and MCL-1 binding after vincristine treatment. c Western blot results of the co-immunoprecipitation between MCL-1 and BAK in control conditions, after 1 nM vincristine treatment and after the sequential combination of 1 nM vincristine and 1 µM S63845 for 36 h. Results showed a significant increase in BAK and MCL-1 binding after vincristine treatment, which was decreased below 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 and BAK inhibition by MCL-1. a Left panel: Western blot results of the unbound fraction after MCL-1 immunoprecipitation. Right panel: MCL-1 levels in the initial cell lysates. High efficiency of MCL-1 immunoprecipitation compared to Rabbit IgG control antibody. b Western blot results of the co-immunoprecipitation between MCL-1 and tBID in control conditions and after 1 nM vincristine treatment for 36 h. Results showed a significant increase in tBID and MCL-1 binding after vincristine treatment. c Western blot results of the co-immunoprecipitation between MCL-1 and BAK in control conditions, after 1 nM vincristine treatment and after the sequential combination of 1 nM vincristine and 1 µM S63845 for 36 h. Results showed a significant increase in BAK and MCL-1 binding after vincristine treatment, which was decreased below 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, Binding Assay

    10) Product Images from "Dynamic Regulation of Oct1 during Mitosis by Phosphorylation and Ubiquitination"

    Article Title: Dynamic Regulation of Oct1 during Mitosis by Phosphorylation and Ubiquitination

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0023872

    Oct1 is present in the spindle matrix and forms a complex with lamin B1 at the midbody in HeLa cells. ( A ) Association of phosphorylated Oct1 with lamin B at the centrosomes and midbody. HeLa cells were fixed and stained with antibodies against lamin B1+B2 and Oct1 pS335 . Mitotic stage is on the left. Asterisk indicates the midbody structure. ( B ) Whole cell extracts from cycling HeLa cells and cells arrested in M-phase using nocodozole were immunoprecipitated using mouse anti-lamin B antibodies. Left panel shows a Western blot using pan-Oct1 antibodies. Black arrow shows predicted Oct1 molecular weight. Asterisk shows the high molecular weight form identified in Fig. 1 . Right panel: the blot was stripped and re-probed using Oct1 pS335 antibodies. ( C ) Spindle matrix preparations generated from Xenopus oocyte extracts (XEE, lane 1) were Western blotted using pan-Oct1, lamin B3, and α-tubulin antibodies. ( D ) IF images of bead spindown preparation. Pan-Oct1 antibodies, and rhodamine-conjugated α-tubulin were used. ( E ) Xenopus Oct1 was immunodepleted using magnetic protein A-coupled beads (see methods ). Oct1 Western blots are shown of the non-specific and Oct1-specific depletions. α-tubulin is shown as a loading control. ( F ) Examples of spindle structures generated using the depleted extracts. Images of structures conforming to the scoring criteria used in (G) are shown. ( G ) Quantification of spindle structures using non-specific of Oct1-specific depletion. Error bars depict standard error of the mean. ( H ) Co-immunoprecipitation Cdk11 with endogenous phospho-Oct1. Mitotic-arrested HeLa whole cell extracts were immunprecipitated using phospho-Oct1 antibodies and probed with anti-Cdk11 or anti-pan-Oct1. Arrest was accomplished with 18 hr treatment with nocodozole. ( I ) HeLa cells were transiently transfected with Lamin B1-specific siRNAs. Cells were incubated for 72 hr, fixed and stained with α-tubulin and pS335 antibodies. Images of cells undergoing abcission are shown. Formaldehyde fixation was used. ( J ) HeLa cells transfected with control siRNAs, or siRNAs against Oct1 or lamin B1 were fixed and stained with lamin B and Oct1 pS335 antibodies. IF images of mitotic HeLa cells undergoing abcission are shown. Arrow indicates position of the midbody. Detail at right shows isolated midbody structures. Formaldehyde fixation was used.
    Figure Legend Snippet: Oct1 is present in the spindle matrix and forms a complex with lamin B1 at the midbody in HeLa cells. ( A ) Association of phosphorylated Oct1 with lamin B at the centrosomes and midbody. HeLa cells were fixed and stained with antibodies against lamin B1+B2 and Oct1 pS335 . Mitotic stage is on the left. Asterisk indicates the midbody structure. ( B ) Whole cell extracts from cycling HeLa cells and cells arrested in M-phase using nocodozole were immunoprecipitated using mouse anti-lamin B antibodies. Left panel shows a Western blot using pan-Oct1 antibodies. Black arrow shows predicted Oct1 molecular weight. Asterisk shows the high molecular weight form identified in Fig. 1 . Right panel: the blot was stripped and re-probed using Oct1 pS335 antibodies. ( C ) Spindle matrix preparations generated from Xenopus oocyte extracts (XEE, lane 1) were Western blotted using pan-Oct1, lamin B3, and α-tubulin antibodies. ( D ) IF images of bead spindown preparation. Pan-Oct1 antibodies, and rhodamine-conjugated α-tubulin were used. ( E ) Xenopus Oct1 was immunodepleted using magnetic protein A-coupled beads (see methods ). Oct1 Western blots are shown of the non-specific and Oct1-specific depletions. α-tubulin is shown as a loading control. ( F ) Examples of spindle structures generated using the depleted extracts. Images of structures conforming to the scoring criteria used in (G) are shown. ( G ) Quantification of spindle structures using non-specific of Oct1-specific depletion. Error bars depict standard error of the mean. ( H ) Co-immunoprecipitation Cdk11 with endogenous phospho-Oct1. Mitotic-arrested HeLa whole cell extracts were immunprecipitated using phospho-Oct1 antibodies and probed with anti-Cdk11 or anti-pan-Oct1. Arrest was accomplished with 18 hr treatment with nocodozole. ( I ) HeLa cells were transiently transfected with Lamin B1-specific siRNAs. Cells were incubated for 72 hr, fixed and stained with α-tubulin and pS335 antibodies. Images of cells undergoing abcission are shown. Formaldehyde fixation was used. ( J ) HeLa cells transfected with control siRNAs, or siRNAs against Oct1 or lamin B1 were fixed and stained with lamin B and Oct1 pS335 antibodies. IF images of mitotic HeLa cells undergoing abcission are shown. Arrow indicates position of the midbody. Detail at right shows isolated midbody structures. Formaldehyde fixation was used.

    Techniques Used: Staining, Immunoprecipitation, Western Blot, Molecular Weight, Generated, Transfection, Incubation, Isolation

    11) Product Images from "Centriolar Satellites Control GABARAP Ubiquitination and GABARAP-Mediated Autophagy"

    Article Title: Centriolar Satellites Control GABARAP Ubiquitination and GABARAP-Mediated Autophagy

    Journal: Current Biology

    doi: 10.1016/j.cub.2017.06.021

    Mib1 E3 Ligase Interacts with and Destabilizes GABARAP and Promotes GABARAP Ubiquitination at Lys13 and Lys23 (A) HEK293A cells expressing FLAG-Mib1 or control vector for 48 hr were analyzed by immunoblot. (B) Quantification of (A). Statistical analysis using unpaired Student’s t test; mean ± SEM; n = 3; ∗ p ≤ 0.001. (C) Anti-GABARAP immunoprecipitate from HEK293A cells analyzed by immunoblotting. Ab, anti-GABARAP antibody. Lys, HEK293A lysate. (D) HEK293A cells expressing FLAG-tagged constructs were incubated with recombinant GST or GST-GABARAP beads and immunoblotted. (E) GFP-TRAP of HEK293A cells expressing the indicated constructs and immunoblot. Immunoprecipitates were stringently washed in denaturing buffer. CS, C985S; GAB, GABARAP; Ponc, Ponceau S. Short and long exposures are shown. ∗ , ∗∗ , ∗∗∗ , mono-, di-, and tri-ubiquitinated GFP-GABARAP, respectively. (F) Immunoprecipitation of U2OS cells expressing the indicated constructs lysed in boiling SDS buffer and immunoblot. Free ubiquitin and ∗ , ∗∗ , ∗∗∗ , mono-, di-, and tri-ubiquitinated GFP-LC3B/GABARAP are indicated, respectively. (G) GFP-TRAP of HEK293A cells expressing the indicated constructs and immunoblot. Immunoprecipitates were washed as in (E). (H) See (G). Low and high exposures are shown. (I) Immunoprecipitation of HEK293A cells expressing the indicated constructs and immunoblot. Cells were treated with MG132 for 5 hr prior to lysis in TNTE buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.5% w/v Triton X-100, 5 mM EDTA) + N-ethylmaleimide. Diubiquitinated GABARAP is indicated with ∗∗ . Immunoglobulin light chain is indicated with an arrow. (J) Immunoprecipitation of HEK293A cells treated with RF or GABARAP siRNA for 72 hr and expressing the indicated constructs and immunoblot. Cells were treated with MG132 for 5 hr prior to lysis in TNTE buffer + N-ethylmaleimide. Di- and tri-ubiquitinated GABARAP is indicated with ∗∗ and ∗∗∗ , respectively. Immunoglobulin light chain is indicated with an arrow. (K) Two GABARAP ubiquitination sites, lysine 13 (K13) and lysine 23 (K23), were identified by mass spectrometry on three different peptides. (Top) Comparison of peak areas for the FVYKEEHPFEK(diGly)R peptide containing K13 ubiquitination site (n = 3 measurements) is shown. (Middle and bottom) The K23 ubiquitination site was detected as two different peptides as a result of missed cleavage. Quantification of peptides K(diGly)KYPDRVPVIVEK (middle) and K(diGly)KYPDR (bottom) showed significantly lower abundance in C985S mutant compared to the WT. (L) Conservation of GABARAP K13 and K23 ( ∗ ) between ATG8 orthologs. See also Figure S5 .
    Figure Legend Snippet: Mib1 E3 Ligase Interacts with and Destabilizes GABARAP and Promotes GABARAP Ubiquitination at Lys13 and Lys23 (A) HEK293A cells expressing FLAG-Mib1 or control vector for 48 hr were analyzed by immunoblot. (B) Quantification of (A). Statistical analysis using unpaired Student’s t test; mean ± SEM; n = 3; ∗ p ≤ 0.001. (C) Anti-GABARAP immunoprecipitate from HEK293A cells analyzed by immunoblotting. Ab, anti-GABARAP antibody. Lys, HEK293A lysate. (D) HEK293A cells expressing FLAG-tagged constructs were incubated with recombinant GST or GST-GABARAP beads and immunoblotted. (E) GFP-TRAP of HEK293A cells expressing the indicated constructs and immunoblot. Immunoprecipitates were stringently washed in denaturing buffer. CS, C985S; GAB, GABARAP; Ponc, Ponceau S. Short and long exposures are shown. ∗ , ∗∗ , ∗∗∗ , mono-, di-, and tri-ubiquitinated GFP-GABARAP, respectively. (F) Immunoprecipitation of U2OS cells expressing the indicated constructs lysed in boiling SDS buffer and immunoblot. Free ubiquitin and ∗ , ∗∗ , ∗∗∗ , mono-, di-, and tri-ubiquitinated GFP-LC3B/GABARAP are indicated, respectively. (G) GFP-TRAP of HEK293A cells expressing the indicated constructs and immunoblot. Immunoprecipitates were washed as in (E). (H) See (G). Low and high exposures are shown. (I) Immunoprecipitation of HEK293A cells expressing the indicated constructs and immunoblot. Cells were treated with MG132 for 5 hr prior to lysis in TNTE buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.5% w/v Triton X-100, 5 mM EDTA) + N-ethylmaleimide. Diubiquitinated GABARAP is indicated with ∗∗ . Immunoglobulin light chain is indicated with an arrow. (J) Immunoprecipitation of HEK293A cells treated with RF or GABARAP siRNA for 72 hr and expressing the indicated constructs and immunoblot. Cells were treated with MG132 for 5 hr prior to lysis in TNTE buffer + N-ethylmaleimide. Di- and tri-ubiquitinated GABARAP is indicated with ∗∗ and ∗∗∗ , respectively. Immunoglobulin light chain is indicated with an arrow. (K) Two GABARAP ubiquitination sites, lysine 13 (K13) and lysine 23 (K23), were identified by mass spectrometry on three different peptides. (Top) Comparison of peak areas for the FVYKEEHPFEK(diGly)R peptide containing K13 ubiquitination site (n = 3 measurements) is shown. (Middle and bottom) The K23 ubiquitination site was detected as two different peptides as a result of missed cleavage. Quantification of peptides K(diGly)KYPDRVPVIVEK (middle) and K(diGly)KYPDR (bottom) showed significantly lower abundance in C985S mutant compared to the WT. (L) Conservation of GABARAP K13 and K23 ( ∗ ) between ATG8 orthologs. See also Figure S5 .

    Techniques Used: Expressing, Plasmid Preparation, Construct, Incubation, Recombinant, Immunoprecipitation, Lysis, Mass Spectrometry, Mutagenesis

    PCM1 Directly Binds GABARAP through a LIR Motif (A) Anti-GABARAP immunoprecipitation from HEK293A cells and immunoblot. Beads + Ab, anti-GABARAP antibody with protein G beads; beads + Lys, HEK293A lysate with protein G beads. (B) HEK293A cells in full medium (FM) or EBSS (ES) for 2 hr prior to lysis, followed by treatment as in (A). (C) HEK293A cells expressing indicated constructs in FM, ES, or EBSS + BAFA (EB) for 2 hr prior to lysis and GFP-TRAP. GFP-GABA, GFP-GABARAP; WT, wild-type. (D) Statistical analysis of (C); one-way ANOVA. NS, non-significant. (E) GFP-TRAP of HEK293A cells expressing the indicated GFP-ATG8 constructs and immunoblot. (F) HEK293A cells expressing the indicated GFP-tagged constructs incubated with GST or GST-GABARAP beads and immunoblotted. 3xAla, LIR mutant. (G) Statistical analysis of (F); unpaired Student’s t test; mean ± SEM; n = 3. ∗∗∗∗ p ≤ 0.0001. (H) 24-mer array of PCM1 peptides covering the LIR motif incubated with GST-GABARAP and immunoblot. Each amino acid position was substituted for every other amino acid.
    Figure Legend Snippet: PCM1 Directly Binds GABARAP through a LIR Motif (A) Anti-GABARAP immunoprecipitation from HEK293A cells and immunoblot. Beads + Ab, anti-GABARAP antibody with protein G beads; beads + Lys, HEK293A lysate with protein G beads. (B) HEK293A cells in full medium (FM) or EBSS (ES) for 2 hr prior to lysis, followed by treatment as in (A). (C) HEK293A cells expressing indicated constructs in FM, ES, or EBSS + BAFA (EB) for 2 hr prior to lysis and GFP-TRAP. GFP-GABA, GFP-GABARAP; WT, wild-type. (D) Statistical analysis of (C); one-way ANOVA. NS, non-significant. (E) GFP-TRAP of HEK293A cells expressing the indicated GFP-ATG8 constructs and immunoblot. (F) HEK293A cells expressing the indicated GFP-tagged constructs incubated with GST or GST-GABARAP beads and immunoblotted. 3xAla, LIR mutant. (G) Statistical analysis of (F); unpaired Student’s t test; mean ± SEM; n = 3. ∗∗∗∗ p ≤ 0.0001. (H) 24-mer array of PCM1 peptides covering the LIR motif incubated with GST-GABARAP and immunoblot. Each amino acid position was substituted for every other amino acid.

    Techniques Used: Immunoprecipitation, Lysis, Expressing, Construct, Incubation, Mutagenesis

    12) Product Images from "DRG2 Deficiency Causes Impaired Microtubule Dynamics in HeLa Cells"

    Article Title: DRG2 Deficiency Causes Impaired Microtubule Dynamics in HeLa Cells

    Journal: Molecules and Cells

    doi: 10.14348/molcells.2018.0129

    DRG2 binds to tau and increases phosphorylation on tau at S202 (A) Immunoprecipitation/western blot assay was performed from extracts of HeLa cells transfected with pEGFP-tau and pcDNA. V5/DRG2.HiSB . For controls, cells were transfected with either pEGFP-N1 or pcDNA.V5.HiSB . (B) Western blot assay was performed with specific antibodies against phospho-tau ( p -tau) S202.
    Figure Legend Snippet: DRG2 binds to tau and increases phosphorylation on tau at S202 (A) Immunoprecipitation/western blot assay was performed from extracts of HeLa cells transfected with pEGFP-tau and pcDNA. V5/DRG2.HiSB . For controls, cells were transfected with either pEGFP-N1 or pcDNA.V5.HiSB . (B) Western blot assay was performed with specific antibodies against phospho-tau ( p -tau) S202.

    Techniques Used: Immunoprecipitation, Western Blot, Transfection

    13) Product Images from "Inefficient TLR4/MD-2 Heterotetramerization by Monophosphoryl Lipid A"

    Article Title: Inefficient TLR4/MD-2 Heterotetramerization by Monophosphoryl Lipid A

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0062622

    Reduced MyD88-associated signaling by sMLA. Mouse BMDC were treated with 100 ng/ml sMLA or sDLA for the indicated times, lysed and either immunoblots (A,C) or immunoprecipitation (B) were performed on the lysates. A) Immunoblots were probed for IRAK1 and β-actin. A representative gel is depicted with a graph that shows the mean +/− SEM from 5 experiments in which IRAK1 levels were normalized to β-actin levels and vehicle control (VC, measured at 15 min.). B) Lysates were immunoprecipitated with IRAK1 antibodies and immunoblotted with antibodies for TRAF6 and IRAK1. The graph shows mean +/− SEM from 8 experiments with at least 5 data points per time point. TRAF6 levels were normalized based on IRAK1 levels and VC (5 min. time point). C) Left, Immunoblots were probed for phosphoERK 1/2 (Thr202/Tyr204), stripped and re-probed for ERK total, then β-actin. Shown are the mean +/− SEM from 5 experiments with levels of pERK1/2 normalized to ERK total and VC (15 min. time point). Right, immunoblots were probed for IκBα stripped and probed for β-actin. Shown are the mean +/− SEM from 4 experiments with levels of IκBα normalized to β-actin and VC (5 min. time point). Two way ANOVAs with Sidak’s multiple comparisons were performed for A and C and two tailed paired T test for B. Asterisks indicate a significant differences between sMLA and sDLA at the indicated time points with * p
    Figure Legend Snippet: Reduced MyD88-associated signaling by sMLA. Mouse BMDC were treated with 100 ng/ml sMLA or sDLA for the indicated times, lysed and either immunoblots (A,C) or immunoprecipitation (B) were performed on the lysates. A) Immunoblots were probed for IRAK1 and β-actin. A representative gel is depicted with a graph that shows the mean +/− SEM from 5 experiments in which IRAK1 levels were normalized to β-actin levels and vehicle control (VC, measured at 15 min.). B) Lysates were immunoprecipitated with IRAK1 antibodies and immunoblotted with antibodies for TRAF6 and IRAK1. The graph shows mean +/− SEM from 8 experiments with at least 5 data points per time point. TRAF6 levels were normalized based on IRAK1 levels and VC (5 min. time point). C) Left, Immunoblots were probed for phosphoERK 1/2 (Thr202/Tyr204), stripped and re-probed for ERK total, then β-actin. Shown are the mean +/− SEM from 5 experiments with levels of pERK1/2 normalized to ERK total and VC (15 min. time point). Right, immunoblots were probed for IκBα stripped and probed for β-actin. Shown are the mean +/− SEM from 4 experiments with levels of IκBα normalized to β-actin and VC (5 min. time point). Two way ANOVAs with Sidak’s multiple comparisons were performed for A and C and two tailed paired T test for B. Asterisks indicate a significant differences between sMLA and sDLA at the indicated time points with * p

    Techniques Used: Western Blot, Immunoprecipitation, Two Tailed Test

    14) Product Images from "Acetylation-dependent regulation of essential iPS-inducing factors: a regulatory crossroad for pluripotency and tumorigenesis"

    Article Title: Acetylation-dependent regulation of essential iPS-inducing factors: a regulatory crossroad for pluripotency and tumorigenesis

    Journal: Cancer Medicine

    doi: 10.1002/cam4.298

    Oct4 and Klf4 are phosphorylated by Akt in vivo. (A) Immunoblot (IB) analysis of whole cell lysates (WCLs) and immunoprecipitates (IPs) derived from 293T cells transfected with HA-tagged Myr-Akt1 and indicated Flag-tagged constructs. Akt-mediated phosphorylation was recognized by an Akt substrate-motif phosphorylation-specific antibody (RxRxxpS/pT). (B) Sequence alignment of the Thr235 putative Akt phosphorylation site in Oct4 among different species. (C) Akt specifically phosphorylated Oct4 at Thr235. (D) Phosphomimetic mutation at Thr235 of Oct4 decreased Oct4 interaction with Sox2. IB analysis and Flag-IP derived from 293T cells transfected with indicated constructs. (E) Phosphorylation of Oct4 on Thr235 led to attenuated Oct4 interaction with Sox2. WCLs of 293T cells transfected with indicated constructs were subjected to immunoprecipitation with Flag antibody. The Flag-IPs and WCLs were immunoblotted with indicated antibodies. (F) Akt phosphorylated Klf4 at Thr399. WCLs of 293T cells transfected with indicated constructs were subjected to immunoprecipitation with Flag antibody. The Flag-IPs and WCLs were immunoblotted with indicated antibodies. (G) Sequence alignment of the Thr429 putative Akt phosphorylation site in Klf4 among different species.
    Figure Legend Snippet: Oct4 and Klf4 are phosphorylated by Akt in vivo. (A) Immunoblot (IB) analysis of whole cell lysates (WCLs) and immunoprecipitates (IPs) derived from 293T cells transfected with HA-tagged Myr-Akt1 and indicated Flag-tagged constructs. Akt-mediated phosphorylation was recognized by an Akt substrate-motif phosphorylation-specific antibody (RxRxxpS/pT). (B) Sequence alignment of the Thr235 putative Akt phosphorylation site in Oct4 among different species. (C) Akt specifically phosphorylated Oct4 at Thr235. (D) Phosphomimetic mutation at Thr235 of Oct4 decreased Oct4 interaction with Sox2. IB analysis and Flag-IP derived from 293T cells transfected with indicated constructs. (E) Phosphorylation of Oct4 on Thr235 led to attenuated Oct4 interaction with Sox2. WCLs of 293T cells transfected with indicated constructs were subjected to immunoprecipitation with Flag antibody. The Flag-IPs and WCLs were immunoblotted with indicated antibodies. (F) Akt phosphorylated Klf4 at Thr399. WCLs of 293T cells transfected with indicated constructs were subjected to immunoprecipitation with Flag antibody. The Flag-IPs and WCLs were immunoblotted with indicated antibodies. (G) Sequence alignment of the Thr429 putative Akt phosphorylation site in Klf4 among different species.

    Techniques Used: In Vivo, Derivative Assay, Transfection, Construct, Sequencing, Mutagenesis, Immunoprecipitation

    Mapping of Klf4 acetylation sites in vivo. (A) Schematic illustration of a series of Klf4 truncation mutations generated. (B) WCLs of 293T cells transfected with HA-p300 and various Klf4 truncation mutants were subjected to immunoprecipitation with Flag antibody. The Flag-IPs and WCLs were immunoblotted with indicated antibodies. (C and D) WCLs of 293T cells transfected with HA-p300 and various Klf4 truncation or K-to-R substitution mutants were subjected to immunoprecipitation with Flag antibody. The Flag-IPs and WCLs were immunoblotted with indicated antibodies. WCLs, whole cell lysates; IP, immunoprecipitation.
    Figure Legend Snippet: Mapping of Klf4 acetylation sites in vivo. (A) Schematic illustration of a series of Klf4 truncation mutations generated. (B) WCLs of 293T cells transfected with HA-p300 and various Klf4 truncation mutants were subjected to immunoprecipitation with Flag antibody. The Flag-IPs and WCLs were immunoblotted with indicated antibodies. (C and D) WCLs of 293T cells transfected with HA-p300 and various Klf4 truncation or K-to-R substitution mutants were subjected to immunoprecipitation with Flag antibody. The Flag-IPs and WCLs were immunoblotted with indicated antibodies. WCLs, whole cell lysates; IP, immunoprecipitation.

    Techniques Used: In Vivo, Generated, Transfection, Immunoprecipitation

    Mapping of Oct4 acetylation sites in vivo. (A) Schematic illustration of a series of generated Oct4 truncation mutations. (B and C) WCLs of 293T cells transfected with HA-p300 and various Oct4 truncation mutants were subjected to immunoprecipitation with Flag antibody. The Flag-IPs and WCLs were immunoblotted with indicated antibodies. (D) WCLs of 293T cells transfected with HA-p300 and various Oct4 K-to-R substitution mutants were subjected to immunoprecipitation with Flag antibody. The Flag-IPs and WCLs were immunoblotted with indicated antibodies. WCLs, whole cell lysates; IP, immunoprecipitation.
    Figure Legend Snippet: Mapping of Oct4 acetylation sites in vivo. (A) Schematic illustration of a series of generated Oct4 truncation mutations. (B and C) WCLs of 293T cells transfected with HA-p300 and various Oct4 truncation mutants were subjected to immunoprecipitation with Flag antibody. The Flag-IPs and WCLs were immunoblotted with indicated antibodies. (D) WCLs of 293T cells transfected with HA-p300 and various Oct4 K-to-R substitution mutants were subjected to immunoprecipitation with Flag antibody. The Flag-IPs and WCLs were immunoblotted with indicated antibodies. WCLs, whole cell lysates; IP, immunoprecipitation.

    Techniques Used: In Vivo, Generated, Transfection, Immunoprecipitation

    Mapping of Sox2 acetylation sites in vivo. (A) Schematic illustration of a series of generated Sox2 truncation mutations. (B) WCLs of 293T cells transfected with various Sox2 mutants were subjected to immunoprecipitation with Flag antibody. The Flag-IPs and WCLs were immunoblotted with Ac-K and Flag antibodies. (C) WCLs of 293T cells transfected with various Sox2 K-to-R substitution mutants were subjected to immunoprecipitation with Flag antibody. The Flag-IPs and WCLs were immunoblotted with Ac-K and Flag antibodies. (D) WCLs of 293T cells transfected with HA-p300 and various Sox2 truncation mutants were subjected to immunoprecipitation with Flag antibody. The Flag-IPs and WCLs were immunoblotted with Ac-K and Flag antibodies. WCLs, whole cell lysates; IP, immunoprecipitation.
    Figure Legend Snippet: Mapping of Sox2 acetylation sites in vivo. (A) Schematic illustration of a series of generated Sox2 truncation mutations. (B) WCLs of 293T cells transfected with various Sox2 mutants were subjected to immunoprecipitation with Flag antibody. The Flag-IPs and WCLs were immunoblotted with Ac-K and Flag antibodies. (C) WCLs of 293T cells transfected with various Sox2 K-to-R substitution mutants were subjected to immunoprecipitation with Flag antibody. The Flag-IPs and WCLs were immunoblotted with Ac-K and Flag antibodies. (D) WCLs of 293T cells transfected with HA-p300 and various Sox2 truncation mutants were subjected to immunoprecipitation with Flag antibody. The Flag-IPs and WCLs were immunoblotted with Ac-K and Flag antibodies. WCLs, whole cell lysates; IP, immunoprecipitation.

    Techniques Used: In Vivo, Generated, Transfection, Immunoprecipitation

    Oct4 and Klf4 are acetylated by p300 in vivo. (A) Whole cell lysates (WCLs) of 293T cells transfected with HA-tagged p300 or CBP with indicated Flag-tagged constructs were subjected to immunoprecipitation (IP) with Flag antibody. The Flag-IPs and WCLs were immunoblotted with indicated antibodies. Acetylation was detected by a Lys-acetylation (Ac-K) antibody. (B) WCLs of 293T cells transfected with indicated constructs were subjected to immunoprecipitation with Flag antibody. The Flag-IPs and WCLs were immunoblotted with indicated antibodies. (C) WCLs of 293T cells transfected with indicated constructs were subjected to immunoprecipitation with HA antibody. The HA-IPs and WCLs were immunoblotted with Flag and HA antibodies.
    Figure Legend Snippet: Oct4 and Klf4 are acetylated by p300 in vivo. (A) Whole cell lysates (WCLs) of 293T cells transfected with HA-tagged p300 or CBP with indicated Flag-tagged constructs were subjected to immunoprecipitation (IP) with Flag antibody. The Flag-IPs and WCLs were immunoblotted with indicated antibodies. Acetylation was detected by a Lys-acetylation (Ac-K) antibody. (B) WCLs of 293T cells transfected with indicated constructs were subjected to immunoprecipitation with Flag antibody. The Flag-IPs and WCLs were immunoblotted with indicated antibodies. (C) WCLs of 293T cells transfected with indicated constructs were subjected to immunoprecipitation with HA antibody. The HA-IPs and WCLs were immunoblotted with Flag and HA antibodies.

    Techniques Used: In Vivo, Transfection, Construct, Immunoprecipitation

    15) Product Images from "E3 Ligase Activity of XIAP RING Domain Is Required for XIAP-Mediated Cancer Cell Migration, but Not for Its RhoGDI Binding Activity"

    Article Title: E3 Ligase Activity of XIAP RING Domain Is Required for XIAP-Mediated Cancer Cell Migration, but Not for Its RhoGDI Binding Activity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0035682

    RhoGDI was involved in XIAP regulation of cell migration and actin polymerization. (A), Lysates from WT and XIAP −/− HCT116 cells were Co-immunoprecipitated with anti-XIAP (mouse) antibody or normal mouse IgG, and immunoprecipitates were then subjected to immunoblotting with anti-RhoGDI (rabbit) or anti-XIAP (rabbit) antibodies. Five percent of lysates was used as input. (B), XIAP −/− (HA-XIAP) cells were transiently transfected with the GFP-RhoGDI or empty vector, GFTP-Vector. Co-immunoprecipitation was performed with anti-GFP antibody-conjugated agarose beads. Immunoprecipitates were then subjected to immunoblotting using antibodies as indicated. (C). Stable transfectants of shRNA-RhoGDI in WT and XIAP −/− cells were identified. Cell migration was determined by wound healing assays at the indicated times between Non-silencing and shRNA-RhoGDI transfectants in WT and XIAP −/− cells respectively. The wound area was quantified using Cell Migration Analysis software, and the quantitative data was shown as indicated (error bar represent S.D, n = 3). The asterisk (*) indicates a significant difference between the indicated cell lines ( p
    Figure Legend Snippet: RhoGDI was involved in XIAP regulation of cell migration and actin polymerization. (A), Lysates from WT and XIAP −/− HCT116 cells were Co-immunoprecipitated with anti-XIAP (mouse) antibody or normal mouse IgG, and immunoprecipitates were then subjected to immunoblotting with anti-RhoGDI (rabbit) or anti-XIAP (rabbit) antibodies. Five percent of lysates was used as input. (B), XIAP −/− (HA-XIAP) cells were transiently transfected with the GFP-RhoGDI or empty vector, GFTP-Vector. Co-immunoprecipitation was performed with anti-GFP antibody-conjugated agarose beads. Immunoprecipitates were then subjected to immunoblotting using antibodies as indicated. (C). Stable transfectants of shRNA-RhoGDI in WT and XIAP −/− cells were identified. Cell migration was determined by wound healing assays at the indicated times between Non-silencing and shRNA-RhoGDI transfectants in WT and XIAP −/− cells respectively. The wound area was quantified using Cell Migration Analysis software, and the quantitative data was shown as indicated (error bar represent S.D, n = 3). The asterisk (*) indicates a significant difference between the indicated cell lines ( p

    Techniques Used: Migration, Immunoprecipitation, Transfection, Plasmid Preparation, shRNA, Software

    XIAP RING Domain was Responsible for RhoGDI Interaction Independent on its E3 Ligase Activity. (A), XIAP −/− cells were transfected with GFP-RhoGDI, along with HA-XIAP, HA-XIAP H467A, HA-XIAP ΔRING, or HA-XIAP ΔBIR. Co-immunoprecipitation was performed with anti-GFP antibody-conjugated agarose beads. Immunoprecipitates were then subjected to immunoblotting for detection of XIAP using HA antibody. (B). WT(Vector), XIAP −/− (Vector) and XIAP −/− (HA-XIAP) HCT116 cells were transfected with constructs of GFP-RhoGDI in combination with Ubiquitin-WT, Ubiquitin-K48R, Ubiquitin-K63R or Ubiquitin-K48R/K63R (KKRR). Forty-eight hours after transfection, cells were lysed and co-immunoprecipitated with anti-GFP antibody, and then immunoblotted with anti-Ub and anti-GFP antibodies. (C), 293T cells were transfected with various constructs as indicated for detection of RhoGDI ubiquitination by anti-Ub and anti-GFP antibodies. (D), A model for XIAP-regulated modulation of cell motility: XIAP binds to RhoGDI through its RING domain and inhibits RhoGDI SUMOylation which results in down-regulation of RhoGDI's function and promotes actin polymerization and cell motility. Or E3 ligase activity of XIAP RING domain could regulate some un-verified factors which subsequently control cell migration independent of RhoGDI binding.
    Figure Legend Snippet: XIAP RING Domain was Responsible for RhoGDI Interaction Independent on its E3 Ligase Activity. (A), XIAP −/− cells were transfected with GFP-RhoGDI, along with HA-XIAP, HA-XIAP H467A, HA-XIAP ΔRING, or HA-XIAP ΔBIR. Co-immunoprecipitation was performed with anti-GFP antibody-conjugated agarose beads. Immunoprecipitates were then subjected to immunoblotting for detection of XIAP using HA antibody. (B). WT(Vector), XIAP −/− (Vector) and XIAP −/− (HA-XIAP) HCT116 cells were transfected with constructs of GFP-RhoGDI in combination with Ubiquitin-WT, Ubiquitin-K48R, Ubiquitin-K63R or Ubiquitin-K48R/K63R (KKRR). Forty-eight hours after transfection, cells were lysed and co-immunoprecipitated with anti-GFP antibody, and then immunoblotted with anti-Ub and anti-GFP antibodies. (C), 293T cells were transfected with various constructs as indicated for detection of RhoGDI ubiquitination by anti-Ub and anti-GFP antibodies. (D), A model for XIAP-regulated modulation of cell motility: XIAP binds to RhoGDI through its RING domain and inhibits RhoGDI SUMOylation which results in down-regulation of RhoGDI's function and promotes actin polymerization and cell motility. Or E3 ligase activity of XIAP RING domain could regulate some un-verified factors which subsequently control cell migration independent of RhoGDI binding.

    Techniques Used: Activity Assay, Transfection, Immunoprecipitation, Plasmid Preparation, Construct, Migration, Binding Assay

    16) Product Images from "Missense mutations near the N-glycosylation site of the A2 domain lead to various intracellular trafficking defects in coagulation factor VIII"

    Article Title: Missense mutations near the N-glycosylation site of the A2 domain lead to various intracellular trafficking defects in coagulation factor VIII

    Journal: Scientific Reports

    doi: 10.1038/srep45033

    Glycosylation status of the A2 domain with missense mutations adjacent to N582 (D580 to W585). ( A ) The amino acid residues of D580 to S584 form a short 3 10 -helix in the structure of B domain deleted FVIII heterodimer. The right side of the panel depicts a hydrogen-bonding network mediated by the sidechains of the residues that comprise the 3 10 helix. W585 is also highlighted on the right panel. Residues and secondary structure elements outside the helix are colored green. The FVIII structure is based on entry 2R7E in the Protein DataBank 35 . ( B ) Extracts of COS-1 cells transiently transfected with constructs expressing WT A2 and the indicated A2 mutants were collected at 36 h after transfection and analyzed by 10% SDS-PAGE and immunoblotting. Arrowhead indicates glycosylated A2 domain and asterisk indicates non-glycosylated A2 domain. Protein levels in the media were detected by immunoprecipitation with an anti-Flag antibody followed by immunoblotting. ( C ) Extracts of COS-1 cells transiently transfected with constructs expressing WT A2 and the indicated A2 mutants were collected at 36 h after transfection and analyzed by 10% SDS-PAGE and immunoblotting. ( D ) Cell extracts of D580H and S584T mutants were treated with PNGase F and analyzed by immunoblotting. ( E ) The N582D mutation was introduced to the D580H and S584T mutants. Cell extracts expressing single or double mutants were analyzed by immunoblotting. Arrowhead denotes N-glycosylated forms and asterisk denotes non-glycosylated form of the A2 domain. All experiments were carried out independently 2–3 times.
    Figure Legend Snippet: Glycosylation status of the A2 domain with missense mutations adjacent to N582 (D580 to W585). ( A ) The amino acid residues of D580 to S584 form a short 3 10 -helix in the structure of B domain deleted FVIII heterodimer. The right side of the panel depicts a hydrogen-bonding network mediated by the sidechains of the residues that comprise the 3 10 helix. W585 is also highlighted on the right panel. Residues and secondary structure elements outside the helix are colored green. The FVIII structure is based on entry 2R7E in the Protein DataBank 35 . ( B ) Extracts of COS-1 cells transiently transfected with constructs expressing WT A2 and the indicated A2 mutants were collected at 36 h after transfection and analyzed by 10% SDS-PAGE and immunoblotting. Arrowhead indicates glycosylated A2 domain and asterisk indicates non-glycosylated A2 domain. Protein levels in the media were detected by immunoprecipitation with an anti-Flag antibody followed by immunoblotting. ( C ) Extracts of COS-1 cells transiently transfected with constructs expressing WT A2 and the indicated A2 mutants were collected at 36 h after transfection and analyzed by 10% SDS-PAGE and immunoblotting. ( D ) Cell extracts of D580H and S584T mutants were treated with PNGase F and analyzed by immunoblotting. ( E ) The N582D mutation was introduced to the D580H and S584T mutants. Cell extracts expressing single or double mutants were analyzed by immunoblotting. Arrowhead denotes N-glycosylated forms and asterisk denotes non-glycosylated form of the A2 domain. All experiments were carried out independently 2–3 times.

    Techniques Used: Transfection, Construct, Expressing, SDS Page, Immunoprecipitation, Mutagenesis

    The A2 domain is not N-glycosylated and N582 mutations result in defective secretion of the A2 domain. ( A ) COS-1 cells were transfected with constructs that express Flag-tagged individual domains (A1, A2, A3 and C) of FVIII. Cell lysates were digested with PNGase F or left untreated, and subsequently immunoblotted with an anti-Flag antibody. ( B ) COS-1 cells were transfected with individual FVIII domain constructs in duplicates. One set of transfected cells were treated with 2 μg/ml tunicamycin for 12 h before lysis. ( C ) COS-1 cells were transfected with a construct expressing WT A2 domain or one of three different A2 domain mutants. A fresh medium change was carried out thirty hours after transfection. At the indicated time after the change of medium, conditioned media were collected and immunoprecipitated with a mouse monoclonal anti-Flag antibody and analyzed by immunoblotting with a rabbit anti-Flag antibody. Thirty-six hours after transfection, cells were lysed and equal amounts of cell extracts were immunoblotted with anti-Flag and anti-β-actin antibodies. ( D ) Six hours after transfection, COS-1 cells were cultured at 28 °C for 24 hours before fresh medium change. Conditioned media were collected at 2 and 4 h and secreted A2 protein was analyzed by immunoprecipitation and immunoblotting. Thirty-six hours after transfection, cells were lysed and equal amounts of cell extracts were immunoblotted with anti-Flag and anti-β-actin antibodies. All experiments were performed independently twice.
    Figure Legend Snippet: The A2 domain is not N-glycosylated and N582 mutations result in defective secretion of the A2 domain. ( A ) COS-1 cells were transfected with constructs that express Flag-tagged individual domains (A1, A2, A3 and C) of FVIII. Cell lysates were digested with PNGase F or left untreated, and subsequently immunoblotted with an anti-Flag antibody. ( B ) COS-1 cells were transfected with individual FVIII domain constructs in duplicates. One set of transfected cells were treated with 2 μg/ml tunicamycin for 12 h before lysis. ( C ) COS-1 cells were transfected with a construct expressing WT A2 domain or one of three different A2 domain mutants. A fresh medium change was carried out thirty hours after transfection. At the indicated time after the change of medium, conditioned media were collected and immunoprecipitated with a mouse monoclonal anti-Flag antibody and analyzed by immunoblotting with a rabbit anti-Flag antibody. Thirty-six hours after transfection, cells were lysed and equal amounts of cell extracts were immunoblotted with anti-Flag and anti-β-actin antibodies. ( D ) Six hours after transfection, COS-1 cells were cultured at 28 °C for 24 hours before fresh medium change. Conditioned media were collected at 2 and 4 h and secreted A2 protein was analyzed by immunoprecipitation and immunoblotting. Thirty-six hours after transfection, cells were lysed and equal amounts of cell extracts were immunoblotted with anti-Flag and anti-β-actin antibodies. All experiments were performed independently twice.

    Techniques Used: Transfection, Construct, Lysis, Expressing, Immunoprecipitation, Cell Culture

    17) Product Images from "Centrosome-intrinsic mechanisms modulate centrosome integrity during fever"

    Article Title: Centrosome-intrinsic mechanisms modulate centrosome integrity during fever

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E15-03-0158

    The centrosome is a substrate organelle for molecular chaperone Hsp70. (See also Supplemental Figures S1–S3.) (A) Left, images of maximum projections from cntrl and HS cells as indicated show that upon HS, Hsp70 accumulates at the centrosome and in the nucleus (inset, arrow points at the centrosome); bar, 10 μm. Semiquantitative analysis of integrated intensity (×10 5 arbitrary units) of centrosomal Hsp70 before and after HS (15–20 centrosomes/sample, mean ± SEM). (B) Superresolution images (OMX) demonstrate loss of γ-tubulin (red) from HS cells and recruitment of Hsp70 (green) as an outer layer in stressed cells; bar, 0.2 μm. (C) Immunoprecipitation (IP) of Hsp70 pulls down PCNT. Immunoglobulin G (IgG), control. (D) Centrosome-targeted Hsp70 (cHsp70) protects γ-tubulin from HS, whereas membrane-targeted Hsp70 (mHsp70) does not. Data are shown as semiquantitative profiles of confocal microscopy images. Right, percentage of cells with centrosomal γ-tubulin after HS in stably expressing centrosome-(cHsp70) or membrane-(mHsp70) targeted Hsp70-expressing cells (four experiments, 500–600 cells/sample, mean ± SD, one-way analysis of variance [ANOVA] combined with Tukey’s multiple comparison test). (E) cHsp70, but not the chaperone-negative mutant cHsp70D10S, protects γ-tubulin signal at the centrosome; semiquantitative analysis of integrated γ-tubulin signal intensity (×10 5 arbitrary units, mean ± SEM). (F) cHsp70 protects centrosomal Centrin2 from HS, whereas mHsp70 does not. Data are shown as semiquantitative profiles of confocal microscopy images. Right, percentage of cells with centrosome-localized Centrin2 after HS in stably expressing cHsp70- or mHsp70-targeted Hsp70 cells (three experiments, 500–600 cells/sample, mean ± SD, one-way ANOVA combined with Tukey’s multiple comparison test).
    Figure Legend Snippet: The centrosome is a substrate organelle for molecular chaperone Hsp70. (See also Supplemental Figures S1–S3.) (A) Left, images of maximum projections from cntrl and HS cells as indicated show that upon HS, Hsp70 accumulates at the centrosome and in the nucleus (inset, arrow points at the centrosome); bar, 10 μm. Semiquantitative analysis of integrated intensity (×10 5 arbitrary units) of centrosomal Hsp70 before and after HS (15–20 centrosomes/sample, mean ± SEM). (B) Superresolution images (OMX) demonstrate loss of γ-tubulin (red) from HS cells and recruitment of Hsp70 (green) as an outer layer in stressed cells; bar, 0.2 μm. (C) Immunoprecipitation (IP) of Hsp70 pulls down PCNT. Immunoglobulin G (IgG), control. (D) Centrosome-targeted Hsp70 (cHsp70) protects γ-tubulin from HS, whereas membrane-targeted Hsp70 (mHsp70) does not. Data are shown as semiquantitative profiles of confocal microscopy images. Right, percentage of cells with centrosomal γ-tubulin after HS in stably expressing centrosome-(cHsp70) or membrane-(mHsp70) targeted Hsp70-expressing cells (four experiments, 500–600 cells/sample, mean ± SD, one-way analysis of variance [ANOVA] combined with Tukey’s multiple comparison test). (E) cHsp70, but not the chaperone-negative mutant cHsp70D10S, protects γ-tubulin signal at the centrosome; semiquantitative analysis of integrated γ-tubulin signal intensity (×10 5 arbitrary units, mean ± SEM). (F) cHsp70 protects centrosomal Centrin2 from HS, whereas mHsp70 does not. Data are shown as semiquantitative profiles of confocal microscopy images. Right, percentage of cells with centrosome-localized Centrin2 after HS in stably expressing cHsp70- or mHsp70-targeted Hsp70 cells (three experiments, 500–600 cells/sample, mean ± SD, one-way ANOVA combined with Tukey’s multiple comparison test).

    Techniques Used: Immunoprecipitation, Confocal Microscopy, Stable Transfection, Expressing, Mutagenesis

    18) Product Images from "Macitentan inhibits the transforming growth factor-β profibrotic action, blocking the signaling mediated by the ETR/TβRI complex in systemic sclerosis dermal fibroblasts"

    Article Title: Macitentan inhibits the transforming growth factor-β profibrotic action, blocking the signaling mediated by the ETR/TβRI complex in systemic sclerosis dermal fibroblasts

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-015-0754-7

    Macitentan (MAC) blocked transforming growth factor beta type I receptor (TβRI) activation. a Protein expression of phospho-Sma and Mad Related (pSMAD)1/5 and pSMAD2/3. Transforming growth factor beta (TGF-β) and endothelin-1 (ET-1) effects induced a significant increase of both SMAD2/3 and SMAD1/5 phosphorylation. MAC significantly blocked both TGF-β and ET-1 effects. The phospho-SMAD levels were significantly higher in systemic sclerosis (SSc) patients. Pictures are representative of all experiments. Protein bands were quantified by densitometry and the values are expressed as protein relative quantification/β actin relative quantification. b TβRI was immunoprecipitated (IP) and its phosphorylation was assessed by Western blot (WB). The immunoprecipitation assay showed a serine phosphorylation in TβRI after TGF-β treatment. MAC significantly inhibited the TβRI phosphorylation. Immunoprecipitated protein bands were quantified by densitometry and the values are expressed as protein relative quantification/TβRI relative quantification. ** p = 0.0002, *** p = 0.0001. UT untreated
    Figure Legend Snippet: Macitentan (MAC) blocked transforming growth factor beta type I receptor (TβRI) activation. a Protein expression of phospho-Sma and Mad Related (pSMAD)1/5 and pSMAD2/3. Transforming growth factor beta (TGF-β) and endothelin-1 (ET-1) effects induced a significant increase of both SMAD2/3 and SMAD1/5 phosphorylation. MAC significantly blocked both TGF-β and ET-1 effects. The phospho-SMAD levels were significantly higher in systemic sclerosis (SSc) patients. Pictures are representative of all experiments. Protein bands were quantified by densitometry and the values are expressed as protein relative quantification/β actin relative quantification. b TβRI was immunoprecipitated (IP) and its phosphorylation was assessed by Western blot (WB). The immunoprecipitation assay showed a serine phosphorylation in TβRI after TGF-β treatment. MAC significantly inhibited the TβRI phosphorylation. Immunoprecipitated protein bands were quantified by densitometry and the values are expressed as protein relative quantification/TβRI relative quantification. ** p = 0.0002, *** p = 0.0001. UT untreated

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

    Transforming growth factor beta type I receptor (TβRI)/endothelin-1 receptor A (ETAR) co-immunoprecipitation. a TβRI was immunoprecipitated (IP) and its association with ETAR was assessed by Western blot (WB). The immunoprecipitation assay showed an association between ETAR and TβRI, independent of both transforming growth factor beta (TGF-β) and endothelin-1 (ET-1) stimulation. In systemic sclerosis (SSc) fibroblasts (FBs), the levels of co-immunoprecipitated receptors were significantly higher when compared with healthy control (HC) FBs. Blot was representative of all the experiments. Co-immunoprecipitated protein bands were quantified by densitometry and the values are expressed as protein relative quantification/TβRI relative quantification. b Western blot of ETAR protein. In SSc FBs, ETAR expression was significantly higher when compared with HC FBs. Blot was representative of all the experiments. The protein bands were quantified by densitometry and the values are expressed as protein relative quantification/β actin relative quantification. c SSc FBs were transfected with specific ETAR siRNA or nontargeting scramble scr-siRNA, and ETAR expression was evaluated by quantitative RT-PCR. The cells transfected with ETAR siRNA showed a decreased expression of ETAR gene, when compared with cells transfected with scr-siRNA. d Western blot of phospho-Sma and Mad Related (pSMAD)1/5 and pSMAD2/3. In SSc FBs treated with scr-siRNA, TGF-β induced a significant increase in SMAD phosphorylation, and macitentan (MAC) inhibited this effect. In SSc FBs treated with ETAR siRNA, TGF-β induced a significant increase in SMAD phosphorylation, and MAC failed to inhibit this effect. Pictures are representative of all experiments. Protein bands were quantified by densitometry and the values were expressed as protein relative quantification/β actin relative quantification. ** p = 0.0002, *** p = 0.0001. UT untreated
    Figure Legend Snippet: Transforming growth factor beta type I receptor (TβRI)/endothelin-1 receptor A (ETAR) co-immunoprecipitation. a TβRI was immunoprecipitated (IP) and its association with ETAR was assessed by Western blot (WB). The immunoprecipitation assay showed an association between ETAR and TβRI, independent of both transforming growth factor beta (TGF-β) and endothelin-1 (ET-1) stimulation. In systemic sclerosis (SSc) fibroblasts (FBs), the levels of co-immunoprecipitated receptors were significantly higher when compared with healthy control (HC) FBs. Blot was representative of all the experiments. Co-immunoprecipitated protein bands were quantified by densitometry and the values are expressed as protein relative quantification/TβRI relative quantification. b Western blot of ETAR protein. In SSc FBs, ETAR expression was significantly higher when compared with HC FBs. Blot was representative of all the experiments. The protein bands were quantified by densitometry and the values are expressed as protein relative quantification/β actin relative quantification. c SSc FBs were transfected with specific ETAR siRNA or nontargeting scramble scr-siRNA, and ETAR expression was evaluated by quantitative RT-PCR. The cells transfected with ETAR siRNA showed a decreased expression of ETAR gene, when compared with cells transfected with scr-siRNA. d Western blot of phospho-Sma and Mad Related (pSMAD)1/5 and pSMAD2/3. In SSc FBs treated with scr-siRNA, TGF-β induced a significant increase in SMAD phosphorylation, and macitentan (MAC) inhibited this effect. In SSc FBs treated with ETAR siRNA, TGF-β induced a significant increase in SMAD phosphorylation, and MAC failed to inhibit this effect. Pictures are representative of all experiments. Protein bands were quantified by densitometry and the values were expressed as protein relative quantification/β actin relative quantification. ** p = 0.0002, *** p = 0.0001. UT untreated

    Techniques Used: Immunoprecipitation, Western Blot, Expressing, Transfection, Quantitative RT-PCR

    19) Product Images from "Small heterodimer partner interacts with NLRP3 and negatively regulates activation of the NLRP3 inflammasome"

    Article Title: Small heterodimer partner interacts with NLRP3 and negatively regulates activation of the NLRP3 inflammasome

    Journal: Nature Communications

    doi: 10.1038/ncomms7115

    SHP interacts with NLRP3 during inflammasome activation. ( a ) Small heterodimer partner (SHP) complexes purified from lipopolysaccharide (LPS)-primed bone marrow-derived macrophages (BMDMs) with or without adenosine triphosphate (ATP, 1 mM) stimulation were subjected to mass spectrometry analysis. Red letters indicate the peptides identified. ( b ) Lipopolysaccharide (LPS)-primed BMDMs were stimulated with ATP for the indicated durations, and subjected to co-immunoprecipitation (co-IP) with antibodies for SHP (left) or NOD-like receptor family, pyrin domain-containing 3 (NLRP3; right), followed by immunoblotting (IB) with antibodies for NLRP3, apoptosis-associated speck-like protein containing a carboxy-terminal CARD (ASC), SHP and actin. ( c ) LPS-primed BMDMs from SHP +/+ and SHP −/− mice were stimulated with ATP for 30 min, fixed, immunostained with antibodies for SHP (Alexa 488) and NLRP3 (Alexa 568), and counterstained with DAPI (blue). Upper immunofluorescence images are representative of three independent replicates; scale bar, 10 μm. ( d ) Schematic diagram of the structures of SHP (Left) and NLRP3 (Right). ( e – g ) 293T cells were co-transfected with a control vector, Flag-NLRP3 or truncated mutants (ΔPYD, ΔNACHT, ΔLRR), together with V5-SHP or its mutants (N-terminal or C-terminal). Cells were subjected to co-IP with antibodies for V5 ( e ) or Flag ( f , g ), followed by IB with antibodies for Flag or V5. ( h ) Schematic diagram of N-terminal deletions of SHP structure. ‘--’ indicates a deleted sequence. ( i ) 293T cells were co-transfected with the indicated constructs, and subjected to co-IP with anti-Flag, followed by IB analysis with antibodies for GFP or Flag. ( j , k ) 293T cells were co-transfected with Flag-NLRP3 or AU1-ASC, together with increasing amounts of V5-SHP, and subjected to co-IP with antibodies for Flag ( j ) or AU1 ( k ), followed by IB analysis with antibodies for Flag, AU1 or V5. Data are representative of at least three independent experiments ( a – c , e – g and i – k ). Protein levels in cell lysates were determined by IB analysis ( e – g and i – k ). U, untreated control.
    Figure Legend Snippet: SHP interacts with NLRP3 during inflammasome activation. ( a ) Small heterodimer partner (SHP) complexes purified from lipopolysaccharide (LPS)-primed bone marrow-derived macrophages (BMDMs) with or without adenosine triphosphate (ATP, 1 mM) stimulation were subjected to mass spectrometry analysis. Red letters indicate the peptides identified. ( b ) Lipopolysaccharide (LPS)-primed BMDMs were stimulated with ATP for the indicated durations, and subjected to co-immunoprecipitation (co-IP) with antibodies for SHP (left) or NOD-like receptor family, pyrin domain-containing 3 (NLRP3; right), followed by immunoblotting (IB) with antibodies for NLRP3, apoptosis-associated speck-like protein containing a carboxy-terminal CARD (ASC), SHP and actin. ( c ) LPS-primed BMDMs from SHP +/+ and SHP −/− mice were stimulated with ATP for 30 min, fixed, immunostained with antibodies for SHP (Alexa 488) and NLRP3 (Alexa 568), and counterstained with DAPI (blue). Upper immunofluorescence images are representative of three independent replicates; scale bar, 10 μm. ( d ) Schematic diagram of the structures of SHP (Left) and NLRP3 (Right). ( e – g ) 293T cells were co-transfected with a control vector, Flag-NLRP3 or truncated mutants (ΔPYD, ΔNACHT, ΔLRR), together with V5-SHP or its mutants (N-terminal or C-terminal). Cells were subjected to co-IP with antibodies for V5 ( e ) or Flag ( f , g ), followed by IB with antibodies for Flag or V5. ( h ) Schematic diagram of N-terminal deletions of SHP structure. ‘--’ indicates a deleted sequence. ( i ) 293T cells were co-transfected with the indicated constructs, and subjected to co-IP with anti-Flag, followed by IB analysis with antibodies for GFP or Flag. ( j , k ) 293T cells were co-transfected with Flag-NLRP3 or AU1-ASC, together with increasing amounts of V5-SHP, and subjected to co-IP with antibodies for Flag ( j ) or AU1 ( k ), followed by IB analysis with antibodies for Flag, AU1 or V5. Data are representative of at least three independent experiments ( a – c , e – g and i – k ). Protein levels in cell lysates were determined by IB analysis ( e – g and i – k ). U, untreated control.

    Techniques Used: Activation Assay, Purification, Derivative Assay, Mass Spectrometry, Immunoprecipitation, Co-Immunoprecipitation Assay, Mouse Assay, Immunofluorescence, Transfection, Plasmid Preparation, Sequencing, Construct

    20) Product Images from "OncomiR Addiction Is Generated by a miR-155 Feedback Loop in Theileria-Transformed Leukocytes"

    Article Title: OncomiR Addiction Is Generated by a miR-155 Feedback Loop in Theileria-Transformed Leukocytes

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003222

    miR-155 stabilized c-Jun by inhibiting its proteasomal degradation. (A) Overexpression of miR-155 or depletion of DET1 in BL3 cells increased the half-life of endogenous c-Jun protein. BL3 cells transiently expressing miR-155 or siDET1 were treated with cycloheximide for the indicated times, followed by immunoblot analysis with a c-Jun antibody and semi-quantification with an αTubulin antibody as a loading control. Relative c-Jun protein levels at time 0 were set as 1 (average ± sd, n = 3). (B) Inhibition by the miR-155 Sponge in TBL3 cells decreased the half-life of endogenous c-Jun. These effects were reversed by siRNA against DET1. TBL3 cells transiently expressing miR-155 Sponge +/− siDET1 were treated with cycloheximide for the indicated times, followed by immunoblot analysis with a c-Jun antibody and semiquantification with α-Tubulin as a loading control. Relative c-Jun levels at time 0 were set as 1 (average ± sd, n = 3). (C) Effect of the miR-155 Sponge on c-Jun protein levels was rescued by treating the proteasome inhibitor MG132. TBL3 cells transiently expressing the miR-155 Sponge were treated with MG132 for 3 h, followed by immunoblot analysis with the c-Jun antibody and semiquantification with α Tubulin as a loading control (average ± sd, n = 3). (D) Overexpression of miR-155 or depletion of DET1 in BL3 cells reduced c-Jun ubiquitination. Transfected cells were treated with MG132 for 3 h, followed by endogenous c-Jun immunoprecipitation and immunoblot analysis with indicated antibodies (average ± sd, n = 3). *p
    Figure Legend Snippet: miR-155 stabilized c-Jun by inhibiting its proteasomal degradation. (A) Overexpression of miR-155 or depletion of DET1 in BL3 cells increased the half-life of endogenous c-Jun protein. BL3 cells transiently expressing miR-155 or siDET1 were treated with cycloheximide for the indicated times, followed by immunoblot analysis with a c-Jun antibody and semi-quantification with an αTubulin antibody as a loading control. Relative c-Jun protein levels at time 0 were set as 1 (average ± sd, n = 3). (B) Inhibition by the miR-155 Sponge in TBL3 cells decreased the half-life of endogenous c-Jun. These effects were reversed by siRNA against DET1. TBL3 cells transiently expressing miR-155 Sponge +/− siDET1 were treated with cycloheximide for the indicated times, followed by immunoblot analysis with a c-Jun antibody and semiquantification with α-Tubulin as a loading control. Relative c-Jun levels at time 0 were set as 1 (average ± sd, n = 3). (C) Effect of the miR-155 Sponge on c-Jun protein levels was rescued by treating the proteasome inhibitor MG132. TBL3 cells transiently expressing the miR-155 Sponge were treated with MG132 for 3 h, followed by immunoblot analysis with the c-Jun antibody and semiquantification with α Tubulin as a loading control (average ± sd, n = 3). (D) Overexpression of miR-155 or depletion of DET1 in BL3 cells reduced c-Jun ubiquitination. Transfected cells were treated with MG132 for 3 h, followed by endogenous c-Jun immunoprecipitation and immunoblot analysis with indicated antibodies (average ± sd, n = 3). *p

    Techniques Used: Over Expression, Expressing, Inhibition, Transfection, Immunoprecipitation

    21) Product Images from "De-acetylation and degradation of HSPA5 is critical for E1A metastasis suppression in breast cancer cells"

    Article Title: De-acetylation and degradation of HSPA5 is critical for E1A metastasis suppression in breast cancer cells

    Journal: Oncotarget

    doi:

    E1A decreases HSPA5 expression through GP78-mediated proteasomal degradation (A) Determination of the protein stability of HSPA5 in 231/V and 231/E1A cells. 231/V and 231/E1A cells were treated with 100 μg/ml cycloheximide (CHX) for the indicated times, and then following by Western blot analysis. Quantification of HSPA5 expression was performed three independent experiments using the Image J system and was normalized to the vehicle control. (B) 231/V and 231/E1A cells were treated with or without the proteasome inhibitor MG132 (5 μM) for 12 h, and HSPA5 expression was analyzed by Western blot analysis. The fold change in the protein expression is shown below the lanes, with the expression levels normalized to lane 1. (C) 231/V and 231/E1A cells were treated with the proteasome inhibitor MG132 (5 μM) for 12 h. Total cell lysates were prepared for in vivo ubiquitination assay. IgG was used as a control for the immunoprecipitation (IP) analysis. (D) 231/E1A cells were transfected with the indicated shRNAs, and HSPA5 expression was measured by Western blot analysis (top) and real-time RT-PCR (bottom). Data shown are mean ± s.e.m. of three independent experiments performed in triplicate. ** p
    Figure Legend Snippet: E1A decreases HSPA5 expression through GP78-mediated proteasomal degradation (A) Determination of the protein stability of HSPA5 in 231/V and 231/E1A cells. 231/V and 231/E1A cells were treated with 100 μg/ml cycloheximide (CHX) for the indicated times, and then following by Western blot analysis. Quantification of HSPA5 expression was performed three independent experiments using the Image J system and was normalized to the vehicle control. (B) 231/V and 231/E1A cells were treated with or without the proteasome inhibitor MG132 (5 μM) for 12 h, and HSPA5 expression was analyzed by Western blot analysis. The fold change in the protein expression is shown below the lanes, with the expression levels normalized to lane 1. (C) 231/V and 231/E1A cells were treated with the proteasome inhibitor MG132 (5 μM) for 12 h. Total cell lysates were prepared for in vivo ubiquitination assay. IgG was used as a control for the immunoprecipitation (IP) analysis. (D) 231/E1A cells were transfected with the indicated shRNAs, and HSPA5 expression was measured by Western blot analysis (top) and real-time RT-PCR (bottom). Data shown are mean ± s.e.m. of three independent experiments performed in triplicate. ** p

    Techniques Used: Expressing, Western Blot, In Vivo, Ubiquitin Assay, Immunoprecipitation, Transfection, Quantitative RT-PCR

    22) Product Images from "A genomic approach to the identification and characterization of HOXA13 functional binding elements"

    Article Title: A genomic approach to the identification and characterization of HOXA13 functional binding elements

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki979

    Characterization of HOXA13-FLAG/EGFP or HOXD13-FLAG/EGFP expressing cells. ( A ) Western blot using HOX-specific antibodies demonstrating HOXA13 expression and anti-FLAG immunoprecipitation from HOXA13-FLAG cell line and absent HOXA13 expression in the HOX (−) cell line ( 1 ); HOXD13-FLAG expression and anti-FLAG immunoprecipitation from HOXD13-FLAG cell line but absent HOXD13 expression in the HOX (−) cell line ( 2 ). ( B ) Immunocytochemistry using Hox specific antibodies and DAPI staining demonstrate expression and nuclear localization of HOXA13 or HOXD13 in their respective cell lines. ( C ) Input RNA using serial dilutions ranging from 156 pg to 10 ng was used in semi-quantitative RT–PCR to look for expression changes of four reported targets. Fhl1 (+6-fold), Enpp2 (+18.8-fold) and M32486 (+2.3) are upregulated in the HOXA13-FLAG (A) and HOXD13-FLAG (D) cell lines and Ngef (−2.4) is downregulated compared to HOX (−). Water (W) was used as a PCR control. Ppic was a loading control and was shown to be unchanged in the HOXA13-FLAG and HOXD13-FLAG cell lines.
    Figure Legend Snippet: Characterization of HOXA13-FLAG/EGFP or HOXD13-FLAG/EGFP expressing cells. ( A ) Western blot using HOX-specific antibodies demonstrating HOXA13 expression and anti-FLAG immunoprecipitation from HOXA13-FLAG cell line and absent HOXA13 expression in the HOX (−) cell line ( 1 ); HOXD13-FLAG expression and anti-FLAG immunoprecipitation from HOXD13-FLAG cell line but absent HOXD13 expression in the HOX (−) cell line ( 2 ). ( B ) Immunocytochemistry using Hox specific antibodies and DAPI staining demonstrate expression and nuclear localization of HOXA13 or HOXD13 in their respective cell lines. ( C ) Input RNA using serial dilutions ranging from 156 pg to 10 ng was used in semi-quantitative RT–PCR to look for expression changes of four reported targets. Fhl1 (+6-fold), Enpp2 (+18.8-fold) and M32486 (+2.3) are upregulated in the HOXA13-FLAG (A) and HOXD13-FLAG (D) cell lines and Ngef (−2.4) is downregulated compared to HOX (−). Water (W) was used as a PCR control. Ppic was a loading control and was shown to be unchanged in the HOXA13-FLAG and HOXD13-FLAG cell lines.

    Techniques Used: Expressing, Western Blot, Immunoprecipitation, Immunocytochemistry, Staining, Quantitative RT-PCR, Polymerase Chain Reaction

    23) Product Images from "PIM-induced phosphorylation of Notch3 promotes breast cancer tumorigenicity in a CSL-independent fashion"

    Article Title: PIM-induced phosphorylation of Notch3 promotes breast cancer tumorigenicity in a CSL-independent fashion

    Journal: The Journal of Biological Chemistry

    doi: 10.1016/j.jbc.2021.100593

    Phosphorylation inhibits CSL-dependent transactivation by N3ICD but promotes tumor growth. A, empty vector (C), GFP-tagged WT, SA, or SE N3ICD and FLAG-tagged CSL were transiently overexpressed in HeLa cells. Interactions of N3ICD and CSL were analyzed by GFP-trap immunoprecipitation, followed by Western blotting with GFP (CSL) and FLAG (N3ICD) antibodies. Shown are average CSL binding values relative to WT N3ICD. B and C, CSL-dependent transactivation assays were performed in MCF-7 cells, their NOTCH3 or CSL -deficient KO derivatives, or in T47D cells, which transiently overexpressed WT, phosphodeficient (SA), or phosphomimicking (SE) N3ICD. Luciferase activities were normalized according to β-galactosidase activities. Shown are averages relative to WT N3ICD from two independent experiments. D, CSL-dependent transactivation assays were also performed in MCF-7 cells transiently overexpressing N1ICD or N3ICD that were treated with DMSO or 10 μM DHPCC-9 for 16 h. E, endogenous NOTCH activity was measured in MCF-7 cells or their NOTCH1 (N1) or NOTCH3 (N3) KO derivatives plated on Delta-like 1 ligands (Dll1) and treated with DMSO, 10 μM PF03084014, or 10 μM AZD-1208. F, RT-quantitative PCR was performed to WT or N1KO MCF-7 cells transiently overexpressing WT or SA N3ICD. NOTCH target gene levels were normalized by NOTCH3 levels after UBC subtraction. Shown are average values relative to DMSO-treated samples. All experiments had three parallel samples. ∗ p
    Figure Legend Snippet: Phosphorylation inhibits CSL-dependent transactivation by N3ICD but promotes tumor growth. A, empty vector (C), GFP-tagged WT, SA, or SE N3ICD and FLAG-tagged CSL were transiently overexpressed in HeLa cells. Interactions of N3ICD and CSL were analyzed by GFP-trap immunoprecipitation, followed by Western blotting with GFP (CSL) and FLAG (N3ICD) antibodies. Shown are average CSL binding values relative to WT N3ICD. B and C, CSL-dependent transactivation assays were performed in MCF-7 cells, their NOTCH3 or CSL -deficient KO derivatives, or in T47D cells, which transiently overexpressed WT, phosphodeficient (SA), or phosphomimicking (SE) N3ICD. Luciferase activities were normalized according to β-galactosidase activities. Shown are averages relative to WT N3ICD from two independent experiments. D, CSL-dependent transactivation assays were also performed in MCF-7 cells transiently overexpressing N1ICD or N3ICD that were treated with DMSO or 10 μM DHPCC-9 for 16 h. E, endogenous NOTCH activity was measured in MCF-7 cells or their NOTCH1 (N1) or NOTCH3 (N3) KO derivatives plated on Delta-like 1 ligands (Dll1) and treated with DMSO, 10 μM PF03084014, or 10 μM AZD-1208. F, RT-quantitative PCR was performed to WT or N1KO MCF-7 cells transiently overexpressing WT or SA N3ICD. NOTCH target gene levels were normalized by NOTCH3 levels after UBC subtraction. Shown are average values relative to DMSO-treated samples. All experiments had three parallel samples. ∗ p

    Techniques Used: Plasmid Preparation, Immunoprecipitation, Western Blot, Binding Assay, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction

    24) Product Images from "Cdx2 homeoprotein inhibits non-homologous end joining in colon cancer but not in leukemia cells"

    Article Title: Cdx2 homeoprotein inhibits non-homologous end joining in colon cancer but not in leukemia cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr1242

    The homeodomain of Cdx2 is necessary for its interaction with Ku70/Ku80. ( A ) Schematic representation of the different Cdx2 deletion mutants tested. TAD: transactivator domain; NLS: nuclear localization signal; HD: homeodomain; SD: stabilization domain. The upper numbers represents the position of the different domains in the 313aa long Cdx2 protein. ( B ) Coimmunoprecipitations of the different mutant proteins with Ku proteins. Co-IP was performed in HCT116 transfected with the indicated mutant plasmids using anti-Flag antibodies. Immunoprecipitates were separated on a SDS–PAGE and analyzed by western blot using anti-Ku70 and anti-Ku80 antibodies (upper panel), anti-Flag antibodies (middle panel). Ku70 and Ku80 proteins present in the protein extracts before immunoprecipitation were revealed by western blot (input, lower panel).
    Figure Legend Snippet: The homeodomain of Cdx2 is necessary for its interaction with Ku70/Ku80. ( A ) Schematic representation of the different Cdx2 deletion mutants tested. TAD: transactivator domain; NLS: nuclear localization signal; HD: homeodomain; SD: stabilization domain. The upper numbers represents the position of the different domains in the 313aa long Cdx2 protein. ( B ) Coimmunoprecipitations of the different mutant proteins with Ku proteins. Co-IP was performed in HCT116 transfected with the indicated mutant plasmids using anti-Flag antibodies. Immunoprecipitates were separated on a SDS–PAGE and analyzed by western blot using anti-Ku70 and anti-Ku80 antibodies (upper panel), anti-Flag antibodies (middle panel). Ku70 and Ku80 proteins present in the protein extracts before immunoprecipitation were revealed by western blot (input, lower panel).

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

    Cdx2 does alter neither Ku proteins and DNA-PKcs recruitment nor DNA-PK autophosphorylation but inhibits DNA-PKcs activity. ( A ) Cdx2 does not interfere with DSB Ku70 binding. HCT116 cells were transfected with pFlag (light gray) or Cdx2 (dark gray) expressing plasmid and Ku70 binding activity was assessed. Results represent the mean of at least four independent experiments. pFlag values were set at 1. ( B ) Cdx2 does not alter DNA-PKcs recruitment. HCT116 were transfected with either pFlag or pFlag-Cdx2 as indicated and immunoprecipitation was performed using anti-Ku70 antibodies in the presence or absence of Ethidium Bromide (EtBr) as indicated at the bottom of the figure. DNA-PKcs, Ku70 or Cdx2 were revealed by western blot. Twenty micrograms of whole-protein extract were loaded on the right line (input). ( C ) Cdx2 does not modify DNA-PKcs autophosphorylation. Time course analysis of the phosphorylation of the DNA-PKcs after etoposide treatment 100 μM for 1 hour of HCT116 cells transfected with pFlag or pFlag-Cdx2. Phospho-DNA-PKcs was revealed by western blot. β-actin was used to normalized the amount of loaded proteins. ( D ) Cdx2 inhibits DNA-PKcs activity. DNA-PKcs activity was assessed in HT29-TW6 or -TG8 (control) cells treated or not with etoposide (VP16) 100 μM or neocarzinostatin 200 ng/μL for 1 hour. Cdx2 expression was induced with doxycyclin when indicated (dark gray bars). Experiments without doxycyclin and etoposide treatment were considered as references and results were set at 1. Results illustrate the mean of at least six experiments and error bars represent SEM. Asterisk indicates a significant difference (** P
    Figure Legend Snippet: Cdx2 does alter neither Ku proteins and DNA-PKcs recruitment nor DNA-PK autophosphorylation but inhibits DNA-PKcs activity. ( A ) Cdx2 does not interfere with DSB Ku70 binding. HCT116 cells were transfected with pFlag (light gray) or Cdx2 (dark gray) expressing plasmid and Ku70 binding activity was assessed. Results represent the mean of at least four independent experiments. pFlag values were set at 1. ( B ) Cdx2 does not alter DNA-PKcs recruitment. HCT116 were transfected with either pFlag or pFlag-Cdx2 as indicated and immunoprecipitation was performed using anti-Ku70 antibodies in the presence or absence of Ethidium Bromide (EtBr) as indicated at the bottom of the figure. DNA-PKcs, Ku70 or Cdx2 were revealed by western blot. Twenty micrograms of whole-protein extract were loaded on the right line (input). ( C ) Cdx2 does not modify DNA-PKcs autophosphorylation. Time course analysis of the phosphorylation of the DNA-PKcs after etoposide treatment 100 μM for 1 hour of HCT116 cells transfected with pFlag or pFlag-Cdx2. Phospho-DNA-PKcs was revealed by western blot. β-actin was used to normalized the amount of loaded proteins. ( D ) Cdx2 inhibits DNA-PKcs activity. DNA-PKcs activity was assessed in HT29-TW6 or -TG8 (control) cells treated or not with etoposide (VP16) 100 μM or neocarzinostatin 200 ng/μL for 1 hour. Cdx2 expression was induced with doxycyclin when indicated (dark gray bars). Experiments without doxycyclin and etoposide treatment were considered as references and results were set at 1. Results illustrate the mean of at least six experiments and error bars represent SEM. Asterisk indicates a significant difference (** P

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

    25) Product Images from "Palmitoylation regulates plasma membrane-nuclear shuttling of R7BP, a novel membrane anchor for the RGS7 family"

    Article Title: Palmitoylation regulates plasma membrane-nuclear shuttling of R7BP, a novel membrane anchor for the RGS7 family

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200502007

    R7BP is palmitoylated at its COOH-terminal cysteine residues. (A) Palmitate labeling of R7BP. HEK293 cells expressing wild type or mutant (C252S, C253S, or C252S/C253S) FLAG-tagged forms of R7BP were metabolically labeled with [ 3 H]palmitic acid or [ 35 S]methionine as a control. FLAG-R7BP proteins were immunoprecipitated and resolved by SDS-PAGE. Radiolabeled R7BP was detected by fluorography ([ 3 H]palmitate; 7-d exposure) or autoradiography ([ 35 S]methionine; 4-h exposure) of dried gels (left). Expression and labeling controls for FLAG-R7BP and the control protein FLAG-ERK2 are shown in the right panel (13-d exposure for 3 H-labeled samples). (B) Palmitate is attached to R7BP by a thioester linkage. HEK293 cells expressing FLAG-R7BP were labeled with [ 3 H]palmitic acid or [ 35 S]methionine followed by immunoprecipitation with FLAG-M2 agarose. Immunoprecipitates resolved on duplicate gels were treated with 0.5 M Tris, pH 7.0, or 0.5 M hydroxylamine, pH 7.0, and analyzed by fluorography (12-d exposure) and autoradiography (16-h exposure). (C) R7BP is labeled with palmitate. HEK293 cells expressing FLAG-R7BP or FLAG-ERK2 proteins were labeled with [ 3 H]palmitic acid, immunoprecipitated, resolved by SDS-PAGE, and excised from the gel. Fatty acids were released from samples by base treatment and analyzed by fractionation on reverse-phase TLC plates relative to the indicated standards (18-d exposure).
    Figure Legend Snippet: R7BP is palmitoylated at its COOH-terminal cysteine residues. (A) Palmitate labeling of R7BP. HEK293 cells expressing wild type or mutant (C252S, C253S, or C252S/C253S) FLAG-tagged forms of R7BP were metabolically labeled with [ 3 H]palmitic acid or [ 35 S]methionine as a control. FLAG-R7BP proteins were immunoprecipitated and resolved by SDS-PAGE. Radiolabeled R7BP was detected by fluorography ([ 3 H]palmitate; 7-d exposure) or autoradiography ([ 35 S]methionine; 4-h exposure) of dried gels (left). Expression and labeling controls for FLAG-R7BP and the control protein FLAG-ERK2 are shown in the right panel (13-d exposure for 3 H-labeled samples). (B) Palmitate is attached to R7BP by a thioester linkage. HEK293 cells expressing FLAG-R7BP were labeled with [ 3 H]palmitic acid or [ 35 S]methionine followed by immunoprecipitation with FLAG-M2 agarose. Immunoprecipitates resolved on duplicate gels were treated with 0.5 M Tris, pH 7.0, or 0.5 M hydroxylamine, pH 7.0, and analyzed by fluorography (12-d exposure) and autoradiography (16-h exposure). (C) R7BP is labeled with palmitate. HEK293 cells expressing FLAG-R7BP or FLAG-ERK2 proteins were labeled with [ 3 H]palmitic acid, immunoprecipitated, resolved by SDS-PAGE, and excised from the gel. Fatty acids were released from samples by base treatment and analyzed by fractionation on reverse-phase TLC plates relative to the indicated standards (18-d exposure).

    Techniques Used: Labeling, Expressing, Mutagenesis, Metabolic Labelling, Immunoprecipitation, SDS Page, Autoradiography, Fractionation, Thin Layer Chromatography

    26) Product Images from "Disrupted-in-Schizophrenia-1 (DISC1) regulates spines of the glutamate synapse via Rac1"

    Article Title: Disrupted-in-Schizophrenia-1 (DISC1) regulates spines of the glutamate synapse via Rac1

    Journal: Nature neuroscience

    doi: 10.1038/nn.2487

    Protein interaction of DISC1/Kal-7 regulates spine morphology in rat primary cortical neurons ( a ) Endogenous interactions of DISC1 with Kal-7 and PSD95 (red asterisks) by co-immunoprecipitation (IP) from primary cortical neurons and rat cerebral cortex. DISC1 did not bind Tiam1 norβPIX. Strong interactions of DISC1/Kal-7 and DISC1/PSD95 are observed in the synaptosomal fractions (double asterisks). Full-length blots are presented in Supplementary Fig. 17 . ( b ) Spine shrinkage and reduced spine density by overexpression (for 2 days) of full length DISC1 (DISC-FL), but not by DISC1 lacking Kal-7 interaction (DISC1-ΔKal-7). Both DISC1-FL and DISC1-ΔKal-7 were localized in the dendritic spine (arrowheads). ( c ) Normalization of DISC1 knockdown-induced spine enlargement by DISC1-FL, but not by DISC1-ΔKal-7. ( d ) Increase in the frequency of mEPSC was normalized by the overexpression of DISC1-FL R , but not by that of DISC1-ΔKal-7 R . Left, representative mEPSC traces; right, mEPSC amplitude and frequency. Bar, s.e.m. * P
    Figure Legend Snippet: Protein interaction of DISC1/Kal-7 regulates spine morphology in rat primary cortical neurons ( a ) Endogenous interactions of DISC1 with Kal-7 and PSD95 (red asterisks) by co-immunoprecipitation (IP) from primary cortical neurons and rat cerebral cortex. DISC1 did not bind Tiam1 norβPIX. Strong interactions of DISC1/Kal-7 and DISC1/PSD95 are observed in the synaptosomal fractions (double asterisks). Full-length blots are presented in Supplementary Fig. 17 . ( b ) Spine shrinkage and reduced spine density by overexpression (for 2 days) of full length DISC1 (DISC-FL), but not by DISC1 lacking Kal-7 interaction (DISC1-ΔKal-7). Both DISC1-FL and DISC1-ΔKal-7 were localized in the dendritic spine (arrowheads). ( c ) Normalization of DISC1 knockdown-induced spine enlargement by DISC1-FL, but not by DISC1-ΔKal-7. ( d ) Increase in the frequency of mEPSC was normalized by the overexpression of DISC1-FL R , but not by that of DISC1-ΔKal-7 R . Left, representative mEPSC traces; right, mEPSC amplitude and frequency. Bar, s.e.m. * P

    Techniques Used: Immunoprecipitation, Over Expression

    27) Product Images from "Reptin Regulates DNA Double Strand Breaks Repair in Human Hepatocellular Carcinoma"

    Article Title: Reptin Regulates DNA Double Strand Breaks Repair in Human Hepatocellular Carcinoma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0123333

    Reptin interacts with DNA-PKcs and regulates its stability. (A) Interaction between Reptin and DNA-PKcs was tested by immunoprecipitation. The migration positions of molecular weight standards (in kDa) are indicated on the left. The faint band seen in the IgG lane with the Reptin antibody corresponds to traces of IgG heavy chains. The picture is representative of 3 similar experiments. (B) Metabolic labeling and pulse chase. HuH7 cells stably expressing a doxycycline-inducible Reptin shRNA were treated (sh Reptin) or not (Control) with doxycycline. After 4 days, they were labeled with EXPRE 35 S 35 S as described in Materials and Methods. Following the indicated periods of chase, DNA-PKcs was immunoprecipitated and the eluates were separated on SDS-PAGE. The top panel shows the autoradiographic image, and the bottom one the Coomassie blue staining of the gel with DNA-PKcs. (C) The graph shows the quantitative analysis of the data following normalization of the autoradiographic signal on the amount of immunoprecipitated DNA-PKcs.
    Figure Legend Snippet: Reptin interacts with DNA-PKcs and regulates its stability. (A) Interaction between Reptin and DNA-PKcs was tested by immunoprecipitation. The migration positions of molecular weight standards (in kDa) are indicated on the left. The faint band seen in the IgG lane with the Reptin antibody corresponds to traces of IgG heavy chains. The picture is representative of 3 similar experiments. (B) Metabolic labeling and pulse chase. HuH7 cells stably expressing a doxycycline-inducible Reptin shRNA were treated (sh Reptin) or not (Control) with doxycycline. After 4 days, they were labeled with EXPRE 35 S 35 S as described in Materials and Methods. Following the indicated periods of chase, DNA-PKcs was immunoprecipitated and the eluates were separated on SDS-PAGE. The top panel shows the autoradiographic image, and the bottom one the Coomassie blue staining of the gel with DNA-PKcs. (C) The graph shows the quantitative analysis of the data following normalization of the autoradiographic signal on the amount of immunoprecipitated DNA-PKcs.

    Techniques Used: Immunoprecipitation, Migration, Molecular Weight, Labeling, Pulse Chase, Stable Transfection, Expressing, shRNA, SDS Page, Staining

    28) Product Images from "EglN2 contributes to triple negative breast tumorigenesis by functioning as a substrate for the FBW7 tumor suppressor"

    Article Title: EglN2 contributes to triple negative breast tumorigenesis by functioning as a substrate for the FBW7 tumor suppressor

    Journal: Oncotarget

    doi: 10.18632/oncotarget.14290

    EglN2 C-terminal Residues Might Mediate Its Negative Regulation by FBW7 A-B . Immunoblot analysis of Hs-578T (A) and MDA-MB-231 (B) cells co-transfected with HA tagged FBW7 or GFP with either HA-tagged GSK3β or Control. Cell lysates were harvested 48 hours post-transfection. C . 293T cells were transfected with either HA tagged EglN2 or control followed by immunoprecipitation with HA agarose beads (3F10, Roche). Coomassie staining was performed to visualize the purified HA EglN2, which was cut followed by mass spectrometry analysis as described previously. EglN2 Ser401 and Thr405 sites were shown to be phosphorylated. D . Immunoblots for 293T cells co-transfected with various FLAG tagged EglN2 constructs (WT, S401A, T405A, ST-AA, ∧TPT or ∧SQPPTPT) and HA tagged GSK3β or Control (-). Equal amount of GFP was transfected to make sure of comparable transfection efficiency. Cell lysates were harvested 48 hours post-transfection. E . Immunoblot (IB) assays of whole cell extract (WCE) and immunoprecipitation (IP) of 293T cells (expressing Ctrl or HA-FBW7) co-transfected with Flag-EglN2 (F-EglN2), Flag-EglN2 ST-AA, Flag-EglN2 ∧SQPPTPT, Flag c-Jun or Flag c-Jun FS-AF mutants. Cells were treated with MG132 (10 μM) for overnight before harvesting at 48 hours post-transfection. F . In vivo ubiquitination assays of immunoprecipitation (IP with Ni-NTA) of 293T cells (expressing Ctrl or HA-FBW7) co-transfected with Flag-EglN2 (WT), Flag-EglN2 S401A, T405A or ST-AA. Cells were treated with MG132 (10 μM) for overnight before harvesting at 48 hours post-transfection.
    Figure Legend Snippet: EglN2 C-terminal Residues Might Mediate Its Negative Regulation by FBW7 A-B . Immunoblot analysis of Hs-578T (A) and MDA-MB-231 (B) cells co-transfected with HA tagged FBW7 or GFP with either HA-tagged GSK3β or Control. Cell lysates were harvested 48 hours post-transfection. C . 293T cells were transfected with either HA tagged EglN2 or control followed by immunoprecipitation with HA agarose beads (3F10, Roche). Coomassie staining was performed to visualize the purified HA EglN2, which was cut followed by mass spectrometry analysis as described previously. EglN2 Ser401 and Thr405 sites were shown to be phosphorylated. D . Immunoblots for 293T cells co-transfected with various FLAG tagged EglN2 constructs (WT, S401A, T405A, ST-AA, ∧TPT or ∧SQPPTPT) and HA tagged GSK3β or Control (-). Equal amount of GFP was transfected to make sure of comparable transfection efficiency. Cell lysates were harvested 48 hours post-transfection. E . Immunoblot (IB) assays of whole cell extract (WCE) and immunoprecipitation (IP) of 293T cells (expressing Ctrl or HA-FBW7) co-transfected with Flag-EglN2 (F-EglN2), Flag-EglN2 ST-AA, Flag-EglN2 ∧SQPPTPT, Flag c-Jun or Flag c-Jun FS-AF mutants. Cells were treated with MG132 (10 μM) for overnight before harvesting at 48 hours post-transfection. F . In vivo ubiquitination assays of immunoprecipitation (IP with Ni-NTA) of 293T cells (expressing Ctrl or HA-FBW7) co-transfected with Flag-EglN2 (WT), Flag-EglN2 S401A, T405A or ST-AA. Cells were treated with MG132 (10 μM) for overnight before harvesting at 48 hours post-transfection.

    Techniques Used: Multiple Displacement Amplification, Transfection, Immunoprecipitation, Staining, Purification, Mass Spectrometry, Western Blot, Construct, Expressing, In Vivo

    29) Product Images from "Suppression of Spry1 inhibits triple-negative breast cancer malignancy by decreasing EGF/EGFR mediated mesenchymal phenotype"

    Article Title: Suppression of Spry1 inhibits triple-negative breast cancer malignancy by decreasing EGF/EGFR mediated mesenchymal phenotype

    Journal: Scientific Reports

    doi: 10.1038/srep23216

    Suppressing Spry1 results in degradation of EGF activated EGFR, and decrease of EGF induced EGFR/Grb2/Gab1/Shp2 cascade complexes formation and downstream signaling, and Snail and Slug induction. ( A ) Time course analysis of Snail, Slug induction, and EGFR protein level upon EGF stimulation. Immunoblotting shows that EGF stimulation induced Snail and Slug expression in NT cells but not in S1kd cells. In NT cells EGF did not change EGFR levels within 6 h, but in S1kd and S4kd cells EGF induced a decrease of EGFR at 2 h and 6 h. The results are representative from at least three independent experiments. The blots were first used for probing pEGFR, Slug or Snail, and then stripped off for reprobing EGFR or Tubulin. ( B ) Quantification to show the trend of EGFR degradation in S1kd cells. ( C ) Immunoprecipitation of EGFR and blotting with Grb2 and EGFR shows decreased EGFR/Grb2 complex formation in S1kd cells compared to NT and S4kd cells. Lysates were incubated with rabbit anti-EGFR overnight, then protein A/G for 1 hr. Beads bound proteins were washed and separated on 8% SDS-PAGE. The same blot was cut between 37 kD and 50 kD markers, and separately probed for EGFR (~185 kD) or Grb2 (~25 kD). ( D ) Quantification of EGF induced EGFR/Grb2 complex formation from three independent experiments. ( E ) Immunoprecipitation of Gab1 and blotting with Grb2, Shp2 and Gab1 shows decreased Gab/Grb2 and Gab1/Shp2 complexes formation in S1kd cells compared to NT and S4kd cells. Lysates were incubated with rabbit anti-Gab1 overnight, then protein A/G for 1 hr. Beads bound proteins were washed and separated on 8% SDS-PAGE. The blot was cut between 37 kD and 50 kD, and between 75 kD and 100 kD, and probed for Grb2 (~25 kD), Shp2 (~72 kD) or Gab1 (~110 kD). ( F ) Quantification of EGFR/Grb2, Gab1/Grb2, Gab1/Shp2 complexes from three independent experiments. ( G ) Representative immunoblotting assay shows decreased EGF mediated pAkt level in S1kd cells compared to NT and S4kd cells. ( H ) Quantification of pAkt/Akt from at least three independent experiments. *p
    Figure Legend Snippet: Suppressing Spry1 results in degradation of EGF activated EGFR, and decrease of EGF induced EGFR/Grb2/Gab1/Shp2 cascade complexes formation and downstream signaling, and Snail and Slug induction. ( A ) Time course analysis of Snail, Slug induction, and EGFR protein level upon EGF stimulation. Immunoblotting shows that EGF stimulation induced Snail and Slug expression in NT cells but not in S1kd cells. In NT cells EGF did not change EGFR levels within 6 h, but in S1kd and S4kd cells EGF induced a decrease of EGFR at 2 h and 6 h. The results are representative from at least three independent experiments. The blots were first used for probing pEGFR, Slug or Snail, and then stripped off for reprobing EGFR or Tubulin. ( B ) Quantification to show the trend of EGFR degradation in S1kd cells. ( C ) Immunoprecipitation of EGFR and blotting with Grb2 and EGFR shows decreased EGFR/Grb2 complex formation in S1kd cells compared to NT and S4kd cells. Lysates were incubated with rabbit anti-EGFR overnight, then protein A/G for 1 hr. Beads bound proteins were washed and separated on 8% SDS-PAGE. The same blot was cut between 37 kD and 50 kD markers, and separately probed for EGFR (~185 kD) or Grb2 (~25 kD). ( D ) Quantification of EGF induced EGFR/Grb2 complex formation from three independent experiments. ( E ) Immunoprecipitation of Gab1 and blotting with Grb2, Shp2 and Gab1 shows decreased Gab/Grb2 and Gab1/Shp2 complexes formation in S1kd cells compared to NT and S4kd cells. Lysates were incubated with rabbit anti-Gab1 overnight, then protein A/G for 1 hr. Beads bound proteins were washed and separated on 8% SDS-PAGE. The blot was cut between 37 kD and 50 kD, and between 75 kD and 100 kD, and probed for Grb2 (~25 kD), Shp2 (~72 kD) or Gab1 (~110 kD). ( F ) Quantification of EGFR/Grb2, Gab1/Grb2, Gab1/Shp2 complexes from three independent experiments. ( G ) Representative immunoblotting assay shows decreased EGF mediated pAkt level in S1kd cells compared to NT and S4kd cells. ( H ) Quantification of pAkt/Akt from at least three independent experiments. *p

    Techniques Used: Expressing, Immunoprecipitation, Incubation, SDS Page

    30) Product Images from "Bcl-xL promotes metastasis independent of its anti-apoptotic activity"

    Article Title: Bcl-xL promotes metastasis independent of its anti-apoptotic activity

    Journal: Nature Communications

    doi: 10.1038/ncomms10384

    Bcl-xL mutants that fail to bind to Bax/Bak promotes migration. ( a ) Schematic diagram of Bcl-xL constructs. BH, Bcl-2 homology domain; TM, transmembrane domain. ( b ) Cell lysates of wt MEFs overexpressing control vector, HA-Bcl-xL (wt), HA-Bcl-xL mt2 and HA-Bcl-xL mt1 were subjected to immunoprecipitation with Bax antibody and then followed by western blot analysis to detect HA-Bcl-xL proteins and Bax. Both Bcl-xL mutants (mt2 and mt1) lost the ability to bind Bax in MEFs. ( c ) wt MEFs overexpressing control vector, HA-Bcl-xL, HA-Bcl-xL-mt2 and HA-Bcl-xL-mt1 were treated with 60-J m −2 UV, and then apoptosis was examined using flow cytometry analysis following Annexin V and PI staining. Only HA-Bcl-xL (wt) prevented UV-induced apoptosis. ** P
    Figure Legend Snippet: Bcl-xL mutants that fail to bind to Bax/Bak promotes migration. ( a ) Schematic diagram of Bcl-xL constructs. BH, Bcl-2 homology domain; TM, transmembrane domain. ( b ) Cell lysates of wt MEFs overexpressing control vector, HA-Bcl-xL (wt), HA-Bcl-xL mt2 and HA-Bcl-xL mt1 were subjected to immunoprecipitation with Bax antibody and then followed by western blot analysis to detect HA-Bcl-xL proteins and Bax. Both Bcl-xL mutants (mt2 and mt1) lost the ability to bind Bax in MEFs. ( c ) wt MEFs overexpressing control vector, HA-Bcl-xL, HA-Bcl-xL-mt2 and HA-Bcl-xL-mt1 were treated with 60-J m −2 UV, and then apoptosis was examined using flow cytometry analysis following Annexin V and PI staining. Only HA-Bcl-xL (wt) prevented UV-induced apoptosis. ** P

    Techniques Used: Migration, Construct, Plasmid Preparation, Immunoprecipitation, Western Blot, Flow Cytometry, Cytometry, Staining

    Bcl-xL mutants defective in anti-apoptotic function retain the effect of wt Bcl-xL on cell migration and EMT in the mouse N134 panNET cell line. ( a ) Cell lysates were subjected to immunoprecipitation with HA magnetic beads and then followed by western blot analysis to detect Bcl-xL and Bax. Both Bcl-xL mutants (mt2 and mt1) lost the ability to bind to Bax in N134. ( b ) N134 tumour cells infected with RCASBP- HA-β-actin , HA-Bcl-xL , mt2 and mt1 were treated with 10-μM etoposide for 24 h, and then apoptosis was examined with flow cytometry analysis following Annexin V and PI staining. Only cells infected with HA-Bcl-xL prevented etoposide-induced apoptosis. * P
    Figure Legend Snippet: Bcl-xL mutants defective in anti-apoptotic function retain the effect of wt Bcl-xL on cell migration and EMT in the mouse N134 panNET cell line. ( a ) Cell lysates were subjected to immunoprecipitation with HA magnetic beads and then followed by western blot analysis to detect Bcl-xL and Bax. Both Bcl-xL mutants (mt2 and mt1) lost the ability to bind to Bax in N134. ( b ) N134 tumour cells infected with RCASBP- HA-β-actin , HA-Bcl-xL , mt2 and mt1 were treated with 10-μM etoposide for 24 h, and then apoptosis was examined with flow cytometry analysis following Annexin V and PI staining. Only cells infected with HA-Bcl-xL prevented etoposide-induced apoptosis. * P

    Techniques Used: Migration, Immunoprecipitation, Magnetic Beads, Western Blot, Infection, Flow Cytometry, Cytometry, Staining

    31) Product Images from "SUMOylation of sPRDM16 promotes the progression of acute myeloid leukemia"

    Article Title: SUMOylation of sPRDM16 promotes the progression of acute myeloid leukemia

    Journal: BMC Cancer

    doi: 10.1186/s12885-015-1844-2

    sPRDM16 was SUMOylated by SUMO1 on lysine-568. a sPRDM16 was SUMOylated in vivo . HEK293T cells were transfected with the indicated plasmids for 36 h. Immunoprecipitation was performed with anti-FLAG M2 agarose beads. The immunoprecipitates (IP) and the original whole-cell lysates (WCL) were analyzed by immunoblotting (IB) with anti-HA or anti-FLAG antibodies. b SENP1 de-SUMOylated sPRDM16. HEK293T cells were transfected with HA-SUMO1, Flag-sPRDM16, RGS-SENP1, or RGS-SENP1m as indicated. Flag-sPRDM16 proteins were pulled down by anti-Flag M2 agarose beads from cell lysates. Bound proteins were blotted with anti-Flag. Cell lysate was immunoblotted (IB) with anti-Flag antibody, anti-HA antibody, or anti-RGS antibody. c K568 was the primary SUMOylation site of sPRDM16. HEK293T cells were transfected with the indicated plasmids. Cell lysates were immunoprecipitated with anti-FLAG M2 agarose beads, followed by Western blot (WB) analysis using anti-HA or anti-FLAG antibodies
    Figure Legend Snippet: sPRDM16 was SUMOylated by SUMO1 on lysine-568. a sPRDM16 was SUMOylated in vivo . HEK293T cells were transfected with the indicated plasmids for 36 h. Immunoprecipitation was performed with anti-FLAG M2 agarose beads. The immunoprecipitates (IP) and the original whole-cell lysates (WCL) were analyzed by immunoblotting (IB) with anti-HA or anti-FLAG antibodies. b SENP1 de-SUMOylated sPRDM16. HEK293T cells were transfected with HA-SUMO1, Flag-sPRDM16, RGS-SENP1, or RGS-SENP1m as indicated. Flag-sPRDM16 proteins were pulled down by anti-Flag M2 agarose beads from cell lysates. Bound proteins were blotted with anti-Flag. Cell lysate was immunoblotted (IB) with anti-Flag antibody, anti-HA antibody, or anti-RGS antibody. c K568 was the primary SUMOylation site of sPRDM16. HEK293T cells were transfected with the indicated plasmids. Cell lysates were immunoprecipitated with anti-FLAG M2 agarose beads, followed by Western blot (WB) analysis using anti-HA or anti-FLAG antibodies

    Techniques Used: In Vivo, Transfection, Immunoprecipitation, Western Blot

    32) Product Images from "Heat-Shock Factor 1 Controls Genome-wide Acetylation in Heat-shocked Cells"

    Article Title: Heat-Shock Factor 1 Controls Genome-wide Acetylation in Heat-shocked Cells

    Journal:

    doi: 10.1091/mbc.E09-04-0295

    HSF1 binding to HDAC 1 and HDAC2 in heat-shocked cells correlates with increased HDAC activities. (A) Increased HDAC1 and 2 activities after heat shock. Immunoprecipitation of transiently transfected Flag-tagged HDAC1 and 2 or Flag tag alone from non-heat-shocked
    Figure Legend Snippet: HSF1 binding to HDAC 1 and HDAC2 in heat-shocked cells correlates with increased HDAC activities. (A) Increased HDAC1 and 2 activities after heat shock. Immunoprecipitation of transiently transfected Flag-tagged HDAC1 and 2 or Flag tag alone from non-heat-shocked

    Techniques Used: Binding Assay, Immunoprecipitation, Transfection, FLAG-tag

    33) Product Images from "Inactivation of ribosomal protein S27-like impairs DNA interstrand cross-link repair by destabilization of FANCD2 and FANCI"

    Article Title: Inactivation of ribosomal protein S27-like impairs DNA interstrand cross-link repair by destabilization of FANCD2 and FANCI

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-020-03082-9

    RPS27L interacts with FANCD2 and FANCI. A Ectopically expressed RPS27L binds to endogenous FANCD2 and FANCI. H1299 cells were transfected with mock vector or FLAG-RPS27L, and subjected to immunoprecipitation (IP) with FLAG beads, followed by immunoblotting (IB) with indicated antibodies (Abs). WCE: whole-cell extract. B – D Endogenous RPS27L binds to endogenous FANCD2 and FANCI. A549 cells were harvested for IP with RPS27L ( B ), FANCD2 ( C ), or FANCI ( D ) Ab, and then IB with indicated Abs. E Schematic diagrams of full length and fragments of FANCD2 and FANCI constructs. The bar graphs were not drawn to scale and the red bar indicates the RPS27L-interacting region. F , G Mapping of FANCD2/FANCI binding to RPS27L. 293 cells were transfected with mock vector, indicated constructs encoding various fragments of FLAG-FANCD2 ( F ) or FLAG-FANCI ( G ) before harvesting for IP with FLAG beads, followed by IB with indicated Abs.
    Figure Legend Snippet: RPS27L interacts with FANCD2 and FANCI. A Ectopically expressed RPS27L binds to endogenous FANCD2 and FANCI. H1299 cells were transfected with mock vector or FLAG-RPS27L, and subjected to immunoprecipitation (IP) with FLAG beads, followed by immunoblotting (IB) with indicated antibodies (Abs). WCE: whole-cell extract. B – D Endogenous RPS27L binds to endogenous FANCD2 and FANCI. A549 cells were harvested for IP with RPS27L ( B ), FANCD2 ( C ), or FANCI ( D ) Ab, and then IB with indicated Abs. E Schematic diagrams of full length and fragments of FANCD2 and FANCI constructs. The bar graphs were not drawn to scale and the red bar indicates the RPS27L-interacting region. F , G Mapping of FANCD2/FANCI binding to RPS27L. 293 cells were transfected with mock vector, indicated constructs encoding various fragments of FLAG-FANCD2 ( F ) or FLAG-FANCI ( G ) before harvesting for IP with FLAG beads, followed by IB with indicated Abs.

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

    34) Product Images from "Disruption of occludin function in polarized epithelial cells activates the extrinsic pathway of apoptosis leading to cell extrusion without loss of transepithelial resistance"

    Article Title: Disruption of occludin function in polarized epithelial cells activates the extrinsic pathway of apoptosis leading to cell extrusion without loss of transepithelial resistance

    Journal: BMC Cell Biology

    doi: 10.1186/1471-2121-10-85

    Co-immunoprecipitation of occludin with FADD after treatment with LYHY . Eph4 cells were treated with 800 μM LHYH for 5 hrs or left untreated after medium change. Cells were lysed and proteins immunoprecipitated with a mouse anti-FADD antibody using protein A/G agarose beads and blotted with a rabbit anti-occludin antibody. A. Western blot for occludin. Lysates of cells that were immunoprecipitated with an anti-FADD antibody. IP-FADD: Two left lanes show IP of lysates from untreated EPH4 cultures and two right hand lanes show IP of lysates from LYHY treated EPH4 cultures. A very small proportion of the total cellular occludin was associated with FADD. B. Quantitation of occludin bands from 3 control and treated cultures. The intensity of stain was variable but higher in the immunoprecipitated fractions from LHYH treated cells (P
    Figure Legend Snippet: Co-immunoprecipitation of occludin with FADD after treatment with LYHY . Eph4 cells were treated with 800 μM LHYH for 5 hrs or left untreated after medium change. Cells were lysed and proteins immunoprecipitated with a mouse anti-FADD antibody using protein A/G agarose beads and blotted with a rabbit anti-occludin antibody. A. Western blot for occludin. Lysates of cells that were immunoprecipitated with an anti-FADD antibody. IP-FADD: Two left lanes show IP of lysates from untreated EPH4 cultures and two right hand lanes show IP of lysates from LYHY treated EPH4 cultures. A very small proportion of the total cellular occludin was associated with FADD. B. Quantitation of occludin bands from 3 control and treated cultures. The intensity of stain was variable but higher in the immunoprecipitated fractions from LHYH treated cells (P

    Techniques Used: Immunoprecipitation, Western Blot, Quantitation Assay, Staining

    35) Product Images from "DNA damage-induced activation of ATM promotes ?-TRCP-mediated Mdm2 ubiquitination and destruction"

    Article Title: DNA damage-induced activation of ATM promotes ?-TRCP-mediated Mdm2 ubiquitination and destruction

    Journal: Oncotarget

    doi:

    ATM phosphorylates Casein Kinase Iδ to regulate its subcellular localization and its ability to interact with Mdm2 (A) Protein sequence illustration of the two putative ATM phosphorylation sites present in the C-terminus of CKIδ across different species. Hyd: hydrophobic amino acids. (B) Immunoblot analysis (IB) of whole cell lysates (WCL) and immunoprecipitates (IP) derived from 293T cells transfected with the indicated GST-CKIδ constructs in the presence or absence of Flag-ATM. Where indicated, the ATM kinase was activated by treatment with etoposide for 30 minutes before harvesting. The phosphorylation status of CKIδ by ATM in vivo was detected with a specific phospho-(S/T) ATM substrate antibody. (C) ATM phosphorylates both Mdm2 and CKIδ in vitro . The ATM kinase was purified by Flag-immunoprecipitation from 293T cells, and then incubated with 5 μg of GST-Mdm2 or the indicated GST-CKIδ proteins (with GST protein as a negative control) in the presence of γ- 32 P-ATP. The kinase reaction products were resolved by SDS-PAGE and phosphorylation was detected by autoradiography. (D) Immunoblot (IB) analysis of whole cell lysates (WCL) and immunoprecipitates (IP) derived from 293T cells transfected with HA-Mdm2 and the indicated GST-CKIδ constructs. Thirty hours post-transfection, cells were pretreated with 10 μM MG132 for 2 hours to block the proteasome pathway and then treated with 25 μM etoposide (or DMSO as control) for 1.5 hours before harvesting.
    Figure Legend Snippet: ATM phosphorylates Casein Kinase Iδ to regulate its subcellular localization and its ability to interact with Mdm2 (A) Protein sequence illustration of the two putative ATM phosphorylation sites present in the C-terminus of CKIδ across different species. Hyd: hydrophobic amino acids. (B) Immunoblot analysis (IB) of whole cell lysates (WCL) and immunoprecipitates (IP) derived from 293T cells transfected with the indicated GST-CKIδ constructs in the presence or absence of Flag-ATM. Where indicated, the ATM kinase was activated by treatment with etoposide for 30 minutes before harvesting. The phosphorylation status of CKIδ by ATM in vivo was detected with a specific phospho-(S/T) ATM substrate antibody. (C) ATM phosphorylates both Mdm2 and CKIδ in vitro . The ATM kinase was purified by Flag-immunoprecipitation from 293T cells, and then incubated with 5 μg of GST-Mdm2 or the indicated GST-CKIδ proteins (with GST protein as a negative control) in the presence of γ- 32 P-ATP. The kinase reaction products were resolved by SDS-PAGE and phosphorylation was detected by autoradiography. (D) Immunoblot (IB) analysis of whole cell lysates (WCL) and immunoprecipitates (IP) derived from 293T cells transfected with HA-Mdm2 and the indicated GST-CKIδ constructs. Thirty hours post-transfection, cells were pretreated with 10 μM MG132 for 2 hours to block the proteasome pathway and then treated with 25 μM etoposide (or DMSO as control) for 1.5 hours before harvesting.

    Techniques Used: Sequencing, Derivative Assay, Transfection, Construct, In Vivo, In Vitro, Purification, Immunoprecipitation, Incubation, Negative Control, SDS Page, Autoradiography, Blocking Assay

    36) Product Images from "Generation of a monoclonal antibody recognizing the CEACAM glycan structure and inhibiting adhesion using cancer tissue-originated spheroid as an antigen"

    Article Title: Generation of a monoclonal antibody recognizing the CEACAM glycan structure and inhibiting adhesion using cancer tissue-originated spheroid as an antigen

    Journal: Scientific Reports

    doi: 10.1038/srep24823

    Pre-incubation with 5G2 mAb inhibited translocation of integrin β4 in CTOSs. (a) Immunocytochemical analysis of CTOSs using integrin β4 antibody. C45 CTOSs under floating conditions (i.e., suspension culture) were pre-incubated with the indicated antibody and cultured for 20 h in Matrigel. (b) CTOS surface coverage with the integrin β4 signal. C45 CTOSs were treated the same as in ( a ). Differences were analysed by the Mann Whitney U-test. IgG, n = 111; 5G2, n = 139. Horizontal bar, median; boxes, 25 th and 75 th percentiles; bars, 10 th and 90 th percentiles. (c) Integrin β4 signal intensity on lateral membranes in CTOSs. C45 CTOSs were treated the same as in ( a ). Data indicate mean ± SD. Differences were analysed by the Mann Whitney U-test. IgG, n = 1512; 5G2, n = 2066. (d) Western blot using phosphor-FAK (pFAK) or total FAK (tFAK) antibody. C45 CTOSs in floating conditions (F) were pre-incubated with the indicated antibody and cultured for 6 h in Matrigel (G). ( e ) Immunoprecipitation analysis of floating cultured C45 CTOSs. Antibodies used for immunoprecipitation (IP) and detection (Blot) are shown.
    Figure Legend Snippet: Pre-incubation with 5G2 mAb inhibited translocation of integrin β4 in CTOSs. (a) Immunocytochemical analysis of CTOSs using integrin β4 antibody. C45 CTOSs under floating conditions (i.e., suspension culture) were pre-incubated with the indicated antibody and cultured for 20 h in Matrigel. (b) CTOS surface coverage with the integrin β4 signal. C45 CTOSs were treated the same as in ( a ). Differences were analysed by the Mann Whitney U-test. IgG, n = 111; 5G2, n = 139. Horizontal bar, median; boxes, 25 th and 75 th percentiles; bars, 10 th and 90 th percentiles. (c) Integrin β4 signal intensity on lateral membranes in CTOSs. C45 CTOSs were treated the same as in ( a ). Data indicate mean ± SD. Differences were analysed by the Mann Whitney U-test. IgG, n = 1512; 5G2, n = 2066. (d) Western blot using phosphor-FAK (pFAK) or total FAK (tFAK) antibody. C45 CTOSs in floating conditions (F) were pre-incubated with the indicated antibody and cultured for 6 h in Matrigel (G). ( e ) Immunoprecipitation analysis of floating cultured C45 CTOSs. Antibodies used for immunoprecipitation (IP) and detection (Blot) are shown.

    Techniques Used: Incubation, Translocation Assay, Cell Culture, MANN-WHITNEY, Western Blot, Immunoprecipitation

    37) Product Images from "FBXL19 recruits CDK-Mediator to CpG islands of developmental genes priming them for activation during lineage commitment"

    Article Title: FBXL19 recruits CDK-Mediator to CpG islands of developmental genes priming them for activation during lineage commitment

    Journal: eLife

    doi: 10.7554/eLife.37084

    FBXL19 interacts with the CDK-Mediator complex in ES cells. ( A ) A representative silver-stained gel for FS2-FBXL19 purification and an empty vector control (EV) purification. Asterisk identifies the band corresponding to FS2-FBXL19 protein. ( B ) In order to visualize FBXL19 protein by western blot in Figure 2—figure supplement 1D and Figure 4B , the Fbxl19 gene was tagged by T7 knock-in in Fbxl19 fl/fl ES cells using the CRISPR Cas9 system. A schematic representation of the generation of the C-terminal T7 knock-in Fbxl19 is shown. HA1/2 indicate the homology arms of the targeting construct. ( C ) Western blot analysis of the expression of T7-FBXL19 from nuclear extract of the generated T7 knock-in ES cell line. ( D ) Western blot analysis of endogenous co-immunoprecipitation (IP) of FBXL19, CDK8 and MED12 from ES cell nuclear extracts. A control IP using a non-specific antibody (α-ΗΑ) was included. ( E ) A schematic illustration of the different FS2-FBXL19 truncation mutants and Western blot analysis of purification of FS2-FBXL19 mutants from HEK293T cells probed with the indicated antibodies. ( F ) Western blot analysis of FS2-FBXL19 and control purifications (EV) probed with the indicated antibodies. ( G ) Western blot analysis of histone extracts generated from Fbxl19 fl/fl (WT) and Fbxl19 ΔCXXC (OHT) ES cells probed with two different antibodies recognizing ubiquitylated H2B K120 (H2Bub1). H4 was used as a loading control. ( H ) Western blot analysis of nuclear extracts from HEK293T cells transiently transfected with empty vector (EV) or Flag-FBXL19-expressing vector without (-) or following MG132 treatment (+). Blots were probed with the indicated antibodies. TBP was used as loading control.
    Figure Legend Snippet: FBXL19 interacts with the CDK-Mediator complex in ES cells. ( A ) A representative silver-stained gel for FS2-FBXL19 purification and an empty vector control (EV) purification. Asterisk identifies the band corresponding to FS2-FBXL19 protein. ( B ) In order to visualize FBXL19 protein by western blot in Figure 2—figure supplement 1D and Figure 4B , the Fbxl19 gene was tagged by T7 knock-in in Fbxl19 fl/fl ES cells using the CRISPR Cas9 system. A schematic representation of the generation of the C-terminal T7 knock-in Fbxl19 is shown. HA1/2 indicate the homology arms of the targeting construct. ( C ) Western blot analysis of the expression of T7-FBXL19 from nuclear extract of the generated T7 knock-in ES cell line. ( D ) Western blot analysis of endogenous co-immunoprecipitation (IP) of FBXL19, CDK8 and MED12 from ES cell nuclear extracts. A control IP using a non-specific antibody (α-ΗΑ) was included. ( E ) A schematic illustration of the different FS2-FBXL19 truncation mutants and Western blot analysis of purification of FS2-FBXL19 mutants from HEK293T cells probed with the indicated antibodies. ( F ) Western blot analysis of FS2-FBXL19 and control purifications (EV) probed with the indicated antibodies. ( G ) Western blot analysis of histone extracts generated from Fbxl19 fl/fl (WT) and Fbxl19 ΔCXXC (OHT) ES cells probed with two different antibodies recognizing ubiquitylated H2B K120 (H2Bub1). H4 was used as a loading control. ( H ) Western blot analysis of nuclear extracts from HEK293T cells transiently transfected with empty vector (EV) or Flag-FBXL19-expressing vector without (-) or following MG132 treatment (+). Blots were probed with the indicated antibodies. TBP was used as loading control.

    Techniques Used: Staining, Purification, Plasmid Preparation, Western Blot, Knock-In, CRISPR, Construct, Expressing, Generated, Immunoprecipitation, Transfection

    38) Product Images from "Binding of ?v?1 and ?v?6 integrins to tenascin-C induces epithelial-mesenchymal transition-like change of breast cancer cells"

    Article Title: Binding of ?v?1 and ?v?6 integrins to tenascin-C induces epithelial-mesenchymal transition-like change of breast cancer cells

    Journal: Oncogenesis

    doi: 10.1038/oncsis.2013.27

    Integrin αvβ1 heterodimers, but not α2, are mobilized to focal adhesions. ( a ) Double immunofluorescence with E-cadherin (ECD) or β-catenin (BCTNN) showing intercellular localization of integrin α2 (P1E6) and β1 (4B7R) subunits. Note localization of integrin αv (P2W7) subunits at cluster surfaces and cell–cell contacts. ( b ) After TNC and TGF-β/TNC treatments, α2 and β1 immunolocalization is dispersed into the cytoplasm of the cells after EMT change and focal adhesion staining is lacking. Scale bars: 20 μm. ( c ) On immunoprecipitation, an isoform of β1 with higher molecular weight was co-precipitated by α2 antibody (HAS3), without apparent change of the amount after the treatments. ( d ) The active β1 subunit (P4G11) is localized in small cell-substratum adhesions projected from cell clusters in the control, without αv (Q-20) colocalization (arrow heads). After TNC and TGF-β/TNC treatments, the subunits accumulated in adhesion plaques of the spreading cells on the substrata, in association with αv subunits (arrows). Scale Bar: 20 μm. ( e ) Immunoprecipitation of β1 showed increased heterodimers with αv subunits in both TNC- and TGF-β/TNC-treated cells, compared with the controls.
    Figure Legend Snippet: Integrin αvβ1 heterodimers, but not α2, are mobilized to focal adhesions. ( a ) Double immunofluorescence with E-cadherin (ECD) or β-catenin (BCTNN) showing intercellular localization of integrin α2 (P1E6) and β1 (4B7R) subunits. Note localization of integrin αv (P2W7) subunits at cluster surfaces and cell–cell contacts. ( b ) After TNC and TGF-β/TNC treatments, α2 and β1 immunolocalization is dispersed into the cytoplasm of the cells after EMT change and focal adhesion staining is lacking. Scale bars: 20 μm. ( c ) On immunoprecipitation, an isoform of β1 with higher molecular weight was co-precipitated by α2 antibody (HAS3), without apparent change of the amount after the treatments. ( d ) The active β1 subunit (P4G11) is localized in small cell-substratum adhesions projected from cell clusters in the control, without αv (Q-20) colocalization (arrow heads). After TNC and TGF-β/TNC treatments, the subunits accumulated in adhesion plaques of the spreading cells on the substrata, in association with αv subunits (arrows). Scale Bar: 20 μm. ( e ) Immunoprecipitation of β1 showed increased heterodimers with αv subunits in both TNC- and TGF-β/TNC-treated cells, compared with the controls.

    Techniques Used: Immunofluorescence, Staining, Immunoprecipitation, Molecular Weight

    Integrin β6 subunits are recruited to αv-positive adhesion plaques. ( a ) Immunofluorescence showed control cells to express αv (P2W7) and β5 integrin subunits, but not the β6 subunit. After TNC only and the TGF-β1/TNC treatment, β6-positive adhesion plaques were observed, more frequently in TGF-β1/TNC-treated cells. ( b ) Double immunofluorescence staining demonstrating colocalization of αv (Q-20) and β6 subunits. Scale bars: 20 μm. ( c ) Immunoprecipitation with anti-αv antibody (P2W7) showing prominent increase of co-precipitated β6 subunit in TGF-β1/TNC-treated cells. SDS-gel electrophoresis was performed under non-reducing conditions. As a negative control, a monoclonal antibody against a viral protein was used instead of anti-integrin antibodies. Whole-cell lysates (Lysate) of TGF-β1/TNC-treated cells were also examined.
    Figure Legend Snippet: Integrin β6 subunits are recruited to αv-positive adhesion plaques. ( a ) Immunofluorescence showed control cells to express αv (P2W7) and β5 integrin subunits, but not the β6 subunit. After TNC only and the TGF-β1/TNC treatment, β6-positive adhesion plaques were observed, more frequently in TGF-β1/TNC-treated cells. ( b ) Double immunofluorescence staining demonstrating colocalization of αv (Q-20) and β6 subunits. Scale bars: 20 μm. ( c ) Immunoprecipitation with anti-αv antibody (P2W7) showing prominent increase of co-precipitated β6 subunit in TGF-β1/TNC-treated cells. SDS-gel electrophoresis was performed under non-reducing conditions. As a negative control, a monoclonal antibody against a viral protein was used instead of anti-integrin antibodies. Whole-cell lysates (Lysate) of TGF-β1/TNC-treated cells were also examined.

    Techniques Used: Immunofluorescence, Double Immunofluorescence Staining, Immunoprecipitation, SDS-Gel, Electrophoresis, Negative Control

    39) Product Images from "Regulation of mATG9 trafficking by Src- and ULK1-mediated phosphorylation in basal and starvation-induced autophagy"

    Article Title: Regulation of mATG9 trafficking by Src- and ULK1-mediated phosphorylation in basal and starvation-induced autophagy

    Journal: Cell Research

    doi: 10.1038/cr.2016.146

    Stimulation of Src kinase activity by EGF promotes retrograde trafficking of mATG9. (A) HeLa cells were serum-starved for 24 h, and then stimulated with 50 ng/ml hEGF for the indicated times. Cells were collected for immunoprecipitation with anti-mATG9 antibody and phospho-mATG9 was assessed by immunoblotting with a specific antibody against Y8. (B) HeLa cells stably expressing mATG9-Myc were transfected with 3×Flag-AP1/2M1, serum-starved for 24 h, and then stimulated with hEGF for the indicated times. Cells were collected for immunoprecipitation with anti-Flag antibody. (C - E) Atg9a KO HeLa cells reconstituted with wild-type mATG9 (C) or the SS1/2 (D) or Y8F (E) mutants were starved of serum for 24 h, and then stimulated with hEGF for the indicated times. Cells were fixed and immunostained with anti-Myc (red) and anti-EGFR (green) antibodies. Nuclei are stained with DAPI (blue). Boxed areas in the left panels are enlarged in the right panels. Scale bar, 10 μm. See also Supplementary information, Figure S3D-S3F .
    Figure Legend Snippet: Stimulation of Src kinase activity by EGF promotes retrograde trafficking of mATG9. (A) HeLa cells were serum-starved for 24 h, and then stimulated with 50 ng/ml hEGF for the indicated times. Cells were collected for immunoprecipitation with anti-mATG9 antibody and phospho-mATG9 was assessed by immunoblotting with a specific antibody against Y8. (B) HeLa cells stably expressing mATG9-Myc were transfected with 3×Flag-AP1/2M1, serum-starved for 24 h, and then stimulated with hEGF for the indicated times. Cells were collected for immunoprecipitation with anti-Flag antibody. (C - E) Atg9a KO HeLa cells reconstituted with wild-type mATG9 (C) or the SS1/2 (D) or Y8F (E) mutants were starved of serum for 24 h, and then stimulated with hEGF for the indicated times. Cells were fixed and immunostained with anti-Myc (red) and anti-EGFR (green) antibodies. Nuclei are stained with DAPI (blue). Boxed areas in the left panels are enlarged in the right panels. Scale bar, 10 μm. See also Supplementary information, Figure S3D-S3F .

    Techniques Used: Activity Assay, Immunoprecipitation, Stable Transfection, Expressing, Transfection, Staining

    ULK1 phosphorylates mATG9 at S14 to promote its trafficking under starvation stress. (A) Alignment of the mATG9 N-terminal sequences from different mammalian species reveals the consensus motif for ULK1 kinase. (B) In vitro ULK1 kinase assay. Immunoprecipitated GFP protein, wild-type ULK1 (WT) or kinase-dead ULK1 (KD) was incubated with purified His-ATG9N-HA peptide (aa1-66) at 30 °C for 30 min in kinase buffer. (C) In vitro ULK1 kinase assay was performed as described in B using His-ATG9N-HA or the indicated point-mutated peptides. (D) HeLa cells were treated with or without EBSS for 2 h in the presence or absence of the PI3K inhibitor Wortmannin (100 nM) or the lysosome inhibitor BA1 (20 nM), and then collected for immunoprecipitation with anti-ULK1 antibody. (E) ULK1 protein produced by an in vitro transcription/translation system was incubated with immobilized His-ATG9N-HA peptide at 4 °C overnight. (F) HeLa cells were transfected with GFP-ULK1 or KD ULK1 for 24 h, and collected for immunoprecipitation with anti-mATG9 antibody. (G) HeLa cells were treated with EBSS for the indicated times, and then collected for immunoprecipitation with anti-mATG9 antibody. The proportion of phospho-mATG9 was assessed by immunoblotting with specific antibodies against Y8 or S14. (H) Parental cells or Ulk1 KO HeLa cells were treated with or without EBSS for 2 h. Cells were collected for immunoprecipitation with anti-mATG9 antibody, and phospho-mATG9 was assessed by immunoblotting with a specific antibody against Y8 or S14. (I , J) HEK293T cells were co-transfected with WT mATG9 or the indicated mutants, 3×Flag-AP1/2M1 and GFP, GFP-ULK1 or KD ULK1 for 24 h, and then collected for immunoprecipitation with anti-Flag antibody. (K , L) HeLa cells were co-transfected with WT mATG9 or the S14A mutant and 3×Flag-AP1/2M1 for 24 h, and then starved in EBSS for 2 h. Cells were collected for immunoprecipitation with anti-Flag antibody. See also Supplementary information, Figure S4 .
    Figure Legend Snippet: ULK1 phosphorylates mATG9 at S14 to promote its trafficking under starvation stress. (A) Alignment of the mATG9 N-terminal sequences from different mammalian species reveals the consensus motif for ULK1 kinase. (B) In vitro ULK1 kinase assay. Immunoprecipitated GFP protein, wild-type ULK1 (WT) or kinase-dead ULK1 (KD) was incubated with purified His-ATG9N-HA peptide (aa1-66) at 30 °C for 30 min in kinase buffer. (C) In vitro ULK1 kinase assay was performed as described in B using His-ATG9N-HA or the indicated point-mutated peptides. (D) HeLa cells were treated with or without EBSS for 2 h in the presence or absence of the PI3K inhibitor Wortmannin (100 nM) or the lysosome inhibitor BA1 (20 nM), and then collected for immunoprecipitation with anti-ULK1 antibody. (E) ULK1 protein produced by an in vitro transcription/translation system was incubated with immobilized His-ATG9N-HA peptide at 4 °C overnight. (F) HeLa cells were transfected with GFP-ULK1 or KD ULK1 for 24 h, and collected for immunoprecipitation with anti-mATG9 antibody. (G) HeLa cells were treated with EBSS for the indicated times, and then collected for immunoprecipitation with anti-mATG9 antibody. The proportion of phospho-mATG9 was assessed by immunoblotting with specific antibodies against Y8 or S14. (H) Parental cells or Ulk1 KO HeLa cells were treated with or without EBSS for 2 h. Cells were collected for immunoprecipitation with anti-mATG9 antibody, and phospho-mATG9 was assessed by immunoblotting with a specific antibody against Y8 or S14. (I , J) HEK293T cells were co-transfected with WT mATG9 or the indicated mutants, 3×Flag-AP1/2M1 and GFP, GFP-ULK1 or KD ULK1 for 24 h, and then collected for immunoprecipitation with anti-Flag antibody. (K , L) HeLa cells were co-transfected with WT mATG9 or the S14A mutant and 3×Flag-AP1/2M1 for 24 h, and then starved in EBSS for 2 h. Cells were collected for immunoprecipitation with anti-Flag antibody. See also Supplementary information, Figure S4 .

    Techniques Used: In Vitro, Kinase Assay, Immunoprecipitation, Incubation, Purification, Produced, Transfection, Mutagenesis

    Src directly phosphorylates the mATG9 N-terminus at Y8. (A) Purified His-ATG9N-HA peptide (aa1-66) was incubated with control lysis buffer or HEK293T cell lysate and incubated at 30 °C for 30 min in kinase buffer. The reaction products were subjected to western blotting using anti-phosphotyrosine and anti-phosphoserine/threonine antibodies to analyze the phosphorylation status of the mATG9 N-terminus. (B) Alignment of mATG9 N-terminal sequences from different mammalian species reveals the conserved consensus motif for Src kinase. The phosphorylated tyrosine (Y) is in red. (C) In vitro Src kinase assay. Immunoprecipitated wild-type (WT) or kinase-dead (KD) Src was incubated with purified His-ATG9N-HA peptide at 30 °C for 30 min in kinase buffer. (D) GST or purified recombinant GST-Src (Sigma) protein was incubated with purified His-ATG9N-HA peptide at 30 °C for 30 min in kinase buffer to assay Src kinase activity in vitro . (E) In vitro Src kinase assay was performed as described in C using His-ATG9N-HA or the indicated point-mutated peptides. (F) HeLa cells were harvested and lysed by NP-40 lysis buffer for immunoprecipitation with anti-mATG9 antibody. (G) HEK293T cell lysate was incubated with immobilized His-ATG9N-HA peptide at 4 °C overnight. (H) HEK293T cells were co-transfected with WT mATG9-Myc or the indicated mutants and Src-GFP or KD Src for 24 h. Total mATG9 was immunoprecipitated with anti-Myc antibody and the phosphotyrosine level was assessed by immunoblotting with anti-phosphotyrosine antibody. See also Supplementary information, Figures S2 and S3A .
    Figure Legend Snippet: Src directly phosphorylates the mATG9 N-terminus at Y8. (A) Purified His-ATG9N-HA peptide (aa1-66) was incubated with control lysis buffer or HEK293T cell lysate and incubated at 30 °C for 30 min in kinase buffer. The reaction products were subjected to western blotting using anti-phosphotyrosine and anti-phosphoserine/threonine antibodies to analyze the phosphorylation status of the mATG9 N-terminus. (B) Alignment of mATG9 N-terminal sequences from different mammalian species reveals the conserved consensus motif for Src kinase. The phosphorylated tyrosine (Y) is in red. (C) In vitro Src kinase assay. Immunoprecipitated wild-type (WT) or kinase-dead (KD) Src was incubated with purified His-ATG9N-HA peptide at 30 °C for 30 min in kinase buffer. (D) GST or purified recombinant GST-Src (Sigma) protein was incubated with purified His-ATG9N-HA peptide at 30 °C for 30 min in kinase buffer to assay Src kinase activity in vitro . (E) In vitro Src kinase assay was performed as described in C using His-ATG9N-HA or the indicated point-mutated peptides. (F) HeLa cells were harvested and lysed by NP-40 lysis buffer for immunoprecipitation with anti-mATG9 antibody. (G) HEK293T cell lysate was incubated with immobilized His-ATG9N-HA peptide at 4 °C overnight. (H) HEK293T cells were co-transfected with WT mATG9-Myc or the indicated mutants and Src-GFP or KD Src for 24 h. Total mATG9 was immunoprecipitated with anti-Myc antibody and the phosphotyrosine level was assessed by immunoblotting with anti-phosphotyrosine antibody. See also Supplementary information, Figures S2 and S3A .

    Techniques Used: Purification, Incubation, Lysis, Western Blot, In Vitro, Kinase Assay, Immunoprecipitation, Recombinant, Activity Assay, Transfection

    Src phosphorylates mATG9 at Y8 to promote constitutive trafficking of mATG9. (A) HeLa cells were transfected with Src-GFP or kinase-dead (KD) Src for 24 h, and collected for immunoprecipitation with anti-mATG9 antibody. (B) HeLa cells were treated with the Src kinase inhibitors PP2 (10 μM) or SU6656 (10 μM) for 12 h and collected for immunoprecipitation with anti-mATG9 antibody. (C , D) HEK293T cells were co-transfected with mATG9-Myc, 3×Flag-AP1/2M1 and GFP vector, Src-GFP or KD Src for 24 h, and then collected for immunoprecipitation with anti-Flag antibody. (E , F) HEK293T cells were co-transfected with WT mATG9 or the Y8F mutant, 3×Flag-AP1/2M1 and Src-GFP for 24 h, and then collected for immunoprecipitation with anti-Flag antibody. (G) HeLa cells were co-transfected with WT mATG9-Myc or the Y8F mutant and 3×Flag-AP1/2M1 for 24 h, and then collected for immunoprecipitation with anti-Flag antibody. (H , I) HeLa cells were co-transfected with mATG9 and 3×Flag-AP1/2M1 for 24 h, and then treated with vehicle or the Src kinase inhibitors PP2 (10 μM) or SU6656 (10 μM) for 12 h. Cells were collected for immunoprecipitation with anti-Flag antibody. (J) U2OS cells were co-transfected with WT mATG9-Myc or the indicated mutants (red) and GFP vector, Src-GFP or KD Src (green) for 24 h, and then fixed and immunostained with anti-Myc antibody. Scale bar, 10 μm. See also Supplementary information, Figure S3B-S3C .
    Figure Legend Snippet: Src phosphorylates mATG9 at Y8 to promote constitutive trafficking of mATG9. (A) HeLa cells were transfected with Src-GFP or kinase-dead (KD) Src for 24 h, and collected for immunoprecipitation with anti-mATG9 antibody. (B) HeLa cells were treated with the Src kinase inhibitors PP2 (10 μM) or SU6656 (10 μM) for 12 h and collected for immunoprecipitation with anti-mATG9 antibody. (C , D) HEK293T cells were co-transfected with mATG9-Myc, 3×Flag-AP1/2M1 and GFP vector, Src-GFP or KD Src for 24 h, and then collected for immunoprecipitation with anti-Flag antibody. (E , F) HEK293T cells were co-transfected with WT mATG9 or the Y8F mutant, 3×Flag-AP1/2M1 and Src-GFP for 24 h, and then collected for immunoprecipitation with anti-Flag antibody. (G) HeLa cells were co-transfected with WT mATG9-Myc or the Y8F mutant and 3×Flag-AP1/2M1 for 24 h, and then collected for immunoprecipitation with anti-Flag antibody. (H , I) HeLa cells were co-transfected with mATG9 and 3×Flag-AP1/2M1 for 24 h, and then treated with vehicle or the Src kinase inhibitors PP2 (10 μM) or SU6656 (10 μM) for 12 h. Cells were collected for immunoprecipitation with anti-Flag antibody. (J) U2OS cells were co-transfected with WT mATG9-Myc or the indicated mutants (red) and GFP vector, Src-GFP or KD Src (green) for 24 h, and then fixed and immunostained with anti-Myc antibody. Scale bar, 10 μm. See also Supplementary information, Figure S3B-S3C .

    Techniques Used: Transfection, Immunoprecipitation, Plasmid Preparation, Mutagenesis

    The mATG9 N-terminus contains two conserved adaptor protein sorting signals. (A) Alignment of mATG9 N-terminus sequences from different mammalian species reveals that two putative AP sorting signals (red color letters) are highly conserved. (B , C) HEK293T cells were transiently co-transfected with wild-type (WT) mATG9-Myc or the indicated mutants and 3×Flag-AP1/2M1. 24 h after transfection, cells were collected for immunoprecipitation with anti-Flag antibody. M1 is the mu subunit of the AP1 and AP2 complexes. (D) U2OS cells were co-transfected with WT mATG9-Myc or the indicated mutants (red) and TGN46-GFP (green). 24 h after transfection, cells were fixed and immunostained with anti-Myc antibody. Cells were counterstained with DAPI (blue). Scale bar, 10 μm. (E) The distribution of mATG9 or the indicated mutants in cells from D was assessed and quantified in a blind fashion (mean ± SEM; n = 100 cells from three independent experiments, ** P
    Figure Legend Snippet: The mATG9 N-terminus contains two conserved adaptor protein sorting signals. (A) Alignment of mATG9 N-terminus sequences from different mammalian species reveals that two putative AP sorting signals (red color letters) are highly conserved. (B , C) HEK293T cells were transiently co-transfected with wild-type (WT) mATG9-Myc or the indicated mutants and 3×Flag-AP1/2M1. 24 h after transfection, cells were collected for immunoprecipitation with anti-Flag antibody. M1 is the mu subunit of the AP1 and AP2 complexes. (D) U2OS cells were co-transfected with WT mATG9-Myc or the indicated mutants (red) and TGN46-GFP (green). 24 h after transfection, cells were fixed and immunostained with anti-Myc antibody. Cells were counterstained with DAPI (blue). Scale bar, 10 μm. (E) The distribution of mATG9 or the indicated mutants in cells from D was assessed and quantified in a blind fashion (mean ± SEM; n = 100 cells from three independent experiments, ** P

    Techniques Used: Transfection, Immunoprecipitation

    Phosphorylation of Y8 and S14 cooperates to regulate mATG9 trafficking and redistribution. (A , B) HEK293T cells were co-transfected with 3×Flag-AP1/2M1 and GFP vector, Src-GFP and/or GFP-ULK1 for 24 h, and then collected for immunoprecipitation with anti-Flag antibody. (C) HEK293T cells were co-transfected with WT mATG9-Myc or the FA mutant, 3×Flag-AP1/2M1, Src-GFP and GFP-ULK1 for 24 h, and then collected for immunoprecipitation with anti-Flag antibody. (D) HEK293T cells were co-transfected with WT mATG9-Myc or the indicated mutants, 3×Flag-AP1/2M1, GFP vector or GFP-ULK1 for 24 h, and then collected for immunoprecipitation with anti-Flag antibody. (E) HeLa cells were co-transfected with WT mATG9-Myc or the indicated mutants and 3×Flag-AP1/2M1 for 24 h, and then starved in EBSS for 2 h. Cells were collected for immunoprecipitation with anti-Flag antibody. (F) HEK293T cells were co-transfected with 3×Flag-TBC1D5 and GFP vector, Src-GFP and/or GFP-ULK1 for 24 h, and then collected for immunoprecipitation with anti-Flag antibody. See also Supplementary information, Figure S5 .
    Figure Legend Snippet: Phosphorylation of Y8 and S14 cooperates to regulate mATG9 trafficking and redistribution. (A , B) HEK293T cells were co-transfected with 3×Flag-AP1/2M1 and GFP vector, Src-GFP and/or GFP-ULK1 for 24 h, and then collected for immunoprecipitation with anti-Flag antibody. (C) HEK293T cells were co-transfected with WT mATG9-Myc or the FA mutant, 3×Flag-AP1/2M1, Src-GFP and GFP-ULK1 for 24 h, and then collected for immunoprecipitation with anti-Flag antibody. (D) HEK293T cells were co-transfected with WT mATG9-Myc or the indicated mutants, 3×Flag-AP1/2M1, GFP vector or GFP-ULK1 for 24 h, and then collected for immunoprecipitation with anti-Flag antibody. (E) HeLa cells were co-transfected with WT mATG9-Myc or the indicated mutants and 3×Flag-AP1/2M1 for 24 h, and then starved in EBSS for 2 h. Cells were collected for immunoprecipitation with anti-Flag antibody. (F) HEK293T cells were co-transfected with 3×Flag-TBC1D5 and GFP vector, Src-GFP and/or GFP-ULK1 for 24 h, and then collected for immunoprecipitation with anti-Flag antibody. See also Supplementary information, Figure S5 .

    Techniques Used: Transfection, Plasmid Preparation, Immunoprecipitation, Mutagenesis

    40) Product Images from "The cAMP sensors, EPAC1 and EPAC2, display distinct subcellular distributions despite sharing a common nuclear pore localisation signal"

    Article Title: The cAMP sensors, EPAC1 and EPAC2, display distinct subcellular distributions despite sharing a common nuclear pore localisation signal

    Journal: Cellular Signalling

    doi: 10.1016/j.cellsig.2015.02.009

    Active EPAC1 is localised to the nucleus in HEK293T cells. HEK293T stably expressing either empty vector or EPAC1-FLAG construct was treated with or without a combination of forskolin and rolipram (F/R, 10 μM, 60′). Cells were fixed for immunofluorescence using anti-EPAC1 (5D3) antibodies. HEK293T cells were transiently transfected with either wild type EPAC1 or EPAC1-R279E constructs and then incubated with F/R (10 μM, 60′), followed by immuno-detection with anti-EPAC1 (5D3). Immunoprecipitation of EPAC1 from stably transfected HEK293T cells. Cell lysates (input) were immunoprecipitated with anti-IgG (mouse), anti-EPAC1 (5D3) or anti-FLAG antibodies (square indicates lane moved for ease of presentation). HEK293T cells were transiently transfected with a EPAC1-HA construct. Cells were then treated with F/R (10 μM, 60′), fixed and probed using anti-EPAC1 (5D3) or anti-HA antibodies as indicated.
    Figure Legend Snippet: Active EPAC1 is localised to the nucleus in HEK293T cells. HEK293T stably expressing either empty vector or EPAC1-FLAG construct was treated with or without a combination of forskolin and rolipram (F/R, 10 μM, 60′). Cells were fixed for immunofluorescence using anti-EPAC1 (5D3) antibodies. HEK293T cells were transiently transfected with either wild type EPAC1 or EPAC1-R279E constructs and then incubated with F/R (10 μM, 60′), followed by immuno-detection with anti-EPAC1 (5D3). Immunoprecipitation of EPAC1 from stably transfected HEK293T cells. Cell lysates (input) were immunoprecipitated with anti-IgG (mouse), anti-EPAC1 (5D3) or anti-FLAG antibodies (square indicates lane moved for ease of presentation). HEK293T cells were transiently transfected with a EPAC1-HA construct. Cells were then treated with F/R (10 μM, 60′), fixed and probed using anti-EPAC1 (5D3) or anti-HA antibodies as indicated.

    Techniques Used: Stable Transfection, Expressing, Plasmid Preparation, Construct, Immunofluorescence, Transfection, Incubation, Immunoprecipitation

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    Article Snippet: .. Quantitative RT-PCR Total RNA from human and murine macrophages was purified using the High Pure RNA Isolation kit from Roche. ..

    Purification:

    Article Title: HSV Infection Induces Production of ROS, which Potentiate Signaling from Pattern Recognition Receptors: Role for S-glutathionylation of TRAF3 and 6
    Article Snippet: .. Quantitative RT-PCR Total RNA from human and murine macrophages was purified using the High Pure RNA Isolation kit from Roche. ..

    Isolation:

    Article Title: HSV Infection Induces Production of ROS, which Potentiate Signaling from Pattern Recognition Receptors: Role for S-glutathionylation of TRAF3 and 6
    Article Snippet: .. Quantitative RT-PCR Total RNA from human and murine macrophages was purified using the High Pure RNA Isolation kit from Roche. ..

    Protease Inhibitor:

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    Article Snippet: .. HEK293T cells were lysed in NP-40 buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1% [vol/vol] NP-40, protease inhibitor cocktail [Roche], and Ser/Thr-phosphatase inhibitor cocktail [Sigma]), followed by centrifugation at 13,000 rpm for 20 min. For GST pull-down assays, postcentrifugation supernatants were mixed with a 50% slurry of glutathione-conjugated Sepharose beads (Amersham Biosciences), and the binding reaction mix was incubated for 3 to 4 h at 4°C. ..

    Article Title: TRAPPC9 Mediates the Interaction between p150Glued and COPII Vesicles at the Target Membrane
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    Article Title: Characterization of Mammalian Selenoprotein O: A Redox-Active Mitochondrial Protein
    Article Snippet: .. 75 Se Metabolic Labeling and Identifying Human SelO HEK293T cells were metabolically labeled with 75 Se for 40 h. The cells were lysed in 0.1% NP40 cell lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5% Glycerol) containing protease inhibitor (Roche Applied Science, Indianapolis, IN, USA) and 20 mM N -ethylmaleimide. ..

    Article Title: Epstein-Barr Virus LF2 Protein Regulates Viral Replication by Altering Rta Subcellular Localization ▿Epstein-Barr Virus LF2 Protein Regulates Viral Replication by Altering Rta Subcellular Localization ▿ †
    Article Snippet: .. Transfected 293T cells were lysed in high-salt NP-40 lysis buffer (1% [vol/vol] Igepal CA-630, 40 mM Tris-HCl [pH 7.5], 300 mM NaCl, and 10 mM MgCl2 ) containing EDTA-free Complete protease inhibitor (Roche, Mannheim, Germany) and cleared by centrifugation at 10,000 × g for 10 min. Supernatants were diluted 1:1 with 1% (vol/vol) NP-40 and precleared with Sepharose (Sigma) for 2 h at 4°C before incubation with anti-Flag M2 agarose (Sigma) overnight at 4°C. ..

    Article Title: Lucanthone and Its Derivative Hycanthone Inhibit Apurinic Endonuclease-1 (APE1) by Direct Protein Binding
    Article Snippet: .. Cleavage of APE1 by lucanthone and CRT0044876 Western blotting was carried out by 7.5% SDS-PAGE of cell extracts (20 µg total protein per lane) either from APE1 overexpresser clone 5 pretreated with 2.5–200 µM concentration of lucanthone/CRT0044876 or recombinant APE1 for 2 h at 37°C in presence of protease inhibitor cocktail (2 tablets (Roche, # 11836153001) containing mixture of several protease inhibitors with broad inhibitory specificity for serine, cysteine and metalloproteases in all systems, dissolved in 20 ml of APE1 buffer). ..

    Article Title: Rab27a controls HIV-1 assembly by regulating plasma membrane levels of phosphatidylinositol 4,5-bisphosphate
    Article Snippet: .. Immunoprecipitation of GFP-CD63 For immunoprecipitation of GFP-CD63, 107 GFP-CD63–transfected cells were lysed in immunoprecipitation buffer (1% Brij 99, 10 mM Tris/HCl, pH 7.4, 150 mM NaCl, and 5 mM EDTA, with Roche protease inhibitor cocktail) for 1 h at 4°C. ..

    Centrifugation:

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    Article Title: Epstein-Barr Virus LF2 Protein Regulates Viral Replication by Altering Rta Subcellular Localization ▿Epstein-Barr Virus LF2 Protein Regulates Viral Replication by Altering Rta Subcellular Localization ▿ †
    Article Snippet: .. Transfected 293T cells were lysed in high-salt NP-40 lysis buffer (1% [vol/vol] Igepal CA-630, 40 mM Tris-HCl [pH 7.5], 300 mM NaCl, and 10 mM MgCl2 ) containing EDTA-free Complete protease inhibitor (Roche, Mannheim, Germany) and cleared by centrifugation at 10,000 × g for 10 min. Supernatants were diluted 1:1 with 1% (vol/vol) NP-40 and precleared with Sepharose (Sigma) for 2 h at 4°C before incubation with anti-Flag M2 agarose (Sigma) overnight at 4°C. ..

    Binding Assay:

    Article Title: Phosphorylation-Mediated Negative Regulation of RIG-I Antiviral Activity ▿
    Article Snippet: .. HEK293T cells were lysed in NP-40 buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1% [vol/vol] NP-40, protease inhibitor cocktail [Roche], and Ser/Thr-phosphatase inhibitor cocktail [Sigma]), followed by centrifugation at 13,000 rpm for 20 min. For GST pull-down assays, postcentrifugation supernatants were mixed with a 50% slurry of glutathione-conjugated Sepharose beads (Amersham Biosciences), and the binding reaction mix was incubated for 3 to 4 h at 4°C. ..

    Incubation:

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    Transfection:

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    Lysis:

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    Labeling:

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    Metabolic Labelling:

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    Western Blot:

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    SDS Page:

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    Concentration Assay:

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    Roche 293t cells
    LF2 binding is required for inhibition of Rta activation of RRE-containing lytic promoters, but Rta sumoylation is not. Reporter assay results from <t>293T</t> cells transfected with luciferase reporter constructs from the BMLF1 (A) or BMRF1 (B) promoter with
    293t Cells, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Roche hek 293 cells
    Pull down experiments of TRPV1 and ARMS in transfected <t>HEK</t> 293 cells and native neuronal tissue A) Co-immunoprecipitation studies were performed in a heterologous (HEK293 cells, left panel) and native (DRG neurons, right panel) expression system. ARMS was precipitated using an antibody directed against ARMS followed by a subsequent detection of TRPV1 using a TRPV1 antibody (solid arrows). BSA-coated agarose beads served as a control. B) Co-immunoprecipitation studies were performed in a heterologous (HEK293 cells, left panel) and native (DRG neurons, right panel) expression system. TRPV1 was precipitated using an antibody directed against TRPV1 followed by a subsequent detection of ARMS using an ARMS antibody (open arrows). BSA-coated agarose beads and control peptide against TRPV1 served as a control. C) Pulldown experiments with Rp-8-AEA-cAMPS-agarose to pull down PKA holoenzyme of TRPV1 and ARMS expressing HEK 293 cells and control proteins of PKA isoforms. Afterwards SDS-PAGE analysis was applied with antibodies directed against TRPV1 (red at 90 kDa) and ARMS (green at 190 kDa). D) Membrane of C was stripped to remove the first set of antibodies and incubated with antibodies against PKA subunits RI (green at 50 kDa) and C (red at 40 kDa). Both samples of transfected cells and control protein samples showed positive fluorescent signals RI and C-subunit of PKA. E) Pull down experiments with control agarose (PKA cannot bind to the coupled beads) of transfected HEK 293 cells and control proteins for PKA isoforms. Afterwards SDS-PAGE was applied and the membrane was treated with antibodies against TRPV1 (red at 90 kDa) and ARMS (green at 190 kDa). F) Membrane of E was stripped to remove the first set of antibodies and incubated with antibodies against PKA subunits RI (green at 50 kDa) and C (red at 40 kDa). Only control protein samples showed fluorescent signals. (B = beads, FT = flow through, I = input IP = immunoprecipitation, nt = not transfected)
    Hek 293 Cells, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche hela cells
    FKBP10 partitions GC to the ERAD pathway, independent of its PPIase or Ca 2+ binding activity. ( A ) Overexpressing FKBP10 or EDEM1 decreases the steady state levels of <t>VSVG-tagged</t> GC in <t>HeLa</t> cells. Both FKBP10 and EDEM1 bind to GC. ( B ) Inhibiting proteasomal
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    Roche endoglycosidase h
    Indirect immunofluorescence microscopy suggests localization of Aut5-Ha at the ER in wild-type cells and in the vacuole and the ER in  pep4 Δ cells. (A to C) Cells grown to stationary phase were starved for 4 h in 1% K acetate. The cells were then fixed with formaldehyde, followed by spheroplasting with Zymolyase. The spheroplasted cells were then incubated with a primary antibody against Ha and afterwards with a secondary Cy3-coupled antibody. Nuclear DNA was further stained with DAPI. From left to right Nomarski optics (Nom), immunofluorescence microscopy (Aut5-Ha), and nuclear staining with DAPI (DNA/DAPI) are shown. (A) Wild-type cells expressing Aut5-Ha from the chromosome under the control of the endogenous promoter. (B)  pep4 Δ cells expressing Aut5-Ha chromosomally, which due to the lack of vacuolar endoproteinase A are defective in vacuolar protein breakdown. Arrowheads point to ER staining. (C) Chromosomal expression of Aut5-Ha in  aut3 Δ  pep4 Δ cells, which are further defective in autophagy. Bar, 20 μm. (D) Pulse-chase analysis indicates a rapid breakdown of Aut5-Ha dependent on vacuolar proteinase A. The indicated strains were grown to log phase in SMD medium, pulse-labeled for 20 min with [ 35 S]methionine and cysteine, and chased in nonradioactive medium as described in Materials and Methods. At the specified times, aliquots were withdrawn and lysates were immunoprecipitated with antibodies to Ha. After deglycosylation with endoglycosidase H, samples were subjected to SDS-PAGE and labeled proteins were visualized using a PhosphorImager. wt, wild type.
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    LF2 binding is required for inhibition of Rta activation of RRE-containing lytic promoters, but Rta sumoylation is not. Reporter assay results from 293T cells transfected with luciferase reporter constructs from the BMLF1 (A) or BMRF1 (B) promoter with

    Journal: Journal of Virology

    Article Title: Epstein-Barr Virus LF2 Protein Regulates Viral Replication by Altering Rta Subcellular Localization ▿Epstein-Barr Virus LF2 Protein Regulates Viral Replication by Altering Rta Subcellular Localization ▿ †

    doi: 10.1128/JVI.00573-10

    Figure Lengend Snippet: LF2 binding is required for inhibition of Rta activation of RRE-containing lytic promoters, but Rta sumoylation is not. Reporter assay results from 293T cells transfected with luciferase reporter constructs from the BMLF1 (A) or BMRF1 (B) promoter with

    Article Snippet: Transfected 293T cells were lysed in high-salt NP-40 lysis buffer (1% [vol/vol] Igepal CA-630, 40 mM Tris-HCl [pH 7.5], 300 mM NaCl, and 10 mM MgCl2 ) containing EDTA-free Complete protease inhibitor (Roche, Mannheim, Germany) and cleared by centrifugation at 10,000 × g for 10 min. Supernatants were diluted 1:1 with 1% (vol/vol) NP-40 and precleared with Sepharose (Sigma) for 2 h at 4°C before incubation with anti-Flag M2 agarose (Sigma) overnight at 4°C.

    Techniques: Binding Assay, Inhibition, Activation Assay, Reporter Assay, Transfection, Luciferase, Construct

    LF2 does not inhibit Rta activation of the BRLF1 or BZLF1 promoter. Reporter assay results from 293T cells transfected with luciferase reporter constructs from the BLRF1 (Rp) (i) or BZLF1 (Zp) promoter (ii) with or without Rta-V5 and LF2-HA are shown.

    Journal: Journal of Virology

    Article Title: Epstein-Barr Virus LF2 Protein Regulates Viral Replication by Altering Rta Subcellular Localization ▿Epstein-Barr Virus LF2 Protein Regulates Viral Replication by Altering Rta Subcellular Localization ▿ †

    doi: 10.1128/JVI.00573-10

    Figure Lengend Snippet: LF2 does not inhibit Rta activation of the BRLF1 or BZLF1 promoter. Reporter assay results from 293T cells transfected with luciferase reporter constructs from the BLRF1 (Rp) (i) or BZLF1 (Zp) promoter (ii) with or without Rta-V5 and LF2-HA are shown.

    Article Snippet: Transfected 293T cells were lysed in high-salt NP-40 lysis buffer (1% [vol/vol] Igepal CA-630, 40 mM Tris-HCl [pH 7.5], 300 mM NaCl, and 10 mM MgCl2 ) containing EDTA-free Complete protease inhibitor (Roche, Mannheim, Germany) and cleared by centrifugation at 10,000 × g for 10 min. Supernatants were diluted 1:1 with 1% (vol/vol) NP-40 and precleared with Sepharose (Sigma) for 2 h at 4°C before incubation with anti-Flag M2 agarose (Sigma) overnight at 4°C.

    Techniques: Activation Assay, Reporter Assay, Transfection, Luciferase, Construct

    Coexpression of LF2 results in redistribution of Rta out of the nucleus to the cytoskeleton. (A) Crude cytoplasmic nuclear fractionation and subsequent NP-40 extraction of the nuclear fraction. 293T cells were transfected with Rta, Flag-LF2, or both,

    Journal: Journal of Virology

    Article Title: Epstein-Barr Virus LF2 Protein Regulates Viral Replication by Altering Rta Subcellular Localization ▿Epstein-Barr Virus LF2 Protein Regulates Viral Replication by Altering Rta Subcellular Localization ▿ †

    doi: 10.1128/JVI.00573-10

    Figure Lengend Snippet: Coexpression of LF2 results in redistribution of Rta out of the nucleus to the cytoskeleton. (A) Crude cytoplasmic nuclear fractionation and subsequent NP-40 extraction of the nuclear fraction. 293T cells were transfected with Rta, Flag-LF2, or both,

    Article Snippet: Transfected 293T cells were lysed in high-salt NP-40 lysis buffer (1% [vol/vol] Igepal CA-630, 40 mM Tris-HCl [pH 7.5], 300 mM NaCl, and 10 mM MgCl2 ) containing EDTA-free Complete protease inhibitor (Roche, Mannheim, Germany) and cleared by centrifugation at 10,000 × g for 10 min. Supernatants were diluted 1:1 with 1% (vol/vol) NP-40 and precleared with Sepharose (Sigma) for 2 h at 4°C before incubation with anti-Flag M2 agarose (Sigma) overnight at 4°C.

    Techniques: Fractionation, Transfection

    LF2 binding to Rta is required for repression and redistribution of Rta. (A) Coimmunoprecipitation assay using 293T cells transfected with V5-tagged full-length Rta (1-605) or Rta with aa 476 to 519 deleted (Δ476-519) in the presence or absence

    Journal: Journal of Virology

    Article Title: Epstein-Barr Virus LF2 Protein Regulates Viral Replication by Altering Rta Subcellular Localization ▿Epstein-Barr Virus LF2 Protein Regulates Viral Replication by Altering Rta Subcellular Localization ▿ †

    doi: 10.1128/JVI.00573-10

    Figure Lengend Snippet: LF2 binding to Rta is required for repression and redistribution of Rta. (A) Coimmunoprecipitation assay using 293T cells transfected with V5-tagged full-length Rta (1-605) or Rta with aa 476 to 519 deleted (Δ476-519) in the presence or absence

    Article Snippet: Transfected 293T cells were lysed in high-salt NP-40 lysis buffer (1% [vol/vol] Igepal CA-630, 40 mM Tris-HCl [pH 7.5], 300 mM NaCl, and 10 mM MgCl2 ) containing EDTA-free Complete protease inhibitor (Roche, Mannheim, Germany) and cleared by centrifugation at 10,000 × g for 10 min. Supernatants were diluted 1:1 with 1% (vol/vol) NP-40 and precleared with Sepharose (Sigma) for 2 h at 4°C before incubation with anti-Flag M2 agarose (Sigma) overnight at 4°C.

    Techniques: Binding Assay, Co-Immunoprecipitation Assay, Transfection

    Rta aa 500 to 526 are sufficient to confer LF2-dependent repression and redistribution onto GAL4-VP16. (A) Reporter assay results from 293T cells transfected with luciferase GAL4 reporter constructs along with GAL4-VP16 or with Rta aa 480 to 501, aa 500

    Journal: Journal of Virology

    Article Title: Epstein-Barr Virus LF2 Protein Regulates Viral Replication by Altering Rta Subcellular Localization ▿Epstein-Barr Virus LF2 Protein Regulates Viral Replication by Altering Rta Subcellular Localization ▿ †

    doi: 10.1128/JVI.00573-10

    Figure Lengend Snippet: Rta aa 500 to 526 are sufficient to confer LF2-dependent repression and redistribution onto GAL4-VP16. (A) Reporter assay results from 293T cells transfected with luciferase GAL4 reporter constructs along with GAL4-VP16 or with Rta aa 480 to 501, aa 500

    Article Snippet: Transfected 293T cells were lysed in high-salt NP-40 lysis buffer (1% [vol/vol] Igepal CA-630, 40 mM Tris-HCl [pH 7.5], 300 mM NaCl, and 10 mM MgCl2 ) containing EDTA-free Complete protease inhibitor (Roche, Mannheim, Germany) and cleared by centrifugation at 10,000 × g for 10 min. Supernatants were diluted 1:1 with 1% (vol/vol) NP-40 and precleared with Sepharose (Sigma) for 2 h at 4°C before incubation with anti-Flag M2 agarose (Sigma) overnight at 4°C.

    Techniques: Reporter Assay, Transfection, Luciferase, Construct

    LF2 mutants defective for Rta redistribution cannot repress Rta. (A) Reporter assay results from 293T cells transfected with a BALF2 promoter-driven luciferase reporter with or without Rta in the presence or absence of HA-tagged full-length LF2 (1-429)

    Journal: Journal of Virology

    Article Title: Epstein-Barr Virus LF2 Protein Regulates Viral Replication by Altering Rta Subcellular Localization ▿Epstein-Barr Virus LF2 Protein Regulates Viral Replication by Altering Rta Subcellular Localization ▿ †

    doi: 10.1128/JVI.00573-10

    Figure Lengend Snippet: LF2 mutants defective for Rta redistribution cannot repress Rta. (A) Reporter assay results from 293T cells transfected with a BALF2 promoter-driven luciferase reporter with or without Rta in the presence or absence of HA-tagged full-length LF2 (1-429)

    Article Snippet: Transfected 293T cells were lysed in high-salt NP-40 lysis buffer (1% [vol/vol] Igepal CA-630, 40 mM Tris-HCl [pH 7.5], 300 mM NaCl, and 10 mM MgCl2 ) containing EDTA-free Complete protease inhibitor (Roche, Mannheim, Germany) and cleared by centrifugation at 10,000 × g for 10 min. Supernatants were diluted 1:1 with 1% (vol/vol) NP-40 and precleared with Sepharose (Sigma) for 2 h at 4°C before incubation with anti-Flag M2 agarose (Sigma) overnight at 4°C.

    Techniques: Reporter Assay, Transfection, Luciferase

    Pull down experiments of TRPV1 and ARMS in transfected HEK 293 cells and native neuronal tissue A) Co-immunoprecipitation studies were performed in a heterologous (HEK293 cells, left panel) and native (DRG neurons, right panel) expression system. ARMS was precipitated using an antibody directed against ARMS followed by a subsequent detection of TRPV1 using a TRPV1 antibody (solid arrows). BSA-coated agarose beads served as a control. B) Co-immunoprecipitation studies were performed in a heterologous (HEK293 cells, left panel) and native (DRG neurons, right panel) expression system. TRPV1 was precipitated using an antibody directed against TRPV1 followed by a subsequent detection of ARMS using an ARMS antibody (open arrows). BSA-coated agarose beads and control peptide against TRPV1 served as a control. C) Pulldown experiments with Rp-8-AEA-cAMPS-agarose to pull down PKA holoenzyme of TRPV1 and ARMS expressing HEK 293 cells and control proteins of PKA isoforms. Afterwards SDS-PAGE analysis was applied with antibodies directed against TRPV1 (red at 90 kDa) and ARMS (green at 190 kDa). D) Membrane of C was stripped to remove the first set of antibodies and incubated with antibodies against PKA subunits RI (green at 50 kDa) and C (red at 40 kDa). Both samples of transfected cells and control protein samples showed positive fluorescent signals RI and C-subunit of PKA. E) Pull down experiments with control agarose (PKA cannot bind to the coupled beads) of transfected HEK 293 cells and control proteins for PKA isoforms. Afterwards SDS-PAGE was applied and the membrane was treated with antibodies against TRPV1 (red at 90 kDa) and ARMS (green at 190 kDa). F) Membrane of E was stripped to remove the first set of antibodies and incubated with antibodies against PKA subunits RI (green at 50 kDa) and C (red at 40 kDa). Only control protein samples showed fluorescent signals. (B = beads, FT = flow through, I = input IP = immunoprecipitation, nt = not transfected)

    Journal: European journal of pain (London, England)

    Article Title: Ankyrin-rich membrane spanning protein as a novel modulator of transient receptor potential vanilloid 1-function in nociceptive neurons

    doi: 10.1002/ejp.1008

    Figure Lengend Snippet: Pull down experiments of TRPV1 and ARMS in transfected HEK 293 cells and native neuronal tissue A) Co-immunoprecipitation studies were performed in a heterologous (HEK293 cells, left panel) and native (DRG neurons, right panel) expression system. ARMS was precipitated using an antibody directed against ARMS followed by a subsequent detection of TRPV1 using a TRPV1 antibody (solid arrows). BSA-coated agarose beads served as a control. B) Co-immunoprecipitation studies were performed in a heterologous (HEK293 cells, left panel) and native (DRG neurons, right panel) expression system. TRPV1 was precipitated using an antibody directed against TRPV1 followed by a subsequent detection of ARMS using an ARMS antibody (open arrows). BSA-coated agarose beads and control peptide against TRPV1 served as a control. C) Pulldown experiments with Rp-8-AEA-cAMPS-agarose to pull down PKA holoenzyme of TRPV1 and ARMS expressing HEK 293 cells and control proteins of PKA isoforms. Afterwards SDS-PAGE analysis was applied with antibodies directed against TRPV1 (red at 90 kDa) and ARMS (green at 190 kDa). D) Membrane of C was stripped to remove the first set of antibodies and incubated with antibodies against PKA subunits RI (green at 50 kDa) and C (red at 40 kDa). Both samples of transfected cells and control protein samples showed positive fluorescent signals RI and C-subunit of PKA. E) Pull down experiments with control agarose (PKA cannot bind to the coupled beads) of transfected HEK 293 cells and control proteins for PKA isoforms. Afterwards SDS-PAGE was applied and the membrane was treated with antibodies against TRPV1 (red at 90 kDa) and ARMS (green at 190 kDa). F) Membrane of E was stripped to remove the first set of antibodies and incubated with antibodies against PKA subunits RI (green at 50 kDa) and C (red at 40 kDa). Only control protein samples showed fluorescent signals. (B = beads, FT = flow through, I = input IP = immunoprecipitation, nt = not transfected)

    Article Snippet: Samples of transfected HEK 293 cells, DRG neurons or SC were lysed in a buffer containing 150 mM NaCl, 50 mM TRIS, 10 mM HEPES, 1 % Triton X-100, 1× Protease Inhibitor Mix (Complete protease inhibitor cocktail, Roche Diagnostics, Switzerland) at pH 7.8.

    Techniques: Transfection, Immunoprecipitation, Expressing, SDS Page, Incubation, Flow Cytometry

    Capsaicin-induced calcium influx in TRPV1- and TRPV1/ARMS-expressing HEK 293 cells A) Mean change of the fura ratio (± SEM, dashed lines) after 1 nM (20 s) and 3 μM (80 s) capsaicin of TRPV1 (black line) and TRPV1/ARMS (red line) expressing cells. B-D) Representative images of the capsaicin-induced (0 nM (B), 1 nM (C) and 3 μM (D)) change of the fluorescence intensity of cells only expressing TRPV1. E-F) Representative images of the capsaicin-induced (0 nM (E), 1 nM (F) and 3 μM (G)) change of the fluorescence intensity of cells co-expressing TRPV1 and ARMS. H) Concentration-response curves of capsaicin-induced calcium influx normalized to the maximum induced response at 3 μM capsaicin of TRPV1- (black line) and TRPV1/ARMS- (red line) expressing cells. Curves are fitted according to the Hill equation. Data are shown as means + S.E.M. (TRPV1 represented by the black curve; n = 74 (1 pM), n = 34 (100 pM), n = 26 (500 pM), n = 41 (1 nM), n = 46 (20 nM), n = 45 (100 nM), n = 62 (200 nM), n = 85 (1 μM), n = 44 (3 μM) or TRPV1/ARMS represented by the red curve; n = 38 (1 pM), n = 59 (100 pM), n = 69 (500 pM), n = 57 (1 nM), n = 141 (20 nM), n = 64 (100 nM), n = 80 (200 nM), n = 46 (1 μM), n = 54 (3 μM)). I) EC 50 values (± 95% CI) generated from capsaicin-induced dose response curves of HEK 293 cells expressing TRPV1 alone (black bar) or TRPV1 and ARMS (empty bar) without pre-treatment or with 20 min H89 pre-treatment (unpaired t-test, **, p

    Journal: European journal of pain (London, England)

    Article Title: Ankyrin-rich membrane spanning protein as a novel modulator of transient receptor potential vanilloid 1-function in nociceptive neurons

    doi: 10.1002/ejp.1008

    Figure Lengend Snippet: Capsaicin-induced calcium influx in TRPV1- and TRPV1/ARMS-expressing HEK 293 cells A) Mean change of the fura ratio (± SEM, dashed lines) after 1 nM (20 s) and 3 μM (80 s) capsaicin of TRPV1 (black line) and TRPV1/ARMS (red line) expressing cells. B-D) Representative images of the capsaicin-induced (0 nM (B), 1 nM (C) and 3 μM (D)) change of the fluorescence intensity of cells only expressing TRPV1. E-F) Representative images of the capsaicin-induced (0 nM (E), 1 nM (F) and 3 μM (G)) change of the fluorescence intensity of cells co-expressing TRPV1 and ARMS. H) Concentration-response curves of capsaicin-induced calcium influx normalized to the maximum induced response at 3 μM capsaicin of TRPV1- (black line) and TRPV1/ARMS- (red line) expressing cells. Curves are fitted according to the Hill equation. Data are shown as means + S.E.M. (TRPV1 represented by the black curve; n = 74 (1 pM), n = 34 (100 pM), n = 26 (500 pM), n = 41 (1 nM), n = 46 (20 nM), n = 45 (100 nM), n = 62 (200 nM), n = 85 (1 μM), n = 44 (3 μM) or TRPV1/ARMS represented by the red curve; n = 38 (1 pM), n = 59 (100 pM), n = 69 (500 pM), n = 57 (1 nM), n = 141 (20 nM), n = 64 (100 nM), n = 80 (200 nM), n = 46 (1 μM), n = 54 (3 μM)). I) EC 50 values (± 95% CI) generated from capsaicin-induced dose response curves of HEK 293 cells expressing TRPV1 alone (black bar) or TRPV1 and ARMS (empty bar) without pre-treatment or with 20 min H89 pre-treatment (unpaired t-test, **, p

    Article Snippet: Samples of transfected HEK 293 cells, DRG neurons or SC were lysed in a buffer containing 150 mM NaCl, 50 mM TRIS, 10 mM HEPES, 1 % Triton X-100, 1× Protease Inhibitor Mix (Complete protease inhibitor cocktail, Roche Diagnostics, Switzerland) at pH 7.8.

    Techniques: Expressing, Fluorescence, Concentration Assay, Generated

    Capsaicin-induced TRPV1 currents in HEK 293 cells expressing TRPV1 and ARMS or only TRPV1 A) Averaged capsaicin-induced (50 nM for 10 s) TRPV1 current traces (± SEM, dashed lines) of cells only expressing TRPV1 (black line) and cell co-expressing TRPV1 and ARMS (red line). B) Mean amplitudes of the capsaicin-induced current of TRPV1- (black bar) and TRPV1/ARMS- (red bar) expressing cells (unpaired t-test, *, p

    Journal: European journal of pain (London, England)

    Article Title: Ankyrin-rich membrane spanning protein as a novel modulator of transient receptor potential vanilloid 1-function in nociceptive neurons

    doi: 10.1002/ejp.1008

    Figure Lengend Snippet: Capsaicin-induced TRPV1 currents in HEK 293 cells expressing TRPV1 and ARMS or only TRPV1 A) Averaged capsaicin-induced (50 nM for 10 s) TRPV1 current traces (± SEM, dashed lines) of cells only expressing TRPV1 (black line) and cell co-expressing TRPV1 and ARMS (red line). B) Mean amplitudes of the capsaicin-induced current of TRPV1- (black bar) and TRPV1/ARMS- (red bar) expressing cells (unpaired t-test, *, p

    Article Snippet: Samples of transfected HEK 293 cells, DRG neurons or SC were lysed in a buffer containing 150 mM NaCl, 50 mM TRIS, 10 mM HEPES, 1 % Triton X-100, 1× Protease Inhibitor Mix (Complete protease inhibitor cocktail, Roche Diagnostics, Switzerland) at pH 7.8.

    Techniques: Expressing

    Capsaicin-induced calcium influx and TRPV1 currents in TRPV1- and TRPV1/ARMS-expressing HEK 293 cells pretreated with protein kinase inhibitors A) Maximum change of the fluorescence intensity induced by 1 nM capsaicin in HEK cells expressing TRPV1 or TRPV1/ARMS without pretreatment, with 10 μM H89-, 1 μM GF 109203X- or 10 μM myr-PKI-pretreatment (2way ANOVA with Bonferroni's post test, **, p

    Journal: European journal of pain (London, England)

    Article Title: Ankyrin-rich membrane spanning protein as a novel modulator of transient receptor potential vanilloid 1-function in nociceptive neurons

    doi: 10.1002/ejp.1008

    Figure Lengend Snippet: Capsaicin-induced calcium influx and TRPV1 currents in TRPV1- and TRPV1/ARMS-expressing HEK 293 cells pretreated with protein kinase inhibitors A) Maximum change of the fluorescence intensity induced by 1 nM capsaicin in HEK cells expressing TRPV1 or TRPV1/ARMS without pretreatment, with 10 μM H89-, 1 μM GF 109203X- or 10 μM myr-PKI-pretreatment (2way ANOVA with Bonferroni's post test, **, p

    Article Snippet: Samples of transfected HEK 293 cells, DRG neurons or SC were lysed in a buffer containing 150 mM NaCl, 50 mM TRIS, 10 mM HEPES, 1 % Triton X-100, 1× Protease Inhibitor Mix (Complete protease inhibitor cocktail, Roche Diagnostics, Switzerland) at pH 7.8.

    Techniques: Expressing, Fluorescence

    FKBP10 partitions GC to the ERAD pathway, independent of its PPIase or Ca 2+ binding activity. ( A ) Overexpressing FKBP10 or EDEM1 decreases the steady state levels of VSVG-tagged GC in HeLa cells. Both FKBP10 and EDEM1 bind to GC. ( B ) Inhibiting proteasomal

    Journal: Chemistry & biology

    Article Title: FKBP10 Depletion Enhances Glucocerebrosidase Proteostasis in Gaucher's Disease Fibroblasts

    doi: 10.1016/j.chembiol.2012.11.014

    Figure Lengend Snippet: FKBP10 partitions GC to the ERAD pathway, independent of its PPIase or Ca 2+ binding activity. ( A ) Overexpressing FKBP10 or EDEM1 decreases the steady state levels of VSVG-tagged GC in HeLa cells. Both FKBP10 and EDEM1 bind to GC. ( B ) Inhibiting proteasomal

    Article Snippet: GC proteins were immunoprecipitated from HeLa cells 48 h post-transfection using the anti-VSVG antibody and the bound proteins were eluted using VSVG peptides (Roche) at 37 °C for 30 min.

    Techniques: Binding Assay, Activity Assay

    Indirect immunofluorescence microscopy suggests localization of Aut5-Ha at the ER in wild-type cells and in the vacuole and the ER in  pep4 Δ cells. (A to C) Cells grown to stationary phase were starved for 4 h in 1% K acetate. The cells were then fixed with formaldehyde, followed by spheroplasting with Zymolyase. The spheroplasted cells were then incubated with a primary antibody against Ha and afterwards with a secondary Cy3-coupled antibody. Nuclear DNA was further stained with DAPI. From left to right Nomarski optics (Nom), immunofluorescence microscopy (Aut5-Ha), and nuclear staining with DAPI (DNA/DAPI) are shown. (A) Wild-type cells expressing Aut5-Ha from the chromosome under the control of the endogenous promoter. (B)  pep4 Δ cells expressing Aut5-Ha chromosomally, which due to the lack of vacuolar endoproteinase A are defective in vacuolar protein breakdown. Arrowheads point to ER staining. (C) Chromosomal expression of Aut5-Ha in  aut3 Δ  pep4 Δ cells, which are further defective in autophagy. Bar, 20 μm. (D) Pulse-chase analysis indicates a rapid breakdown of Aut5-Ha dependent on vacuolar proteinase A. The indicated strains were grown to log phase in SMD medium, pulse-labeled for 20 min with [ 35 S]methionine and cysteine, and chased in nonradioactive medium as described in Materials and Methods. At the specified times, aliquots were withdrawn and lysates were immunoprecipitated with antibodies to Ha. After deglycosylation with endoglycosidase H, samples were subjected to SDS-PAGE and labeled proteins were visualized using a PhosphorImager. wt, wild type.

    Journal: Journal of Bacteriology

    Article Title: Aut5/Cvt17p, a Putative Lipase Essential for Disintegration of Autophagic Bodies inside the Vacuole

    doi: 10.1128/JB.183.20.5942-5955.2001

    Figure Lengend Snippet: Indirect immunofluorescence microscopy suggests localization of Aut5-Ha at the ER in wild-type cells and in the vacuole and the ER in pep4 Δ cells. (A to C) Cells grown to stationary phase were starved for 4 h in 1% K acetate. The cells were then fixed with formaldehyde, followed by spheroplasting with Zymolyase. The spheroplasted cells were then incubated with a primary antibody against Ha and afterwards with a secondary Cy3-coupled antibody. Nuclear DNA was further stained with DAPI. From left to right Nomarski optics (Nom), immunofluorescence microscopy (Aut5-Ha), and nuclear staining with DAPI (DNA/DAPI) are shown. (A) Wild-type cells expressing Aut5-Ha from the chromosome under the control of the endogenous promoter. (B) pep4 Δ cells expressing Aut5-Ha chromosomally, which due to the lack of vacuolar endoproteinase A are defective in vacuolar protein breakdown. Arrowheads point to ER staining. (C) Chromosomal expression of Aut5-Ha in aut3 Δ pep4 Δ cells, which are further defective in autophagy. Bar, 20 μm. (D) Pulse-chase analysis indicates a rapid breakdown of Aut5-Ha dependent on vacuolar proteinase A. The indicated strains were grown to log phase in SMD medium, pulse-labeled for 20 min with [ 35 S]methionine and cysteine, and chased in nonradioactive medium as described in Materials and Methods. At the specified times, aliquots were withdrawn and lysates were immunoprecipitated with antibodies to Ha. After deglycosylation with endoglycosidase H, samples were subjected to SDS-PAGE and labeled proteins were visualized using a PhosphorImager. wt, wild type.

    Article Snippet: To test this, we treated cell extracts with endoglycosidase H (Fig. D).

    Techniques: Immunofluorescence, Microscopy, Incubation, Staining, Expressing, Pulse Chase, Labeling, Immunoprecipitation, SDS Page

    Aut5-Ha is a glycosylated integral membrane protein. (A) Aut5-Ha 3 is specifically detected in immunoblots using an antibody to Ha. Crude extracts of stationary-phase cells were subjected to SDS-PAGE, blotted on PVDF membranes, and analyzed with antibodies to Ha. Lane 1, wild type; 2, wild-type ( wt ) cells expressing Aut5-Ha from the chromosome under the control of the native AUT5 promoter; 3, pep4 Δ cells expressing Aut5-Ha from the chromosome; 4, aut5 Δ cells carrying a centromeric Aut5-Ha plasmid ( cen ); 5, aut5 Δ cells expressing Aut5-Ha from a 2μm (2μ) plasmid; 6, aut5 Δ pep4 Δ cells with a 2μm Aut5-Ha plasmid; 7, aut5 Δ cells expressing from a 2μm plasmid a mutated version of Aut5-Ha in which serine 332 was replaced by alanine; 8, aut5 Δ pep4 Δ cells expressing from a 2μm plasmid a mutated version of Aut5-Ha in which serine 332 was replaced by alanine. Aut5-Ha shows a molecular mass of ∼75 kDa; note the smear of the protein to a higher molecular mass. ∗, cross-reacting material. (B) Western blotting of aminopeptidase I. The maturation of aminopeptidase I, which is similar to that of the wild type in strains expressing Aut5-Ha either from the chromosome or from plasmids, indicates the biological activity of the tagged protein (lanes 1, 2, 4, and 5). Replacement of serine 332 with alanine by site-directed mutagenesis impairs the maturation of aminopeptidase I, indicating an impaired biological activity of the mutated protein. The PVDF membrane shown in panel A was stripped and reprobed with antibodies to aminopeptidase I. pAPI, precursor aminopeptidase I; mAPI, mature aminopeptidase I. (C) Aut5-Ha is an integral membrane protein. pep4 Δ cells, starved for 4 h in 1% K acetate (K-Ac), were lysed with glass beads, and the homogenate was separated by centrifugation at 100,000 × g for 45 min into pellet (P) and supernatant (S) fractions. Fractions were subjected to SDS-electrophoresis, blotted onto a PVDF membrane, and analyzed using Ha antibodies. As indicated, the cell lysate was incubated with 1 M potassium acetate, 0.1 M Na 2 CO 3 , 2.5 M urea, or 1% Triton X-100 (Tx100) prior to centrifugation. As a control, the membrane was stripped and reprobed successively with antibodies against Vph1p (an integral membrane protein), Vma2p (peripheral membrane protein), and PGK as a soluble protein. (D) Aut5-Ha is glycosylated. Cells starved for nitrogen for 4 h in 1% K acetate were lysed with glass beads; Aut5-Ha was immunoprecipitated with Ha antibodies and deglycosylated by treatment with endoglycosidase H (Endo H). After SDS-PAGE and being blotted onto a PVDF membrane, the precipitates were analyzed with Ha antibodies. Aut5-Ha∗ refers to the deglycosylated species.

    Journal: Journal of Bacteriology

    Article Title: Aut5/Cvt17p, a Putative Lipase Essential for Disintegration of Autophagic Bodies inside the Vacuole

    doi: 10.1128/JB.183.20.5942-5955.2001

    Figure Lengend Snippet: Aut5-Ha is a glycosylated integral membrane protein. (A) Aut5-Ha 3 is specifically detected in immunoblots using an antibody to Ha. Crude extracts of stationary-phase cells were subjected to SDS-PAGE, blotted on PVDF membranes, and analyzed with antibodies to Ha. Lane 1, wild type; 2, wild-type ( wt ) cells expressing Aut5-Ha from the chromosome under the control of the native AUT5 promoter; 3, pep4 Δ cells expressing Aut5-Ha from the chromosome; 4, aut5 Δ cells carrying a centromeric Aut5-Ha plasmid ( cen ); 5, aut5 Δ cells expressing Aut5-Ha from a 2μm (2μ) plasmid; 6, aut5 Δ pep4 Δ cells with a 2μm Aut5-Ha plasmid; 7, aut5 Δ cells expressing from a 2μm plasmid a mutated version of Aut5-Ha in which serine 332 was replaced by alanine; 8, aut5 Δ pep4 Δ cells expressing from a 2μm plasmid a mutated version of Aut5-Ha in which serine 332 was replaced by alanine. Aut5-Ha shows a molecular mass of ∼75 kDa; note the smear of the protein to a higher molecular mass. ∗, cross-reacting material. (B) Western blotting of aminopeptidase I. The maturation of aminopeptidase I, which is similar to that of the wild type in strains expressing Aut5-Ha either from the chromosome or from plasmids, indicates the biological activity of the tagged protein (lanes 1, 2, 4, and 5). Replacement of serine 332 with alanine by site-directed mutagenesis impairs the maturation of aminopeptidase I, indicating an impaired biological activity of the mutated protein. The PVDF membrane shown in panel A was stripped and reprobed with antibodies to aminopeptidase I. pAPI, precursor aminopeptidase I; mAPI, mature aminopeptidase I. (C) Aut5-Ha is an integral membrane protein. pep4 Δ cells, starved for 4 h in 1% K acetate (K-Ac), were lysed with glass beads, and the homogenate was separated by centrifugation at 100,000 × g for 45 min into pellet (P) and supernatant (S) fractions. Fractions were subjected to SDS-electrophoresis, blotted onto a PVDF membrane, and analyzed using Ha antibodies. As indicated, the cell lysate was incubated with 1 M potassium acetate, 0.1 M Na 2 CO 3 , 2.5 M urea, or 1% Triton X-100 (Tx100) prior to centrifugation. As a control, the membrane was stripped and reprobed successively with antibodies against Vph1p (an integral membrane protein), Vma2p (peripheral membrane protein), and PGK as a soluble protein. (D) Aut5-Ha is glycosylated. Cells starved for nitrogen for 4 h in 1% K acetate were lysed with glass beads; Aut5-Ha was immunoprecipitated with Ha antibodies and deglycosylated by treatment with endoglycosidase H (Endo H). After SDS-PAGE and being blotted onto a PVDF membrane, the precipitates were analyzed with Ha antibodies. Aut5-Ha∗ refers to the deglycosylated species.

    Article Snippet: To test this, we treated cell extracts with endoglycosidase H (Fig. D).

    Techniques: Western Blot, SDS Page, Expressing, Plasmid Preparation, Activity Assay, Mutagenesis, Centrifugation, Electrophoresis, Incubation, Immunoprecipitation