co immunoprecipitation buffer  (Millipore)

 
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    Name:
    Ethylenediaminetetraacetic acid
    Description:
    Ethylenediaminetetraacetic acid EDTA is a hydrophilic metal chelating agent that transforms metal ions into inactive cyclic metal complexes Hence it has industrial application for resolving metal contamination
    Catalog Number:
    E9884
    Price:
    None
    Applications:
    Ethylenediaminetetraacetic acid has been used: . as a component of labeling buffer, which is used in the washing step during cell fractionation{155) . for investigating the effect of papain enzyme on wound debridement and other skin issues . for harvesting rat arterial smooth muscle cell after its pre-treatment for its use in flow cytometry analysis
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    Structured Review

    Millipore co immunoprecipitation buffer
    Ethylenediaminetetraacetic acid
    Ethylenediaminetetraacetic acid EDTA is a hydrophilic metal chelating agent that transforms metal ions into inactive cyclic metal complexes Hence it has industrial application for resolving metal contamination
    https://www.bioz.com/result/co immunoprecipitation buffer/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    co immunoprecipitation buffer - by Bioz Stars, 2021-05
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    Images

    1) Product Images from "The targetable role of herpes virus-associated ubiquitin-specific protease (HAUSP) in p190 BCR-ABL leukemia"

    Article Title: The targetable role of herpes virus-associated ubiquitin-specific protease (HAUSP) in p190 BCR-ABL leukemia

    Journal: Oncology Letters

    doi: 10.3892/ol.2016.5073

    (A) Western immunoblotting using anti-pTyr antibody. HEK 293T cells were transfected with the HAUSP WT or HAUSP TM (Y243F/Y878F/Y947F) in the presence of p190 BCR-ABL. Immunoprecipitation was performed with anti-Myc antibodies (HAUSP). (B) Immunoprecipitation of Myc-HAUSP and anti-pTyr immunoblot following treatment with 1 or 5 µM imatinib for 24 h; (C) Left panel: Western immunoblot of HEK 293T cells expressing p190 BCR-ABL and Myc-HAUSP. Cells were treated with P5091 and imatinib for 24 h at concentration of 4.2 and 5 µM respectively, and were subsequently analyzed for expression of p53 and p-p53. Right panel: Quantification and statistical analysis of p-p53 protein (**P=0.0018 and *P=0.0371). P-values were calculated by a two-tailed t-test. p-, phosphorylated; Tyr, tyrosine; HAUSP, herpesvirus-associated ubiquitin-specific protease; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IP, immunoprecipitation; WT, wild-type; TM, triple mutant; IM, imatinib.
    Figure Legend Snippet: (A) Western immunoblotting using anti-pTyr antibody. HEK 293T cells were transfected with the HAUSP WT or HAUSP TM (Y243F/Y878F/Y947F) in the presence of p190 BCR-ABL. Immunoprecipitation was performed with anti-Myc antibodies (HAUSP). (B) Immunoprecipitation of Myc-HAUSP and anti-pTyr immunoblot following treatment with 1 or 5 µM imatinib for 24 h; (C) Left panel: Western immunoblot of HEK 293T cells expressing p190 BCR-ABL and Myc-HAUSP. Cells were treated with P5091 and imatinib for 24 h at concentration of 4.2 and 5 µM respectively, and were subsequently analyzed for expression of p53 and p-p53. Right panel: Quantification and statistical analysis of p-p53 protein (**P=0.0018 and *P=0.0371). P-values were calculated by a two-tailed t-test. p-, phosphorylated; Tyr, tyrosine; HAUSP, herpesvirus-associated ubiquitin-specific protease; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IP, immunoprecipitation; WT, wild-type; TM, triple mutant; IM, imatinib.

    Techniques Used: Western Blot, Transfection, Immunoprecipitation, Expressing, Concentration Assay, Two Tailed Test, Mutagenesis

    2) Product Images from "Stress-induced TRBP phosphorylation enhances its interaction with PKR to regulate cellular survival"

    Article Title: Stress-induced TRBP phosphorylation enhances its interaction with PKR to regulate cellular survival

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-19360-8

    TRBP phosphorylation strengthens PKR-TRBP interaction and weakens TRBP-TRBP interaction. ( A ) Phospho-mimic TRBP mutant interacts stronger with PKR compared to the phospho-defective TRBP mutant in yeast two-hybrid assay. PKR/pGAD424 and either AAAA TRBP/pGBKT7, DDDD TRBP/pGBKT7, or wt TRBP/pGBKT7 were co-transformed into AH109 yeast cells and selected on SD double dropout media (-tryptophan, - leucine). Ten microliters of transformed yeast cells (OD 600 = 10, 1, 0.1, 0.01) were spotted on SD triple dropout media (-tryptophan, - leucine, - histidine) containing 10 mM 3-amino-1,2,4-triazole (3-AT). Plates were incubated for 3 days at 30 °C. Transformation of PKR in pGAD424 and pGBKT7 empty vector served as a negative control. ( B ) Phosphomimic TRBP mutant shows stronger heteromeric interaction with PKR compared to the phosphodefective TRBP mutant in mammalian cells. HeLa cells were transfected with Flag K296R PKR/pcDNA 3.1 − and either myc TRBP AAAA/pcDNA 3.1 − , myc wt TRBP/pcDNA 3.1 − , or myc TRBP DDDD/pcDNA 3.1 − . The cells were harvested 24 hours after transfection, and myc AAAA, DDDD or wt TRBP was immunoprecipitated using anti-myc monoclonal antibody conjugated agarose beads. Co-immunoprecipitated Flag PKR was analyzed by western blot analysis with an anti-Flag antibody (IP: x Flag (PKR) panel). The blot was subsequently re-probed with anti-myc antibody to ensure equal myc TRBP immunoprecipitation from each sample (IP: x myc (TRBP) panel). Equal Flag PKR and myc TRBP expression in all samples was tested by western blot analysis of equal amounts of total cell lysate with anti-myc, and anti-Flag antibodies (input: x Flag (PKR) and x myc (TRBP) panels). ( C ) Changes in TRBP association with PKR. Flag TRBP overexpressing cells were treated with 25 μM sodium arsenite for the indicated time points. Cell extracts were prepared in the presence of a phosphatase inhibitor and 25 μg of cell extract was incubated with 500 ng of pure recombinant hexahistidine (His)-tagged PKR immobilized on Ni 2+ -agarose beads. After washing the beads, PKR-associated Flag TRBP was analyzed by SDS polyacrylamide gel electrophoresis followed by western blot analysis with anti-Flag antibody. Western blot analysis was also performed with anti-His antibody to ensure equal His- PKR in each sample. 25 μg of cell extract was also analyzed by western blot analysis with anti-Flag and anti-GAPDH antibodies to ensure equal addition of cell lysate for each pull down (Input). Quantification of TRBP-PKR pull down: Band intensities were quantified using ImageQuant TL Software, and the ratios of bound TRBP to bound PKR across all samples were calculated and normalized to the band intensities of Flag-TRBP input for each sample. Bound TRBP/his-PKR ratios for all samples were all expressed relative to the control sample (Lane 2). Averages from three independent experiments are plotted as bar graphs ± S.D. One-way ANOVA followed by post-hoc Tukey test was performed, asterisk *p value 0.0000012 and double asterisk **p value 0.0066374. ( D ) Phosphomimic TRBP mutant shows stronger homomeric interaction compared to the phosphodefective TRBP mutant in yeast two-hybrid assay. AAAA TRBP or DDDD TRBP point mutants in pGADT7 and pGBKT7 were co-transformed into AH109 yeast cells and selected on SD double dropout media (-tryptophan, -leucine). Ten microliters of transformed yeast cells (OD 600 = 10, 1, 0.1, 0.01) were spotted on SD triple dropout media plate (tryptophan, -leucine, -histidine) containing 10 mM 3-amino-1,2,4-triazole (3-AT). Plates were incubated for 5 days at 30 °C. Transformation of pGADT7 and pGBKT7 empty vectors served as a negative control. ( E ) Phosphomimic TRBP mutant shows stronger homomeric interaction compared to the phosphodefective TRBP mutant in mammalian cells. HeLa cells were transfected with either myc TRBP DDDD/pcDNA 3.1 − and Flag TRBP DDDD/pcDNA 3.1 − or Flag TRBP AAAA/pcDNA 3.1 − and myc TRBP AAAA/pcDNA 3.1 − . The cells were harvested 24 hours after transfection, and Flag TRBP AAAA or DDDD was immunoprecipitated using anti-Flag monoclonal antibody conjugated agarose beads. The co-immunoprecipitation of myc-TRBP was analyzed by western blot analysis with an anti-myc antibody (IP: x Myc panel). Blot was subsequently stripped and re-probed with anti-Flag antibody to ensure equal Flag-TRBP immunoprecipitation from each sample (IP: x Flag panel). Equal AAAA TRBP and DDDD TRBP expression in all samples was tested by western blot analysis of equal amounts of total cell lysate with anti-myc, and anti-Flag antibodies (Input: x Myc and x Flag panels).
    Figure Legend Snippet: TRBP phosphorylation strengthens PKR-TRBP interaction and weakens TRBP-TRBP interaction. ( A ) Phospho-mimic TRBP mutant interacts stronger with PKR compared to the phospho-defective TRBP mutant in yeast two-hybrid assay. PKR/pGAD424 and either AAAA TRBP/pGBKT7, DDDD TRBP/pGBKT7, or wt TRBP/pGBKT7 were co-transformed into AH109 yeast cells and selected on SD double dropout media (-tryptophan, - leucine). Ten microliters of transformed yeast cells (OD 600 = 10, 1, 0.1, 0.01) were spotted on SD triple dropout media (-tryptophan, - leucine, - histidine) containing 10 mM 3-amino-1,2,4-triazole (3-AT). Plates were incubated for 3 days at 30 °C. Transformation of PKR in pGAD424 and pGBKT7 empty vector served as a negative control. ( B ) Phosphomimic TRBP mutant shows stronger heteromeric interaction with PKR compared to the phosphodefective TRBP mutant in mammalian cells. HeLa cells were transfected with Flag K296R PKR/pcDNA 3.1 − and either myc TRBP AAAA/pcDNA 3.1 − , myc wt TRBP/pcDNA 3.1 − , or myc TRBP DDDD/pcDNA 3.1 − . The cells were harvested 24 hours after transfection, and myc AAAA, DDDD or wt TRBP was immunoprecipitated using anti-myc monoclonal antibody conjugated agarose beads. Co-immunoprecipitated Flag PKR was analyzed by western blot analysis with an anti-Flag antibody (IP: x Flag (PKR) panel). The blot was subsequently re-probed with anti-myc antibody to ensure equal myc TRBP immunoprecipitation from each sample (IP: x myc (TRBP) panel). Equal Flag PKR and myc TRBP expression in all samples was tested by western blot analysis of equal amounts of total cell lysate with anti-myc, and anti-Flag antibodies (input: x Flag (PKR) and x myc (TRBP) panels). ( C ) Changes in TRBP association with PKR. Flag TRBP overexpressing cells were treated with 25 μM sodium arsenite for the indicated time points. Cell extracts were prepared in the presence of a phosphatase inhibitor and 25 μg of cell extract was incubated with 500 ng of pure recombinant hexahistidine (His)-tagged PKR immobilized on Ni 2+ -agarose beads. After washing the beads, PKR-associated Flag TRBP was analyzed by SDS polyacrylamide gel electrophoresis followed by western blot analysis with anti-Flag antibody. Western blot analysis was also performed with anti-His antibody to ensure equal His- PKR in each sample. 25 μg of cell extract was also analyzed by western blot analysis with anti-Flag and anti-GAPDH antibodies to ensure equal addition of cell lysate for each pull down (Input). Quantification of TRBP-PKR pull down: Band intensities were quantified using ImageQuant TL Software, and the ratios of bound TRBP to bound PKR across all samples were calculated and normalized to the band intensities of Flag-TRBP input for each sample. Bound TRBP/his-PKR ratios for all samples were all expressed relative to the control sample (Lane 2). Averages from three independent experiments are plotted as bar graphs ± S.D. One-way ANOVA followed by post-hoc Tukey test was performed, asterisk *p value 0.0000012 and double asterisk **p value 0.0066374. ( D ) Phosphomimic TRBP mutant shows stronger homomeric interaction compared to the phosphodefective TRBP mutant in yeast two-hybrid assay. AAAA TRBP or DDDD TRBP point mutants in pGADT7 and pGBKT7 were co-transformed into AH109 yeast cells and selected on SD double dropout media (-tryptophan, -leucine). Ten microliters of transformed yeast cells (OD 600 = 10, 1, 0.1, 0.01) were spotted on SD triple dropout media plate (tryptophan, -leucine, -histidine) containing 10 mM 3-amino-1,2,4-triazole (3-AT). Plates were incubated for 5 days at 30 °C. Transformation of pGADT7 and pGBKT7 empty vectors served as a negative control. ( E ) Phosphomimic TRBP mutant shows stronger homomeric interaction compared to the phosphodefective TRBP mutant in mammalian cells. HeLa cells were transfected with either myc TRBP DDDD/pcDNA 3.1 − and Flag TRBP DDDD/pcDNA 3.1 − or Flag TRBP AAAA/pcDNA 3.1 − and myc TRBP AAAA/pcDNA 3.1 − . The cells were harvested 24 hours after transfection, and Flag TRBP AAAA or DDDD was immunoprecipitated using anti-Flag monoclonal antibody conjugated agarose beads. The co-immunoprecipitation of myc-TRBP was analyzed by western blot analysis with an anti-myc antibody (IP: x Myc panel). Blot was subsequently stripped and re-probed with anti-Flag antibody to ensure equal Flag-TRBP immunoprecipitation from each sample (IP: x Flag panel). Equal AAAA TRBP and DDDD TRBP expression in all samples was tested by western blot analysis of equal amounts of total cell lysate with anti-myc, and anti-Flag antibodies (Input: x Myc and x Flag panels).

    Techniques Used: Mutagenesis, Y2H Assay, Transformation Assay, Incubation, Plasmid Preparation, Negative Control, Transfection, Immunoprecipitation, Western Blot, Expressing, Recombinant, Polyacrylamide Gel Electrophoresis, Software

    3) Product Images from "LASP-1 – A nuclear hub for the UHRF1-DNMT1-G9a-Snail1 complex"

    Article Title: LASP-1 – A nuclear hub for the UHRF1-DNMT1-G9a-Snail1 complex

    Journal: Oncogene

    doi: 10.1038/onc.2015.166

    LASP-1 associates with epigenetic proteins UHRF1, DNMT1, G9a and Snail1 A) LASP-1 co-immunoprecipitates with UHRF1 and DNMT1. 100 μg of the nuclear extracts from MDA-Bone-Un cells stimulated with vehicle (0 min) or 500 ng /ml of CXCL12 for 15 min were incubated with control mouse monoclonal IgG (Mock) or with mouse anti-LASP-1 antibody. LASP1-bound proteins were eluted and analyzed for endogenous LASP-1 and associated proteins UHRF1 and DNMT1. Bottom panel: 15 μg of total cell lysates from MDA-Bone-Un cells that were stimulated with vehicle (0 min) or CXCL12 for 15 min. LASP-1 lysate blot was obtained after stripping and re-probing for LASP-1 after the detection of Snail1 first. The experiment was repeated twice and the representative blot was quantified and shown. NS – Non-silenced control; KD – LASP-1 knock down. The band intensities of proteins were quantified by using Image J analysis and normalized to their respective non-stimulated control (bands in -CXCL12 lane). The fold change was given above the bands to track the level of the immunoprecipitated proteins with respect to their protein levels in the lysates. B) LASP-1 co-immunoprecipitates with G9a and Snail1. 100 μg of the nuclear extracts from MDA-Bone-Un cells stimulated with vehicle (0 min and Mock) or 500 ng /ml of CXCL12 for 15 min were incubated with control mouse monoclonal IgG (Mock) or with mouse anti-LASP-1 antibody. Bound proteins were eluted and analyzed for endogenous LASP-1 and associated G9a and Snail1. NS – Non-silenced control; KD – LASP-1 knock down. Bottom panel: 15 μg of total cell lysates from MDA-Bone-Un cells that were stimulated with vehicle (0 min) or CXCL12 for 15 min. LASP-1 lysate blot was obtained after stripping and re-probing for LASP-1 after the detection of Snail1 first. The experiment was repeated twice and the representative blot was quantified and shown. NS – Non-silenced control; KD – LASP-1 knock down. The band intensities of proteins were quantified by using Image J analysis and normalized to their respective non-stimulated control (bands in -CXCL12 lane). The fold-change was given above the bands to track the level of the immunoprecipitated proteins with respect to their protein levels in the lysates. C) LASP-1 co-immunoprecipitates with Di-, Tri-methylated histone H3 . Total cell lysates from MDA-Bone-Un cells stimulated with vehicle (0 min) or 200 ng /ml of CXCL12 for 5, 10, 15 and 30 min were incubated with control mouse monoclonal IgG (Mock) or with mouse anti-LASP-1 antibody. The proteins were eluted and analyzed for endogenous LASP-1 and associated di-, tri-methylated histone H3. Bottom panel: 15 μg of total cell lysates from MDA-Bone-Un cells that were stimulated with vehicle (0 min) or CXCL12 for 5, 10, 15 and 30 min were blotted for LASP-1 and di-, tri-methylated histone H3. The experiment was repeated thrice and the representative blot was shown. D) LASP-1 associates with UHRF1, DNMT1 and G9a in the GST pull down assay. Left panel: Purification of GST, full length and different domains of LASP-1fused to GST. 20 μg purified GST and GST-LASP1 (full length) and different domains of LASP-1 fused to GST were separated by 10% SDS-PAGE and stained by colloidal Coomassie blue. Lane1 – M.W. markers; Lane 2 – GST; Lane 3 – GST-LASP1; Lane 4 – GST-LIM; Lane 5 – GST-LIM-NR; Lane 6 – GST-NR-SH3. Right top panel: LASP-1 associates with UHRF1, DNMT1 and G9a in the GST pull down assay . 200 μg purified GST and GST-LASP1 proteins were mixed with 1–1.5 mg of total lysate from MDA-Bone-Un cells. Bound proteins were eluted and analyzed for association of LASP-1 with endogenous UHRF1, DNMT1 and G9a. The experiment was repeated thrice and the representative blot was shown. Lane 1 – 15 μg of lysate; Lane 2 – GST; Lane 3 – GST-LASP1; Lane 4 – GST-LIM; Lane 5 – GST-LIM-NR; Lane 6 – GST-NR-SH3. Right bottom panel: LASP-1 appears to associate with ubiquitinated UHRF1 . In some GST-pull down experiments, associated UHRF1 appeared smeared but distinct; a lower and a higher exposure of the same blot were shown. Lane 1 – 15 μg of lysate; Lane 2 – GST; Lane 3 – GST-LASP1; Lane 4 – GST-LIM; Lane 5 – GST-LIM-NR; Lane 6 – GST-NR-SH3. E) Stable knockdown of LASP-1. MDA-Bone-Un cells employed in the co-immunoprecipitation (Co-IP) experiments were re-analyzed again to know the level of stable knock down of LASP-1 at the time of Co-IP experiment by Western blotting of total cell lysates (30 μg) for LASP-1. β-actin served as the loading control. F) and G) LASP-1 associates with G9a and Snail1 in situ in a CXCL12-dependent manner. MDA-Bone-Un breast cancer cells that were grown on glass coverslips were stimulated with and without CXCL12 for 15 min. The cells were fixed and subjected to proximity ligation assay. The experiment was repeated twice and the representative images are shown.
    Figure Legend Snippet: LASP-1 associates with epigenetic proteins UHRF1, DNMT1, G9a and Snail1 A) LASP-1 co-immunoprecipitates with UHRF1 and DNMT1. 100 μg of the nuclear extracts from MDA-Bone-Un cells stimulated with vehicle (0 min) or 500 ng /ml of CXCL12 for 15 min were incubated with control mouse monoclonal IgG (Mock) or with mouse anti-LASP-1 antibody. LASP1-bound proteins were eluted and analyzed for endogenous LASP-1 and associated proteins UHRF1 and DNMT1. Bottom panel: 15 μg of total cell lysates from MDA-Bone-Un cells that were stimulated with vehicle (0 min) or CXCL12 for 15 min. LASP-1 lysate blot was obtained after stripping and re-probing for LASP-1 after the detection of Snail1 first. The experiment was repeated twice and the representative blot was quantified and shown. NS – Non-silenced control; KD – LASP-1 knock down. The band intensities of proteins were quantified by using Image J analysis and normalized to their respective non-stimulated control (bands in -CXCL12 lane). The fold change was given above the bands to track the level of the immunoprecipitated proteins with respect to their protein levels in the lysates. B) LASP-1 co-immunoprecipitates with G9a and Snail1. 100 μg of the nuclear extracts from MDA-Bone-Un cells stimulated with vehicle (0 min and Mock) or 500 ng /ml of CXCL12 for 15 min were incubated with control mouse monoclonal IgG (Mock) or with mouse anti-LASP-1 antibody. Bound proteins were eluted and analyzed for endogenous LASP-1 and associated G9a and Snail1. NS – Non-silenced control; KD – LASP-1 knock down. Bottom panel: 15 μg of total cell lysates from MDA-Bone-Un cells that were stimulated with vehicle (0 min) or CXCL12 for 15 min. LASP-1 lysate blot was obtained after stripping and re-probing for LASP-1 after the detection of Snail1 first. The experiment was repeated twice and the representative blot was quantified and shown. NS – Non-silenced control; KD – LASP-1 knock down. The band intensities of proteins were quantified by using Image J analysis and normalized to their respective non-stimulated control (bands in -CXCL12 lane). The fold-change was given above the bands to track the level of the immunoprecipitated proteins with respect to their protein levels in the lysates. C) LASP-1 co-immunoprecipitates with Di-, Tri-methylated histone H3 . Total cell lysates from MDA-Bone-Un cells stimulated with vehicle (0 min) or 200 ng /ml of CXCL12 for 5, 10, 15 and 30 min were incubated with control mouse monoclonal IgG (Mock) or with mouse anti-LASP-1 antibody. The proteins were eluted and analyzed for endogenous LASP-1 and associated di-, tri-methylated histone H3. Bottom panel: 15 μg of total cell lysates from MDA-Bone-Un cells that were stimulated with vehicle (0 min) or CXCL12 for 5, 10, 15 and 30 min were blotted for LASP-1 and di-, tri-methylated histone H3. The experiment was repeated thrice and the representative blot was shown. D) LASP-1 associates with UHRF1, DNMT1 and G9a in the GST pull down assay. Left panel: Purification of GST, full length and different domains of LASP-1fused to GST. 20 μg purified GST and GST-LASP1 (full length) and different domains of LASP-1 fused to GST were separated by 10% SDS-PAGE and stained by colloidal Coomassie blue. Lane1 – M.W. markers; Lane 2 – GST; Lane 3 – GST-LASP1; Lane 4 – GST-LIM; Lane 5 – GST-LIM-NR; Lane 6 – GST-NR-SH3. Right top panel: LASP-1 associates with UHRF1, DNMT1 and G9a in the GST pull down assay . 200 μg purified GST and GST-LASP1 proteins were mixed with 1–1.5 mg of total lysate from MDA-Bone-Un cells. Bound proteins were eluted and analyzed for association of LASP-1 with endogenous UHRF1, DNMT1 and G9a. The experiment was repeated thrice and the representative blot was shown. Lane 1 – 15 μg of lysate; Lane 2 – GST; Lane 3 – GST-LASP1; Lane 4 – GST-LIM; Lane 5 – GST-LIM-NR; Lane 6 – GST-NR-SH3. Right bottom panel: LASP-1 appears to associate with ubiquitinated UHRF1 . In some GST-pull down experiments, associated UHRF1 appeared smeared but distinct; a lower and a higher exposure of the same blot were shown. Lane 1 – 15 μg of lysate; Lane 2 – GST; Lane 3 – GST-LASP1; Lane 4 – GST-LIM; Lane 5 – GST-LIM-NR; Lane 6 – GST-NR-SH3. E) Stable knockdown of LASP-1. MDA-Bone-Un cells employed in the co-immunoprecipitation (Co-IP) experiments were re-analyzed again to know the level of stable knock down of LASP-1 at the time of Co-IP experiment by Western blotting of total cell lysates (30 μg) for LASP-1. β-actin served as the loading control. F) and G) LASP-1 associates with G9a and Snail1 in situ in a CXCL12-dependent manner. MDA-Bone-Un breast cancer cells that were grown on glass coverslips were stimulated with and without CXCL12 for 15 min. The cells were fixed and subjected to proximity ligation assay. The experiment was repeated twice and the representative images are shown.

    Techniques Used: Multiple Displacement Amplification, Incubation, Stripping Membranes, Immunoprecipitation, Methylation, Pull Down Assay, Purification, SDS Page, Staining, Co-Immunoprecipitation Assay, Western Blot, In Situ, Proximity Ligation Assay

    4) Product Images from "Regulation of cargo transfer between ESCRT-0 and ESCRT-I complexes by flotillin-1 during endosomal sorting of ubiquitinated cargo"

    Article Title: Regulation of cargo transfer between ESCRT-0 and ESCRT-I complexes by flotillin-1 during endosomal sorting of ubiquitinated cargo

    Journal: Oncogenesis

    doi: 10.1038/oncsis.2017.47

    Flotillins directly interact with Hrs, and flotillin-1 is required for Hrs interaction with ubiquitin.( a ) Co-immunoprecipitation of endogenous flotillin-1 with Hrs from HeLa cells stimulated with EGF for 30 min. Flotillin-1-knockout cells were used as a control to identify flotillin-1 bands. Control immunoprecipitation from HeLa cells with an isotype-matched antibody does not show any co-precipitation. ( b ) Purified Hrs-GST proteins were used to pull down flotillins from HeLa lysates for the mapping of the interaction sites of flotillins in Hrs. Flotillin interaction with Hrs is not dependent on the UIM domain in Hrs, whereas mutation of the PSAP motif into LSAL or deletion of the VHS domain partially abrogates flotillin interaction. VHS domain alone shows only a minor binding to flotillins. Deletion of the C-terminal region including the core/coiled-coil/GAT domains abolishes Hrs interaction with flotillins, and deletion of the C-terminal region beyond these domains results in decreased binding. Deletion of the clathrin-binding motif (CB) in Hrs C terminus also abrogates flotillin binding. ( c ) Structure of the constructs used in a . ( d ) Hrs and flotillins directly interact. GST pulldown was performed with recombinant, purified proteins. GST-tag in Hrs was removed before pulldown using thrombin digestion. ( e ) Interaction of endogenous Hrs with ubiquitin in an ubiquitin-GST-pulldown assay is reduced in EGF-stimulated (30 min) flotillin-1-knockdown cells, but can be rescued by the addition of purified recombinant flotillin-1. The pulldown samples were analysed by western blot using Hrs, flotillin-1 and GAPDH antibodies. ( f ) Quantification of Hrs binding to ubiquitin-GST upon 30 min EGF. Statistical analysis was performed using one-way analysis of variance (ANOVA) with Bonferroni post-test. The values shown represent mean±s.d. from three independent experiments. * P
    Figure Legend Snippet: Flotillins directly interact with Hrs, and flotillin-1 is required for Hrs interaction with ubiquitin.( a ) Co-immunoprecipitation of endogenous flotillin-1 with Hrs from HeLa cells stimulated with EGF for 30 min. Flotillin-1-knockout cells were used as a control to identify flotillin-1 bands. Control immunoprecipitation from HeLa cells with an isotype-matched antibody does not show any co-precipitation. ( b ) Purified Hrs-GST proteins were used to pull down flotillins from HeLa lysates for the mapping of the interaction sites of flotillins in Hrs. Flotillin interaction with Hrs is not dependent on the UIM domain in Hrs, whereas mutation of the PSAP motif into LSAL or deletion of the VHS domain partially abrogates flotillin interaction. VHS domain alone shows only a minor binding to flotillins. Deletion of the C-terminal region including the core/coiled-coil/GAT domains abolishes Hrs interaction with flotillins, and deletion of the C-terminal region beyond these domains results in decreased binding. Deletion of the clathrin-binding motif (CB) in Hrs C terminus also abrogates flotillin binding. ( c ) Structure of the constructs used in a . ( d ) Hrs and flotillins directly interact. GST pulldown was performed with recombinant, purified proteins. GST-tag in Hrs was removed before pulldown using thrombin digestion. ( e ) Interaction of endogenous Hrs with ubiquitin in an ubiquitin-GST-pulldown assay is reduced in EGF-stimulated (30 min) flotillin-1-knockdown cells, but can be rescued by the addition of purified recombinant flotillin-1. The pulldown samples were analysed by western blot using Hrs, flotillin-1 and GAPDH antibodies. ( f ) Quantification of Hrs binding to ubiquitin-GST upon 30 min EGF. Statistical analysis was performed using one-way analysis of variance (ANOVA) with Bonferroni post-test. The values shown represent mean±s.d. from three independent experiments. * P

    Techniques Used: Immunoprecipitation, Knock-Out, Purification, Mutagenesis, Binding Assay, Construct, Recombinant, GST Pulldown Assay, Western Blot

    5) Product Images from "MISC-1/OGC Links Mitochondrial Metabolism, Apoptosis and Insulin Secretion"

    Article Title: MISC-1/OGC Links Mitochondrial Metabolism, Apoptosis and Insulin Secretion

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0017827

    OGC interacts with Bcl-x L and ANT. We performed a co-immunoprecipitation experiment in HEK293 cells using an antibody against ANT, which is an integral component of the MPTP. We were able to detect Bcl-X L , OGC and ANT in the co-immuno precipitant. As a control protein, we chose subunit 1 of complex IV of the electron transport chain, which is also localized to the inner mitochondrial membrane. This control protein did not co-immunoprecipitate with Bcl-x L , OGC and ANT, but was present in the supernatant (input). We obtained similar results by immunoprecipitation with an antibody against Bcl-x L (data not shown). Co-IP: co-immunoprecipitation. WB: western blot.
    Figure Legend Snippet: OGC interacts with Bcl-x L and ANT. We performed a co-immunoprecipitation experiment in HEK293 cells using an antibody against ANT, which is an integral component of the MPTP. We were able to detect Bcl-X L , OGC and ANT in the co-immuno precipitant. As a control protein, we chose subunit 1 of complex IV of the electron transport chain, which is also localized to the inner mitochondrial membrane. This control protein did not co-immunoprecipitate with Bcl-x L , OGC and ANT, but was present in the supernatant (input). We obtained similar results by immunoprecipitation with an antibody against Bcl-x L (data not shown). Co-IP: co-immunoprecipitation. WB: western blot.

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

    6) Product Images from "EP2 Induces p38 Phosphorylation via the Activation of Src in HEK 293 Cells"

    Article Title: EP2 Induces p38 Phosphorylation via the Activation of Src in HEK 293 Cells

    Journal: Biomolecules & Therapeutics

    doi: 10.4062/biomolther.2015.043

    β-Arrestin further increases overexpression of EP2-induced p38 phosphorylation and forms a complex with EP2 and Src. (A) EP2 and Src interact with each other. (Left) HEK 293 cells were transfected empty vector or pcDNA3.1(−) FLAG-EP2 and pcDNA3.1 (−) Myc-Src. Forty-eight hours after transfection, cell lysates were immunopreciptated for FLAG with FLAG antibody-agarose. Immunoprecipitated proteins were subjected to western blot analysis for Myc. (Right) HEK 293 cells were transfected empty vector or pcDNA3.1(−) Myc-Src and pcDNA3.1 (−) FLAG-EP2. Forty-eight hours after transfection, cells lysates were immunopreciptated for Myc with Myc antibody. Immunoprecipitated proteins were subjected to western blot analysis for FLAG. (B) The overexpression of β-arrestin 1 or 2 further increases overexpression of EP2-induced p38 phosphorylation. HEK 293 cells were co-transfected with (i) empty vector and pCMV-FLAG-p38, (ii) pcDNA3.1 (−) HA-EP2 and pCMV-FLAG-p38, (iii) pcDNA3.1 (−) ARRB1-Myc and pCMV-FLAG-p38, (iv) pcDNA3.1 (−) ARRB2-Myc and pCMV-FLAG-p38, (v) pcDNA3.1 (−) HA-EP2, pcDNA3.1 (−) ARRB1-Myc, and pCMV-FLAG-p38, and (vi) pcDNA3.1 (−) HA-EP2, pcDNA3.1 (−) ARRB2-Myc, and pCMV-FLAG-p38. Forty-eight hours later, cell lysates were subjected to western blot analysis for p-p38. Membranes were stripped and sequentially reprobed for FLAG, HA, and Myc to confirm equal expression of p38, EP2, and β-arrestins. (C) Experimental scheme of sequential co-immunoprecipitation. (D) Src forms a complex with EP2 and β-arrestin. HEK 293 cells were co-transfected with (i) pcDNA3.1 (−) FLAG-EP2 and pcDNA3.1 (−) HA-Src, (ii) pcDNA3.1 (−) ARRB1-Myc and pcDNA3.1 (−) HA-Src, (iii) pcDNA3.1 (−) FLAG-EP2, pcDNA3.1 (−) ARRB1-Myc, and pcDNA3.1 (−) HA-Src, (iv) pcDNA3.1 (−) ARRB2-Myc and pcDNA3.1 (−) HA-Src, and (v) pcDNA3.1 (−) FLAG-EP2, pcD-NA3.1 (−) ARRB2-Myc, and pcDNA3.1 (−) HA-Src. Forty-eight hours after transfection, cell lysates were immunopreciptated for FLAG using FLAG antibody-agarose. Immunoprecipitated proteins were then eluted under non-denaturing condition using FLAG peptide. FLAG peptide-eluted proteins were immunoprecipitated with Myc antibody. Myc-immunopreciptated proteins were subjected to western blot analysis for HA, FLAG, and Myc (right panel). The lysates used for sequential co-immunoprecipitation were included to confirm equal expression of EP2, Src, and β-arrestins (left panel). The location of β-arrestin 1 and 2 were shown (arrows).
    Figure Legend Snippet: β-Arrestin further increases overexpression of EP2-induced p38 phosphorylation and forms a complex with EP2 and Src. (A) EP2 and Src interact with each other. (Left) HEK 293 cells were transfected empty vector or pcDNA3.1(−) FLAG-EP2 and pcDNA3.1 (−) Myc-Src. Forty-eight hours after transfection, cell lysates were immunopreciptated for FLAG with FLAG antibody-agarose. Immunoprecipitated proteins were subjected to western blot analysis for Myc. (Right) HEK 293 cells were transfected empty vector or pcDNA3.1(−) Myc-Src and pcDNA3.1 (−) FLAG-EP2. Forty-eight hours after transfection, cells lysates were immunopreciptated for Myc with Myc antibody. Immunoprecipitated proteins were subjected to western blot analysis for FLAG. (B) The overexpression of β-arrestin 1 or 2 further increases overexpression of EP2-induced p38 phosphorylation. HEK 293 cells were co-transfected with (i) empty vector and pCMV-FLAG-p38, (ii) pcDNA3.1 (−) HA-EP2 and pCMV-FLAG-p38, (iii) pcDNA3.1 (−) ARRB1-Myc and pCMV-FLAG-p38, (iv) pcDNA3.1 (−) ARRB2-Myc and pCMV-FLAG-p38, (v) pcDNA3.1 (−) HA-EP2, pcDNA3.1 (−) ARRB1-Myc, and pCMV-FLAG-p38, and (vi) pcDNA3.1 (−) HA-EP2, pcDNA3.1 (−) ARRB2-Myc, and pCMV-FLAG-p38. Forty-eight hours later, cell lysates were subjected to western blot analysis for p-p38. Membranes were stripped and sequentially reprobed for FLAG, HA, and Myc to confirm equal expression of p38, EP2, and β-arrestins. (C) Experimental scheme of sequential co-immunoprecipitation. (D) Src forms a complex with EP2 and β-arrestin. HEK 293 cells were co-transfected with (i) pcDNA3.1 (−) FLAG-EP2 and pcDNA3.1 (−) HA-Src, (ii) pcDNA3.1 (−) ARRB1-Myc and pcDNA3.1 (−) HA-Src, (iii) pcDNA3.1 (−) FLAG-EP2, pcDNA3.1 (−) ARRB1-Myc, and pcDNA3.1 (−) HA-Src, (iv) pcDNA3.1 (−) ARRB2-Myc and pcDNA3.1 (−) HA-Src, and (v) pcDNA3.1 (−) FLAG-EP2, pcD-NA3.1 (−) ARRB2-Myc, and pcDNA3.1 (−) HA-Src. Forty-eight hours after transfection, cell lysates were immunopreciptated for FLAG using FLAG antibody-agarose. Immunoprecipitated proteins were then eluted under non-denaturing condition using FLAG peptide. FLAG peptide-eluted proteins were immunoprecipitated with Myc antibody. Myc-immunopreciptated proteins were subjected to western blot analysis for HA, FLAG, and Myc (right panel). The lysates used for sequential co-immunoprecipitation were included to confirm equal expression of EP2, Src, and β-arrestins (left panel). The location of β-arrestin 1 and 2 were shown (arrows).

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Expressing

    7) Product Images from "EP2 Induces p38 Phosphorylation via the Activation of Src in HEK 293 Cells"

    Article Title: EP2 Induces p38 Phosphorylation via the Activation of Src in HEK 293 Cells

    Journal: Biomolecules & Therapeutics

    doi: 10.4062/biomolther.2015.043

    β-Arrestin further increases overexpression of EP2-induced p38 phosphorylation and forms a complex with EP2 and Src. (A) EP2 and Src interact with each other. (Left) HEK 293 cells were transfected empty vector or pcDNA3.1(−) FLAG-EP2 and pcDNA3.1 (−) Myc-Src. Forty-eight hours after transfection, cell lysates were immunopreciptated for FLAG with FLAG antibody-agarose. Immunoprecipitated proteins were subjected to western blot analysis for Myc. (Right) HEK 293 cells were transfected empty vector or pcDNA3.1(−) Myc-Src and pcDNA3.1 (−) FLAG-EP2. Forty-eight hours after transfection, cells lysates were immunopreciptated for Myc with Myc antibody. Immunoprecipitated proteins were subjected to western blot analysis for FLAG. (B) The overexpression of β-arrestin 1 or 2 further increases overexpression of EP2-induced p38 phosphorylation. HEK 293 cells were co-transfected with (i) empty vector and pCMV-FLAG-p38, (ii) pcDNA3.1 (−) HA-EP2 and pCMV-FLAG-p38, (iii) pcDNA3.1 (−) ARRB1-Myc and pCMV-FLAG-p38, (iv) pcDNA3.1 (−) ARRB2-Myc and pCMV-FLAG-p38, (v) pcDNA3.1 (−) HA-EP2, pcDNA3.1 (−) ARRB1-Myc, and pCMV-FLAG-p38, and (vi) pcDNA3.1 (−) HA-EP2, pcDNA3.1 (−) ARRB2-Myc, and pCMV-FLAG-p38. Forty-eight hours later, cell lysates were subjected to western blot analysis for p-p38. Membranes were stripped and sequentially reprobed for FLAG, HA, and Myc to confirm equal expression of p38, EP2, and β-arrestins. (C) Experimental scheme of sequential co-immunoprecipitation. (D) Src forms a complex with EP2 and β-arrestin. HEK 293 cells were co-transfected with (i) pcDNA3.1 (−) FLAG-EP2 and pcDNA3.1 (−) HA-Src, (ii) pcDNA3.1 (−) ARRB1-Myc and pcDNA3.1 (−) HA-Src, (iii) pcDNA3.1 (−) FLAG-EP2, pcDNA3.1 (−) ARRB1-Myc, and pcDNA3.1 (−) HA-Src, (iv) pcDNA3.1 (−) ARRB2-Myc and pcDNA3.1 (−) HA-Src, and (v) pcDNA3.1 (−) FLAG-EP2, pcD-NA3.1 (−) ARRB2-Myc, and pcDNA3.1 (−) HA-Src. Forty-eight hours after transfection, cell lysates were immunopreciptated for FLAG using FLAG antibody-agarose. Immunoprecipitated proteins were then eluted under non-denaturing condition using FLAG peptide. FLAG peptide-eluted proteins were immunoprecipitated with Myc antibody. Myc-immunopreciptated proteins were subjected to western blot analysis for HA, FLAG, and Myc (right panel). The lysates used for sequential co-immunoprecipitation were included to confirm equal expression of EP2, Src, and β-arrestins (left panel). The location of β-arrestin 1 and 2 were shown (arrows).
    Figure Legend Snippet: β-Arrestin further increases overexpression of EP2-induced p38 phosphorylation and forms a complex with EP2 and Src. (A) EP2 and Src interact with each other. (Left) HEK 293 cells were transfected empty vector or pcDNA3.1(−) FLAG-EP2 and pcDNA3.1 (−) Myc-Src. Forty-eight hours after transfection, cell lysates were immunopreciptated for FLAG with FLAG antibody-agarose. Immunoprecipitated proteins were subjected to western blot analysis for Myc. (Right) HEK 293 cells were transfected empty vector or pcDNA3.1(−) Myc-Src and pcDNA3.1 (−) FLAG-EP2. Forty-eight hours after transfection, cells lysates were immunopreciptated for Myc with Myc antibody. Immunoprecipitated proteins were subjected to western blot analysis for FLAG. (B) The overexpression of β-arrestin 1 or 2 further increases overexpression of EP2-induced p38 phosphorylation. HEK 293 cells were co-transfected with (i) empty vector and pCMV-FLAG-p38, (ii) pcDNA3.1 (−) HA-EP2 and pCMV-FLAG-p38, (iii) pcDNA3.1 (−) ARRB1-Myc and pCMV-FLAG-p38, (iv) pcDNA3.1 (−) ARRB2-Myc and pCMV-FLAG-p38, (v) pcDNA3.1 (−) HA-EP2, pcDNA3.1 (−) ARRB1-Myc, and pCMV-FLAG-p38, and (vi) pcDNA3.1 (−) HA-EP2, pcDNA3.1 (−) ARRB2-Myc, and pCMV-FLAG-p38. Forty-eight hours later, cell lysates were subjected to western blot analysis for p-p38. Membranes were stripped and sequentially reprobed for FLAG, HA, and Myc to confirm equal expression of p38, EP2, and β-arrestins. (C) Experimental scheme of sequential co-immunoprecipitation. (D) Src forms a complex with EP2 and β-arrestin. HEK 293 cells were co-transfected with (i) pcDNA3.1 (−) FLAG-EP2 and pcDNA3.1 (−) HA-Src, (ii) pcDNA3.1 (−) ARRB1-Myc and pcDNA3.1 (−) HA-Src, (iii) pcDNA3.1 (−) FLAG-EP2, pcDNA3.1 (−) ARRB1-Myc, and pcDNA3.1 (−) HA-Src, (iv) pcDNA3.1 (−) ARRB2-Myc and pcDNA3.1 (−) HA-Src, and (v) pcDNA3.1 (−) FLAG-EP2, pcD-NA3.1 (−) ARRB2-Myc, and pcDNA3.1 (−) HA-Src. Forty-eight hours after transfection, cell lysates were immunopreciptated for FLAG using FLAG antibody-agarose. Immunoprecipitated proteins were then eluted under non-denaturing condition using FLAG peptide. FLAG peptide-eluted proteins were immunoprecipitated with Myc antibody. Myc-immunopreciptated proteins were subjected to western blot analysis for HA, FLAG, and Myc (right panel). The lysates used for sequential co-immunoprecipitation were included to confirm equal expression of EP2, Src, and β-arrestins (left panel). The location of β-arrestin 1 and 2 were shown (arrows).

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Expressing

    8) Product Images from "Adaptor Protein2 (AP2) orchestrates CXCR2-mediated cell migration"

    Article Title: Adaptor Protein2 (AP2) orchestrates CXCR2-mediated cell migration

    Journal: Traffic (Copenhagen, Denmark)

    doi: 10.1111/tra.12154

    Patch 1 and Patch 2 residues of the AP2-μ2 subunit that bind to PIP 2 lipid are not critical for the binding of the AP2 complex to CXCR2 A) Non-silencing (NS), HEK-293-μ2-KD cells and HEK-293-μ2-KD cells with transiently over-expressed AP2-μ2 mutants (P1, P1+P2, P1P2T and WT) were serum starved, stimulated with 100 ng /ml CXCL8 and cross-linked with DSP. The cells were lysed and a CXCR2 co-immunoprecipitation assay was performed. The CXCR2 associated proteins were eluted with 50 mM DTT and separated by 10%SDS-PAGE. The CXCR2 associated AP2 complex was probed with an anti-β2 antibody. Experiments were repeated 3 times and the mean band densities as normalized to co-immunoprecipitation with CXCR2 ±S. D. are shown. B) HA-AP2-μ2 associates with endogenous α2 subunit of AP-2. HEK-293-μ2-KD cells with transiently over-expressed HA-AP2-μ2 mutants (P1, P1+P2, T, P1P2T and WT), were lysed subjected to Western blot analysis for α2 and HA-μ2 subunits (upper panel). Each HA-tagged μ2 subunit was immunoprecipitated with anti-HA-agarose and blotted for endogenous α2 and for HA-μ2 (upper panel). A representative blot from 3 individual experiments is shown. C) Functional AP-2 complexes successfully incorporate transiently expressed HA-AP2-μ2 as shown by a reciprocal co-immunoprecipitation. A reciprocal co-immunoprecipitation of the endogenous AP2-β2 from 1.5 mg of total lysate shows that the functional AP-2 complexes contain both endogenous AP2-α2 and overexpressed HA-AP2-μ2. One thirtieth (1/30) of the total lysate input for co-immunoprecipitation was used for Western analysis of the total lysates. The top panel shows co-immunoprecipitation and the bottom panel shows the lysates. A representative blot from 2 individual experiments is shown.
    Figure Legend Snippet: Patch 1 and Patch 2 residues of the AP2-μ2 subunit that bind to PIP 2 lipid are not critical for the binding of the AP2 complex to CXCR2 A) Non-silencing (NS), HEK-293-μ2-KD cells and HEK-293-μ2-KD cells with transiently over-expressed AP2-μ2 mutants (P1, P1+P2, P1P2T and WT) were serum starved, stimulated with 100 ng /ml CXCL8 and cross-linked with DSP. The cells were lysed and a CXCR2 co-immunoprecipitation assay was performed. The CXCR2 associated proteins were eluted with 50 mM DTT and separated by 10%SDS-PAGE. The CXCR2 associated AP2 complex was probed with an anti-β2 antibody. Experiments were repeated 3 times and the mean band densities as normalized to co-immunoprecipitation with CXCR2 ±S. D. are shown. B) HA-AP2-μ2 associates with endogenous α2 subunit of AP-2. HEK-293-μ2-KD cells with transiently over-expressed HA-AP2-μ2 mutants (P1, P1+P2, T, P1P2T and WT), were lysed subjected to Western blot analysis for α2 and HA-μ2 subunits (upper panel). Each HA-tagged μ2 subunit was immunoprecipitated with anti-HA-agarose and blotted for endogenous α2 and for HA-μ2 (upper panel). A representative blot from 3 individual experiments is shown. C) Functional AP-2 complexes successfully incorporate transiently expressed HA-AP2-μ2 as shown by a reciprocal co-immunoprecipitation. A reciprocal co-immunoprecipitation of the endogenous AP2-β2 from 1.5 mg of total lysate shows that the functional AP-2 complexes contain both endogenous AP2-α2 and overexpressed HA-AP2-μ2. One thirtieth (1/30) of the total lysate input for co-immunoprecipitation was used for Western analysis of the total lysates. The top panel shows co-immunoprecipitation and the bottom panel shows the lysates. A representative blot from 2 individual experiments is shown.

    Techniques Used: Binding Assay, Co-Immunoprecipitation Assay, SDS Page, Immunoprecipitation, Western Blot, Functional Assay

    AP2 is essential for CXCR2-mediated chemotaxis, but β-arrestin1 is dispensable A) Top panel: LLKIL motif in CTDs of human CXC chemokine receptors is conserved. The CTDs of CXCR2 (45 residues), CXCR1 (44 residues), CXCR3 (49 residues) and CXCR4 (47 residues) were aligned with CLUSTALW (1.83) multiple sequence alignment program. The LLKIL functional motif of CXCR2 and similar putative motifs in other CXC receptor CTDs are in bold. Also, the serine residues known to be phosphorylated in CXCR2 CTD in response to CXCL8 stimulation are in bold. Bottom panel: The mutations in CXCR2 important for binding of AP2 and β-arrestin are illustrated. B) Decreased association of CXCR2 mutants with AP2 and/or β-arrestin1 after stimulation with CXCL8. dHL-60 cells stably expressing CXCR2-WT, 4A or CXCR2-4A/IL mutants were stimulated with or without CXCL8. CXCR2 was immunoprecipitated with anti-CXCR2 antibody, and blotted for AP2-β2 subunit or β-arrestin1. The blot was stripped and re-blotted for CXCR2. The relative values of fold increase in response to CXCL8 stimulation for each cell line calculated from 3 independent experiments is shown under the western blots (fold ± S.E.M.). One tenth of the total lysate input for co-immunoprecipitation was used for Western analysis of the total lysates. C) CXCL8-mediated internalization of CXCR2 is abolished in 4A/IL mutant of CXCR2, but only partially attenuated in 4A-CXCR2 mutant. The internalization of CXCR2 was performed by following the internalization of 125 I-CXCL8 in dHL60-CXCR2 cells stably expressing CXCR2-WT, 4A or 4A/IL mutant. Error bars are S.E.M and the experiments were repeated 3 times with duplicates for each treatment. ANOVA: 2 min – 4A vs. 4A/IL, p
    Figure Legend Snippet: AP2 is essential for CXCR2-mediated chemotaxis, but β-arrestin1 is dispensable A) Top panel: LLKIL motif in CTDs of human CXC chemokine receptors is conserved. The CTDs of CXCR2 (45 residues), CXCR1 (44 residues), CXCR3 (49 residues) and CXCR4 (47 residues) were aligned with CLUSTALW (1.83) multiple sequence alignment program. The LLKIL functional motif of CXCR2 and similar putative motifs in other CXC receptor CTDs are in bold. Also, the serine residues known to be phosphorylated in CXCR2 CTD in response to CXCL8 stimulation are in bold. Bottom panel: The mutations in CXCR2 important for binding of AP2 and β-arrestin are illustrated. B) Decreased association of CXCR2 mutants with AP2 and/or β-arrestin1 after stimulation with CXCL8. dHL-60 cells stably expressing CXCR2-WT, 4A or CXCR2-4A/IL mutants were stimulated with or without CXCL8. CXCR2 was immunoprecipitated with anti-CXCR2 antibody, and blotted for AP2-β2 subunit or β-arrestin1. The blot was stripped and re-blotted for CXCR2. The relative values of fold increase in response to CXCL8 stimulation for each cell line calculated from 3 independent experiments is shown under the western blots (fold ± S.E.M.). One tenth of the total lysate input for co-immunoprecipitation was used for Western analysis of the total lysates. C) CXCL8-mediated internalization of CXCR2 is abolished in 4A/IL mutant of CXCR2, but only partially attenuated in 4A-CXCR2 mutant. The internalization of CXCR2 was performed by following the internalization of 125 I-CXCL8 in dHL60-CXCR2 cells stably expressing CXCR2-WT, 4A or 4A/IL mutant. Error bars are S.E.M and the experiments were repeated 3 times with duplicates for each treatment. ANOVA: 2 min – 4A vs. 4A/IL, p

    Techniques Used: Chemotaxis Assay, Sequencing, Functional Assay, Binding Assay, Stable Transfection, Expressing, Immunoprecipitation, Western Blot, Mutagenesis

    9) Product Images from "Kaposi Sarcoma-associated Herpesvirus vIRF-3 Protein Binds to F-box of Skp2 Protein and Acts as a Regulator of c-Myc Protein Function and Stability *"

    Article Title: Kaposi Sarcoma-associated Herpesvirus vIRF-3 Protein Binds to F-box of Skp2 Protein and Acts as a Regulator of c-Myc Protein Function and Stability *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.335216

    vIRF-3 interacts with F-box of Skp2. A , schematic representation of Skp2 deletion mutants tagged with Myc tag. The shaded box indicates the vIRF-3-binding domain. LRR , leucine-rich region. B , co-immunoprecipitation of Skp2 and vIRF-3 in PEL cells, BC-3
    Figure Legend Snippet: vIRF-3 interacts with F-box of Skp2. A , schematic representation of Skp2 deletion mutants tagged with Myc tag. The shaded box indicates the vIRF-3-binding domain. LRR , leucine-rich region. B , co-immunoprecipitation of Skp2 and vIRF-3 in PEL cells, BC-3

    Techniques Used: Binding Assay, Immunoprecipitation

    10) Product Images from "MISC-1/OGC Links Mitochondrial Metabolism, Apoptosis and Insulin Secretion"

    Article Title: MISC-1/OGC Links Mitochondrial Metabolism, Apoptosis and Insulin Secretion

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0017827

    OGC interacts with Bcl-x L and ANT. We performed a co-immunoprecipitation experiment in HEK293 cells using an antibody against ANT, which is an integral component of the MPTP. We were able to detect Bcl-X L , OGC and ANT in the co-immuno precipitant. As a control protein, we chose subunit 1 of complex IV of the electron transport chain, which is also localized to the inner mitochondrial membrane. This control protein did not co-immunoprecipitate with Bcl-x L , OGC and ANT, but was present in the supernatant (input). We obtained similar results by immunoprecipitation with an antibody against Bcl-x L (data not shown). Co-IP: co-immunoprecipitation. WB: western blot.
    Figure Legend Snippet: OGC interacts with Bcl-x L and ANT. We performed a co-immunoprecipitation experiment in HEK293 cells using an antibody against ANT, which is an integral component of the MPTP. We were able to detect Bcl-X L , OGC and ANT in the co-immuno precipitant. As a control protein, we chose subunit 1 of complex IV of the electron transport chain, which is also localized to the inner mitochondrial membrane. This control protein did not co-immunoprecipitate with Bcl-x L , OGC and ANT, but was present in the supernatant (input). We obtained similar results by immunoprecipitation with an antibody against Bcl-x L (data not shown). Co-IP: co-immunoprecipitation. WB: western blot.

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

    11) Product Images from "Structure of the Tuberous Sclerosis Complex 2 (TSC2) N Terminus Provides Insight into Complex Assembly and Tuberous Sclerosis Pathogenesis *"

    Article Title: Structure of the Tuberous Sclerosis Complex 2 (TSC2) N Terminus Provides Insight into Complex Assembly and Tuberous Sclerosis Pathogenesis *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.732446

    Interaction mapping of the TSC complex. a , schematic representation of the domain architecture of TSC1 and TSC2 from C. thermophilum and human hamartin and tuberin. b , reconstitution of the complex between TSC1 and TSC2-N with recombinant proteins. His 6 -SUMO (HS)-TSC1 and GST-TSC2 were co-expressed in E. coli and purified via GSH affinity chromatography. TSC2-N binds TSC1-fl, but not TSC1-N. Note that TSC1 is partially degraded when expressed alone (marked by an asterisk ), and the yield of TSC2-N is reduced when purified in a complex. c , the N terminus of TSC2 is sufficient for binding to TSC1. Full-length FLAG-tagged TSC1 was co-transfected with HA-TSC2 constructs, and their interaction was probed by co-immunoprecipitation and Western blotting analysis. d , TSC2 binds to the C terminus of TSC1. FLAG-TSC1 full-length and truncations were co-transfected with HA-TSC2, and binding was probed by co-immunoprecipitation and Western blotting analysis. e , tuberin ( tub ) N terminus binds the hamartin ( ham ) C terminus. FLAG-hamartin C-terminal half was co-transfected with HA-tuberin full-length and N terminus, and binding was probed by co-immunoprecipitation and Western blotting analysis. IP , immunoprecipitation.
    Figure Legend Snippet: Interaction mapping of the TSC complex. a , schematic representation of the domain architecture of TSC1 and TSC2 from C. thermophilum and human hamartin and tuberin. b , reconstitution of the complex between TSC1 and TSC2-N with recombinant proteins. His 6 -SUMO (HS)-TSC1 and GST-TSC2 were co-expressed in E. coli and purified via GSH affinity chromatography. TSC2-N binds TSC1-fl, but not TSC1-N. Note that TSC1 is partially degraded when expressed alone (marked by an asterisk ), and the yield of TSC2-N is reduced when purified in a complex. c , the N terminus of TSC2 is sufficient for binding to TSC1. Full-length FLAG-tagged TSC1 was co-transfected with HA-TSC2 constructs, and their interaction was probed by co-immunoprecipitation and Western blotting analysis. d , TSC2 binds to the C terminus of TSC1. FLAG-TSC1 full-length and truncations were co-transfected with HA-TSC2, and binding was probed by co-immunoprecipitation and Western blotting analysis. e , tuberin ( tub ) N terminus binds the hamartin ( ham ) C terminus. FLAG-hamartin C-terminal half was co-transfected with HA-tuberin full-length and N terminus, and binding was probed by co-immunoprecipitation and Western blotting analysis. IP , immunoprecipitation.

    Techniques Used: Recombinant, Purification, Affinity Chromatography, Binding Assay, Transfection, Construct, Immunoprecipitation, Western Blot

    Conserved interaction interface on TSC2-N. a , co-immunoprecipitation of tuberin-N ( tub ) point mutations with the C terminus of hamartin ( ham ). The pathogenic surface mutation E75G (equivalent L122G in Ct TSC2) and the structural mutation R261P (equivalent to S341P) show impaired interaction, whereas R261W (equivalent to S341W) binding is comparable with wild type. b , quantification of the co-immunoprecipitation experiment from three biological repeats. c , residues that affect TSC1 binding (colored magenta ) map to the base of HR 2 and 3 in TSC2-N. The locations of mutations that showed no effect in our assay are shown in lime green. IP , immunoprecipitation.
    Figure Legend Snippet: Conserved interaction interface on TSC2-N. a , co-immunoprecipitation of tuberin-N ( tub ) point mutations with the C terminus of hamartin ( ham ). The pathogenic surface mutation E75G (equivalent L122G in Ct TSC2) and the structural mutation R261P (equivalent to S341P) show impaired interaction, whereas R261W (equivalent to S341W) binding is comparable with wild type. b , quantification of the co-immunoprecipitation experiment from three biological repeats. c , residues that affect TSC1 binding (colored magenta ) map to the base of HR 2 and 3 in TSC2-N. The locations of mutations that showed no effect in our assay are shown in lime green. IP , immunoprecipitation.

    Techniques Used: Immunoprecipitation, Mutagenesis, Binding Assay

    12) Product Images from "PRMT5: A novel regulator of Hepatitis B virus replication and an arginine methylase of HBV core"

    Article Title: PRMT5: A novel regulator of Hepatitis B virus replication and an arginine methylase of HBV core

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0186982

    Mass spectrometry analysis of HBc and its PTMs. (A) MS analysis detected high molecular structures of HBc protein. Protein lysates isolated from HBc-HA transfected HepG2_hNTCP cells were subjected to immunoprecipitation with anti-HA magnetic beads. The immunoprecipitated HBc protein was resolved by SDS-PAGE and stained by Coomassie blue (lane 1), visualized by Western blot with anti-HBc (lane 2) or anti-Ub (lane 3) antibodies. The Coomassie stained gel was cut into 14 slices and resulting bands were analyzed by LC-MS/MS. The column “No. of HBc peptides” corresponds to number of peptides spectra identified with confidence higher than 95% as assigned by ProteinPilot. (B) Tandem mass spectra of peptides derived from wt HBc showing ubiquitin conjugation with di-Gly modification (upper panel) of specific HBc residues. The lower panel represents the spectrum for dimethyl modification of wt HBc at R150. (C) MS analysis of immunoprecipitated wt HBc revealed multiple ubiquitinated ( U ), arginine dimethylated ( D ) and phosphorylated ( P ) sites. I, II, III, IV–four arginine-rich domains. (D) Potential PTMs of HBc detected by MS analysis of HBc mutant proteins (I, I_II_III, I_II_IV, II_III_IV, I_II_III_IV and I*_II_III_IV). The amino acid sequence is derived from HBc mutant I_II_III_IV containing mutations of all four arginine-rich clusters. U , ubiquitination; P , phosphorylation; M , arginine-monomethylation. (E) Tandem mass spectra of peptides derived from HBc mutant (I_II_III) showing ubiquitin conjugation with di-Gly modification at K7 (upper panel). The lower panel represents spectrum of monomethyl modification of HBc mutant (I*_II_III_IV) at R156.
    Figure Legend Snippet: Mass spectrometry analysis of HBc and its PTMs. (A) MS analysis detected high molecular structures of HBc protein. Protein lysates isolated from HBc-HA transfected HepG2_hNTCP cells were subjected to immunoprecipitation with anti-HA magnetic beads. The immunoprecipitated HBc protein was resolved by SDS-PAGE and stained by Coomassie blue (lane 1), visualized by Western blot with anti-HBc (lane 2) or anti-Ub (lane 3) antibodies. The Coomassie stained gel was cut into 14 slices and resulting bands were analyzed by LC-MS/MS. The column “No. of HBc peptides” corresponds to number of peptides spectra identified with confidence higher than 95% as assigned by ProteinPilot. (B) Tandem mass spectra of peptides derived from wt HBc showing ubiquitin conjugation with di-Gly modification (upper panel) of specific HBc residues. The lower panel represents the spectrum for dimethyl modification of wt HBc at R150. (C) MS analysis of immunoprecipitated wt HBc revealed multiple ubiquitinated ( U ), arginine dimethylated ( D ) and phosphorylated ( P ) sites. I, II, III, IV–four arginine-rich domains. (D) Potential PTMs of HBc detected by MS analysis of HBc mutant proteins (I, I_II_III, I_II_IV, II_III_IV, I_II_III_IV and I*_II_III_IV). The amino acid sequence is derived from HBc mutant I_II_III_IV containing mutations of all four arginine-rich clusters. U , ubiquitination; P , phosphorylation; M , arginine-monomethylation. (E) Tandem mass spectra of peptides derived from HBc mutant (I_II_III) showing ubiquitin conjugation with di-Gly modification at K7 (upper panel). The lower panel represents spectrum of monomethyl modification of HBc mutant (I*_II_III_IV) at R156.

    Techniques Used: Mass Spectrometry, Isolation, Transfection, Immunoprecipitation, Magnetic Beads, SDS Page, Staining, Western Blot, Liquid Chromatography with Mass Spectroscopy, Derivative Assay, Conjugation Assay, Modification, Mutagenesis, Sequencing

    Symmetric dimethylation and monomethylation of arginine residues at the C-terminal domain of HBc protein. (A) Detection of monomethylation and symmetric dimethylation of HBc protein. HepG2_hNTCP cells were transfected with HBc-HA (full length, aa 1–185), HBc-ΔC-HA (C-terminal deletion, aa 1–149), or an empty vector (pcDNA). Forty-eight hours after transfection, cell lysates (400 μg) were precipitated with anti-HA antibodies (HBc and HBc-ΔC), and the precipitated complexes were analyzed by Western blot with monomethyl-arginine (MM-R, top panel) or symmetric-dimethyl-arginine (SDM-R, middle panel) antibodies. The quality/efficiency of anti-HA immunoprecipitation is shown by Western blot with anti-HA or anti-HBc antibodies in the bottom panels. *, non-specific WB signal. Red and green arrows show HBc protein fragments of 30 kDa and 48 kDa, respectively. (B) Cell lysates (400 μg) of HepG2.2.15 cells were precipitated with HBc antibodies and the precipitated complexes were analyzed by Western blot with monomethyl-arginine (MM-R, top panel) or symmetric-dimethyl-arginine (SDM-R, middle panel) antibodies. The expression level of HBc protein is shown in the bottom panel. *, non-specific. (C) Symmetric dimethylation of HBc is stimulated by PRMT5, PRMT7 and MEP50. HEK293T cells were co-transfected with a constant amount of HBc-HA expression plasmid in combination with expression plasmids encoding Flag-tagged PRMT1, PRMT3, PRMT5 (v1, v2), PRMT7, PRMT9, MEP50, or an empty vector. Forty-eight hours after transfection, cell lysates (400 μg) were precipitated with anti-HA antibodies (HBc), and the precipitated complexes were analyzed by Western blot with SDM-R antibodies. The relative levels of transfected HBc-HA and Flag-PRMTs or Flag-MEP50 in 40 μg of cell lysates were estimated by Western blots (10% input). The intensity of the symmetric dimethylation signal was quantified using the ImageQuant TL Array software and normalized to the level of HBc expression. The level of HBc methylation in mock (pcDNA) transfected cells was set to 1 (Quant. and graph below). Asterisks indicate statistically significant differences between the control (pcDNA) and PRMTs groups determined by ANOVA (*** P
    Figure Legend Snippet: Symmetric dimethylation and monomethylation of arginine residues at the C-terminal domain of HBc protein. (A) Detection of monomethylation and symmetric dimethylation of HBc protein. HepG2_hNTCP cells were transfected with HBc-HA (full length, aa 1–185), HBc-ΔC-HA (C-terminal deletion, aa 1–149), or an empty vector (pcDNA). Forty-eight hours after transfection, cell lysates (400 μg) were precipitated with anti-HA antibodies (HBc and HBc-ΔC), and the precipitated complexes were analyzed by Western blot with monomethyl-arginine (MM-R, top panel) or symmetric-dimethyl-arginine (SDM-R, middle panel) antibodies. The quality/efficiency of anti-HA immunoprecipitation is shown by Western blot with anti-HA or anti-HBc antibodies in the bottom panels. *, non-specific WB signal. Red and green arrows show HBc protein fragments of 30 kDa and 48 kDa, respectively. (B) Cell lysates (400 μg) of HepG2.2.15 cells were precipitated with HBc antibodies and the precipitated complexes were analyzed by Western blot with monomethyl-arginine (MM-R, top panel) or symmetric-dimethyl-arginine (SDM-R, middle panel) antibodies. The expression level of HBc protein is shown in the bottom panel. *, non-specific. (C) Symmetric dimethylation of HBc is stimulated by PRMT5, PRMT7 and MEP50. HEK293T cells were co-transfected with a constant amount of HBc-HA expression plasmid in combination with expression plasmids encoding Flag-tagged PRMT1, PRMT3, PRMT5 (v1, v2), PRMT7, PRMT9, MEP50, or an empty vector. Forty-eight hours after transfection, cell lysates (400 μg) were precipitated with anti-HA antibodies (HBc), and the precipitated complexes were analyzed by Western blot with SDM-R antibodies. The relative levels of transfected HBc-HA and Flag-PRMTs or Flag-MEP50 in 40 μg of cell lysates were estimated by Western blots (10% input). The intensity of the symmetric dimethylation signal was quantified using the ImageQuant TL Array software and normalized to the level of HBc expression. The level of HBc methylation in mock (pcDNA) transfected cells was set to 1 (Quant. and graph below). Asterisks indicate statistically significant differences between the control (pcDNA) and PRMTs groups determined by ANOVA (*** P

    Techniques Used: Transfection, Plasmid Preparation, Western Blot, Immunoprecipitation, Expressing, Software, Methylation

    Nuclear versus cytoplasmic accumulation of monomethylated and symmetrically dimethylated HBc wt and ARD mutants. (A) HepG2_hNTCP cells were transfected with HA-tagged wt HBc or an empty vector, pcDNA, as indicated. Forty-eight hours after transfection, the cells were fractionated into nuclear and cytoplasmic extracts. The cell lysates (400 μg) were analyzed by immunoprecipitation (IP) with anti-HA-specific antibodies followed by Western blotting with SDM-R and MM-R antibodies. The relative levels of transfected HBc-HA in 40 μg of cell lysates were estimated by Western blots (bottom panel). *, non-specific WB signal. (B) HepG2_hNTCP cells were transfected with HA-tagged wt HBc and selected ARD mutants, as indicated. Forty-eight hours after transfection, the cells were fractionated into cytoplasmic (left), nuclear (middle) or whole-cell extracts (WCE; right). The cell lysates (400 μg) were analyzed by immunoprecipitation (IP) with anti-HA-specific antibodies followed by Western blotting with SDM-R antibodies. The relative levels of transfected HBc-HA in 40 μg of cell lysates were estimated by Western blots (bottom panel). *, non-specific WB signal. The intensity of methylation signal was quantified using the ImageQuant TL Array software and normalized to the level of HBc expression. The levels representing nuclear methylation of wt and individual ARD mutants were plotted against the corresponding levels of cytoplasmic methylation. (C) Down-regulation of PRMT5 and MEP50 leads to reduced levels of HBc protein in nuclei of transfected cells. HepG2_hNTCP_HBc-Flag stable cell line was transfected with PRMT5-, MEP50-specific siRNAs or control siRNA. Forty-eight hours after transfection, the cells were fractionated into nuclear and cytoplasmic extracts and analyzed by Western blot with anti-PRMT5, MEP50 and Flag antibodies. The intensity of HBc signal was quantified using the ImageQuant TL Array software and normalized to the level of Histone 3 expression. (D) A graphical representation of localization tendencies of different monomethylated, symmetrically dimethylated, phosphorylated and poly-ubiquitinated forms of HBc protein.
    Figure Legend Snippet: Nuclear versus cytoplasmic accumulation of monomethylated and symmetrically dimethylated HBc wt and ARD mutants. (A) HepG2_hNTCP cells were transfected with HA-tagged wt HBc or an empty vector, pcDNA, as indicated. Forty-eight hours after transfection, the cells were fractionated into nuclear and cytoplasmic extracts. The cell lysates (400 μg) were analyzed by immunoprecipitation (IP) with anti-HA-specific antibodies followed by Western blotting with SDM-R and MM-R antibodies. The relative levels of transfected HBc-HA in 40 μg of cell lysates were estimated by Western blots (bottom panel). *, non-specific WB signal. (B) HepG2_hNTCP cells were transfected with HA-tagged wt HBc and selected ARD mutants, as indicated. Forty-eight hours after transfection, the cells were fractionated into cytoplasmic (left), nuclear (middle) or whole-cell extracts (WCE; right). The cell lysates (400 μg) were analyzed by immunoprecipitation (IP) with anti-HA-specific antibodies followed by Western blotting with SDM-R antibodies. The relative levels of transfected HBc-HA in 40 μg of cell lysates were estimated by Western blots (bottom panel). *, non-specific WB signal. The intensity of methylation signal was quantified using the ImageQuant TL Array software and normalized to the level of HBc expression. The levels representing nuclear methylation of wt and individual ARD mutants were plotted against the corresponding levels of cytoplasmic methylation. (C) Down-regulation of PRMT5 and MEP50 leads to reduced levels of HBc protein in nuclei of transfected cells. HepG2_hNTCP_HBc-Flag stable cell line was transfected with PRMT5-, MEP50-specific siRNAs or control siRNA. Forty-eight hours after transfection, the cells were fractionated into nuclear and cytoplasmic extracts and analyzed by Western blot with anti-PRMT5, MEP50 and Flag antibodies. The intensity of HBc signal was quantified using the ImageQuant TL Array software and normalized to the level of Histone 3 expression. (D) A graphical representation of localization tendencies of different monomethylated, symmetrically dimethylated, phosphorylated and poly-ubiquitinated forms of HBc protein.

    Techniques Used: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Methylation, Software, Expressing, Stable Transfection

    HBc protein interacts with PRMT3, PRMT5 and MEP50. (A) Schematic representation of HBc (Genotype A, subtype adw2) and its C-terminal deletion mutant (HBc-ΔC). ARD, Arginine-rich domain; S, serine residues modified by phosphorylation. (B) Flag-tagged PRMT1, PRMT3, PRMT5v1 and MEP50 were in vitro translated using TNT T7 quick coupled transcription/translation system and incubated with HBc fused to GST or GST alone immobilized on glutathione-Sepharose beads. The bound proteins were eluted and resolved on 10% SDS-PAGE followed by Western blot with anti-Flag antibodies. Five % of PRMTs´ protein input is shown below (5% input). (C) HBc protein interacts with PRMT3, PRMT5 and MEP50 via its C-terminal domain. HEK293T cells were transfected with equal amounts of Flag-tagged PRMT1, PRMT3, PRMT5 (v1, v2) and MEP50 expression constructs. Forty-eight hours after transfection, the cell lysates were incubated with HBc (aa 1–185), HBc-ΔC (aa 1–149) fused to GST or GST alone immobilized on glutathione-Sepharose beads. The bound proteins were eluted and resolved on 10% SDS-PAGE followed by Western blot with anti-Flag antibodies. Five % of PRMTs´ protein input is shown below (5% input). (D) Coomassie blue staining of purified GST, GST-HBc, and GST-HBc-ΔC used in GST pull-down experiments. (E) Co-immunoprecipitation of HBc and PRMTs in transfected HEK293T cells. HEK293T cells were transfected with Flag-tagged PRMTs’ expression plasmids and HBc-V5/AP expression plasmid, as indicated. Forty-eight hours after transfection, the cells were harvested and protein lysates were prepared. Protein lysates (400 μg) were immunoprecipitated (IP) with anti-Flag antibodies, and the immunoprecipitated complexes were analyzed by Western blot (WB) with NeutrAvidin conjugated to HRP. The relative levels of PRMTs and HBc in 40 μg of protein lysates are shown for comparison (10% input). (F) Co-immunoprecipitation of HBc and endogenous PRMT5 in HepG2.2.15 cells. Protein lysates (400 μg) isolated from HepG2.2.15 cells were immunoprecipitated (IP) with anti-HBc or control (IgG) antibodies. The immunoprecipitated complexes were analyzed by Western blot (WB) with anti-PRMT5 and anti-HBc antibodies. The relative levels of PRMT5 and HBc in 40 μg of protein lysates are shown in bottom panels (10% input).
    Figure Legend Snippet: HBc protein interacts with PRMT3, PRMT5 and MEP50. (A) Schematic representation of HBc (Genotype A, subtype adw2) and its C-terminal deletion mutant (HBc-ΔC). ARD, Arginine-rich domain; S, serine residues modified by phosphorylation. (B) Flag-tagged PRMT1, PRMT3, PRMT5v1 and MEP50 were in vitro translated using TNT T7 quick coupled transcription/translation system and incubated with HBc fused to GST or GST alone immobilized on glutathione-Sepharose beads. The bound proteins were eluted and resolved on 10% SDS-PAGE followed by Western blot with anti-Flag antibodies. Five % of PRMTs´ protein input is shown below (5% input). (C) HBc protein interacts with PRMT3, PRMT5 and MEP50 via its C-terminal domain. HEK293T cells were transfected with equal amounts of Flag-tagged PRMT1, PRMT3, PRMT5 (v1, v2) and MEP50 expression constructs. Forty-eight hours after transfection, the cell lysates were incubated with HBc (aa 1–185), HBc-ΔC (aa 1–149) fused to GST or GST alone immobilized on glutathione-Sepharose beads. The bound proteins were eluted and resolved on 10% SDS-PAGE followed by Western blot with anti-Flag antibodies. Five % of PRMTs´ protein input is shown below (5% input). (D) Coomassie blue staining of purified GST, GST-HBc, and GST-HBc-ΔC used in GST pull-down experiments. (E) Co-immunoprecipitation of HBc and PRMTs in transfected HEK293T cells. HEK293T cells were transfected with Flag-tagged PRMTs’ expression plasmids and HBc-V5/AP expression plasmid, as indicated. Forty-eight hours after transfection, the cells were harvested and protein lysates were prepared. Protein lysates (400 μg) were immunoprecipitated (IP) with anti-Flag antibodies, and the immunoprecipitated complexes were analyzed by Western blot (WB) with NeutrAvidin conjugated to HRP. The relative levels of PRMTs and HBc in 40 μg of protein lysates are shown for comparison (10% input). (F) Co-immunoprecipitation of HBc and endogenous PRMT5 in HepG2.2.15 cells. Protein lysates (400 μg) isolated from HepG2.2.15 cells were immunoprecipitated (IP) with anti-HBc or control (IgG) antibodies. The immunoprecipitated complexes were analyzed by Western blot (WB) with anti-PRMT5 and anti-HBc antibodies. The relative levels of PRMT5 and HBc in 40 μg of protein lysates are shown in bottom panels (10% input).

    Techniques Used: Mutagenesis, Modification, In Vitro, Incubation, SDS Page, Western Blot, Transfection, Expressing, Construct, Staining, Purification, Immunoprecipitation, Plasmid Preparation, Isolation

    HBc protein is modified by ubiquitination on K7 and by methylation on R150 and R156. (A) HepG2_hNTCP cells were transfected with wt HBc tagged with Flag or an empty vector, pcDNA. Forty-eight hours after transfection, cell lysates (400 μg) were precipitated with anti-Flag antibodies (HBc), and the precipitated complexes were analyzed by Western blot with SDM-R, MM-R and Ub antibodies. The quality/efficiency of anti-Flag immunoprecipitation is shown in the bottom panel. (B) HepG2_hNTCP cells were transfected with wt Ub-HA together with wt HBc or K-to-R single and double mutants (HBc-K7R, HBc-K96R and HBc-K7/96R) tagged with Flag, as indicated. Forty-eight hours after transfection, cell lysates (400 μg) were precipitated with anti-Flag antibodies (HBc), and the precipitated complexes were analyzed by Western blot with Ub antibodies. The quality/efficiency of anti-Flag immunoprecipitation is shown in the bottom panel. (C) HepG2_hNTCP cells were transfected with wt HBc or R-to-A single and double mutants (HBc-R150A, HBc-R156A and HBc-R150/156A) tagged with HA, as indicated. Forty-eight hours after transfection, cell lysates (400 μg) were precipitated with anti-HA antibodies (HBc), and the precipitated complexes were analyzed by Western blot with SDM-R, MM-R and phospho-serine antibodies. The relative levels of transfected HBc-HA in 40 μg of cell lysates were estimated by Western blots (bottom panel). *, non-specific WB signal.
    Figure Legend Snippet: HBc protein is modified by ubiquitination on K7 and by methylation on R150 and R156. (A) HepG2_hNTCP cells were transfected with wt HBc tagged with Flag or an empty vector, pcDNA. Forty-eight hours after transfection, cell lysates (400 μg) were precipitated with anti-Flag antibodies (HBc), and the precipitated complexes were analyzed by Western blot with SDM-R, MM-R and Ub antibodies. The quality/efficiency of anti-Flag immunoprecipitation is shown in the bottom panel. (B) HepG2_hNTCP cells were transfected with wt Ub-HA together with wt HBc or K-to-R single and double mutants (HBc-K7R, HBc-K96R and HBc-K7/96R) tagged with Flag, as indicated. Forty-eight hours after transfection, cell lysates (400 μg) were precipitated with anti-Flag antibodies (HBc), and the precipitated complexes were analyzed by Western blot with Ub antibodies. The quality/efficiency of anti-Flag immunoprecipitation is shown in the bottom panel. (C) HepG2_hNTCP cells were transfected with wt HBc or R-to-A single and double mutants (HBc-R150A, HBc-R156A and HBc-R150/156A) tagged with HA, as indicated. Forty-eight hours after transfection, cell lysates (400 μg) were precipitated with anti-HA antibodies (HBc), and the precipitated complexes were analyzed by Western blot with SDM-R, MM-R and phospho-serine antibodies. The relative levels of transfected HBc-HA in 40 μg of cell lysates were estimated by Western blots (bottom panel). *, non-specific WB signal.

    Techniques Used: Modification, Methylation, Transfection, Plasmid Preparation, Western Blot, Immunoprecipitation

    13) Product Images from "Stress-induced TRBP phosphorylation enhances its interaction with PKR to regulate cellular survival"

    Article Title: Stress-induced TRBP phosphorylation enhances its interaction with PKR to regulate cellular survival

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-19360-8

    TRBP phosphorylation strengthens PKR-TRBP interaction and weakens TRBP-TRBP interaction. ( A ) Phospho-mimic TRBP mutant interacts stronger with PKR compared to the phospho-defective TRBP mutant in yeast two-hybrid assay. PKR/pGAD424 and either AAAA TRBP/pGBKT7, DDDD TRBP/pGBKT7, or wt TRBP/pGBKT7 were co-transformed into AH109 yeast cells and selected on SD double dropout media (-tryptophan, - leucine). Ten microliters of transformed yeast cells (OD 600 = 10, 1, 0.1, 0.01) were spotted on SD triple dropout media (-tryptophan, - leucine, - histidine) containing 10 mM 3-amino-1,2,4-triazole (3-AT). Plates were incubated for 3 days at 30 °C. Transformation of PKR in pGAD424 and pGBKT7 empty vector served as a negative control. ( B ) Phosphomimic TRBP mutant shows stronger heteromeric interaction with PKR compared to the phosphodefective TRBP mutant in mammalian cells. HeLa cells were transfected with Flag K296R PKR/pcDNA 3.1 − and either myc TRBP AAAA/pcDNA 3.1 − , myc wt TRBP/pcDNA 3.1 − , or myc TRBP DDDD/pcDNA 3.1 − . The cells were harvested 24 hours after transfection, and myc AAAA, DDDD or wt TRBP was immunoprecipitated using anti-myc monoclonal antibody conjugated agarose beads. Co-immunoprecipitated Flag PKR was analyzed by western blot analysis with an anti-Flag antibody (IP: x Flag (PKR) panel). The blot was subsequently re-probed with anti-myc antibody to ensure equal myc TRBP immunoprecipitation from each sample (IP: x myc (TRBP) panel). Equal Flag PKR and myc TRBP expression in all samples was tested by western blot analysis of equal amounts of total cell lysate with anti-myc, and anti-Flag antibodies (input: x Flag (PKR) and x myc (TRBP) panels). ( C ) Changes in TRBP association with PKR. Flag TRBP overexpressing cells were treated with 25 μM sodium arsenite for the indicated time points. Cell extracts were prepared in the presence of a phosphatase inhibitor and 25 μg of cell extract was incubated with 500 ng of pure recombinant hexahistidine (His)-tagged PKR immobilized on Ni 2+ -agarose beads. After washing the beads, PKR-associated Flag TRBP was analyzed by SDS polyacrylamide gel electrophoresis followed by western blot analysis with anti-Flag antibody. Western blot analysis was also performed with anti-His antibody to ensure equal His- PKR in each sample. 25 μg of cell extract was also analyzed by western blot analysis with anti-Flag and anti-GAPDH antibodies to ensure equal addition of cell lysate for each pull down (Input). Quantification of TRBP-PKR pull down: Band intensities were quantified using ImageQuant TL Software, and the ratios of bound TRBP to bound PKR across all samples were calculated and normalized to the band intensities of Flag-TRBP input for each sample. Bound TRBP/his-PKR ratios for all samples were all expressed relative to the control sample (Lane 2). Averages from three independent experiments are plotted as bar graphs ± S.D. One-way ANOVA followed by post-hoc Tukey test was performed, asterisk *p value 0.0000012 and double asterisk **p value 0.0066374. ( D ) Phosphomimic TRBP mutant shows stronger homomeric interaction compared to the phosphodefective TRBP mutant in yeast two-hybrid assay. AAAA TRBP or DDDD TRBP point mutants in pGADT7 and pGBKT7 were co-transformed into AH109 yeast cells and selected on SD double dropout media (-tryptophan, -leucine). Ten microliters of transformed yeast cells (OD 600 = 10, 1, 0.1, 0.01) were spotted on SD triple dropout media plate (tryptophan, -leucine, -histidine) containing 10 mM 3-amino-1,2,4-triazole (3-AT). Plates were incubated for 5 days at 30 °C. Transformation of pGADT7 and pGBKT7 empty vectors served as a negative control. ( E ) Phosphomimic TRBP mutant shows stronger homomeric interaction compared to the phosphodefective TRBP mutant in mammalian cells. HeLa cells were transfected with either myc TRBP DDDD/pcDNA 3.1 − and Flag TRBP DDDD/pcDNA 3.1 − or Flag TRBP AAAA/pcDNA 3.1 − and myc TRBP AAAA/pcDNA 3.1 − . The cells were harvested 24 hours after transfection, and Flag TRBP AAAA or DDDD was immunoprecipitated using anti-Flag monoclonal antibody conjugated agarose beads. The co-immunoprecipitation of myc-TRBP was analyzed by western blot analysis with an anti-myc antibody (IP: x Myc panel). Blot was subsequently stripped and re-probed with anti-Flag antibody to ensure equal Flag-TRBP immunoprecipitation from each sample (IP: x Flag panel). Equal AAAA TRBP and DDDD TRBP expression in all samples was tested by western blot analysis of equal amounts of total cell lysate with anti-myc, and anti-Flag antibodies (Input: x Myc and x Flag panels).
    Figure Legend Snippet: TRBP phosphorylation strengthens PKR-TRBP interaction and weakens TRBP-TRBP interaction. ( A ) Phospho-mimic TRBP mutant interacts stronger with PKR compared to the phospho-defective TRBP mutant in yeast two-hybrid assay. PKR/pGAD424 and either AAAA TRBP/pGBKT7, DDDD TRBP/pGBKT7, or wt TRBP/pGBKT7 were co-transformed into AH109 yeast cells and selected on SD double dropout media (-tryptophan, - leucine). Ten microliters of transformed yeast cells (OD 600 = 10, 1, 0.1, 0.01) were spotted on SD triple dropout media (-tryptophan, - leucine, - histidine) containing 10 mM 3-amino-1,2,4-triazole (3-AT). Plates were incubated for 3 days at 30 °C. Transformation of PKR in pGAD424 and pGBKT7 empty vector served as a negative control. ( B ) Phosphomimic TRBP mutant shows stronger heteromeric interaction with PKR compared to the phosphodefective TRBP mutant in mammalian cells. HeLa cells were transfected with Flag K296R PKR/pcDNA 3.1 − and either myc TRBP AAAA/pcDNA 3.1 − , myc wt TRBP/pcDNA 3.1 − , or myc TRBP DDDD/pcDNA 3.1 − . The cells were harvested 24 hours after transfection, and myc AAAA, DDDD or wt TRBP was immunoprecipitated using anti-myc monoclonal antibody conjugated agarose beads. Co-immunoprecipitated Flag PKR was analyzed by western blot analysis with an anti-Flag antibody (IP: x Flag (PKR) panel). The blot was subsequently re-probed with anti-myc antibody to ensure equal myc TRBP immunoprecipitation from each sample (IP: x myc (TRBP) panel). Equal Flag PKR and myc TRBP expression in all samples was tested by western blot analysis of equal amounts of total cell lysate with anti-myc, and anti-Flag antibodies (input: x Flag (PKR) and x myc (TRBP) panels). ( C ) Changes in TRBP association with PKR. Flag TRBP overexpressing cells were treated with 25 μM sodium arsenite for the indicated time points. Cell extracts were prepared in the presence of a phosphatase inhibitor and 25 μg of cell extract was incubated with 500 ng of pure recombinant hexahistidine (His)-tagged PKR immobilized on Ni 2+ -agarose beads. After washing the beads, PKR-associated Flag TRBP was analyzed by SDS polyacrylamide gel electrophoresis followed by western blot analysis with anti-Flag antibody. Western blot analysis was also performed with anti-His antibody to ensure equal His- PKR in each sample. 25 μg of cell extract was also analyzed by western blot analysis with anti-Flag and anti-GAPDH antibodies to ensure equal addition of cell lysate for each pull down (Input). Quantification of TRBP-PKR pull down: Band intensities were quantified using ImageQuant TL Software, and the ratios of bound TRBP to bound PKR across all samples were calculated and normalized to the band intensities of Flag-TRBP input for each sample. Bound TRBP/his-PKR ratios for all samples were all expressed relative to the control sample (Lane 2). Averages from three independent experiments are plotted as bar graphs ± S.D. One-way ANOVA followed by post-hoc Tukey test was performed, asterisk *p value 0.0000012 and double asterisk **p value 0.0066374. ( D ) Phosphomimic TRBP mutant shows stronger homomeric interaction compared to the phosphodefective TRBP mutant in yeast two-hybrid assay. AAAA TRBP or DDDD TRBP point mutants in pGADT7 and pGBKT7 were co-transformed into AH109 yeast cells and selected on SD double dropout media (-tryptophan, -leucine). Ten microliters of transformed yeast cells (OD 600 = 10, 1, 0.1, 0.01) were spotted on SD triple dropout media plate (tryptophan, -leucine, -histidine) containing 10 mM 3-amino-1,2,4-triazole (3-AT). Plates were incubated for 5 days at 30 °C. Transformation of pGADT7 and pGBKT7 empty vectors served as a negative control. ( E ) Phosphomimic TRBP mutant shows stronger homomeric interaction compared to the phosphodefective TRBP mutant in mammalian cells. HeLa cells were transfected with either myc TRBP DDDD/pcDNA 3.1 − and Flag TRBP DDDD/pcDNA 3.1 − or Flag TRBP AAAA/pcDNA 3.1 − and myc TRBP AAAA/pcDNA 3.1 − . The cells were harvested 24 hours after transfection, and Flag TRBP AAAA or DDDD was immunoprecipitated using anti-Flag monoclonal antibody conjugated agarose beads. The co-immunoprecipitation of myc-TRBP was analyzed by western blot analysis with an anti-myc antibody (IP: x Myc panel). Blot was subsequently stripped and re-probed with anti-Flag antibody to ensure equal Flag-TRBP immunoprecipitation from each sample (IP: x Flag panel). Equal AAAA TRBP and DDDD TRBP expression in all samples was tested by western blot analysis of equal amounts of total cell lysate with anti-myc, and anti-Flag antibodies (Input: x Myc and x Flag panels).

    Techniques Used: Mutagenesis, Y2H Assay, Transformation Assay, Incubation, Plasmid Preparation, Negative Control, Transfection, Immunoprecipitation, Western Blot, Expressing, Recombinant, Polyacrylamide Gel Electrophoresis, Software

    14) Product Images from "Angiotensin-(1-7) Prevents Lipopolysaccharide-Induced Autophagy via the Mas Receptor in Skeletal Muscle"

    Article Title: Angiotensin-(1-7) Prevents Lipopolysaccharide-Induced Autophagy via the Mas Receptor in Skeletal Muscle

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21249344

    Ang-(1-7) prevented the LPS-induced disassembly of Beclin1/BCL-2 complex C2C12 cells. ( A ) C2C12 cells were incubated with LPS (500 ng/mL) at different times from 0 to 4 h. The interaction between Beclin1 and BCL-2 was analyzed using immunoprecipitation with anti-BCL-2. From the eluate, the Beclin1 and BCL-2 protein levels were detected by Western blot. The protein levels of GAPDH are shown as loading control. Molecular weights are shown in kDa. ( B ) Quantification of three independent experiments represented in (A). The Beclin1/BCL-2 ratio from the eluate was normalized to GAPDH and expressed as the mean ± S.E. (fold of change relative to time 0. *, p
    Figure Legend Snippet: Ang-(1-7) prevented the LPS-induced disassembly of Beclin1/BCL-2 complex C2C12 cells. ( A ) C2C12 cells were incubated with LPS (500 ng/mL) at different times from 0 to 4 h. The interaction between Beclin1 and BCL-2 was analyzed using immunoprecipitation with anti-BCL-2. From the eluate, the Beclin1 and BCL-2 protein levels were detected by Western blot. The protein levels of GAPDH are shown as loading control. Molecular weights are shown in kDa. ( B ) Quantification of three independent experiments represented in (A). The Beclin1/BCL-2 ratio from the eluate was normalized to GAPDH and expressed as the mean ± S.E. (fold of change relative to time 0. *, p

    Techniques Used: Incubation, Immunoprecipitation, Western Blot

    15) Product Images from "The PAX-SIX-EYA-DACH network modulates GATA-FOG function in fly hematopoiesis and human erythropoiesis"

    Article Title: The PAX-SIX-EYA-DACH network modulates GATA-FOG function in fly hematopoiesis and human erythropoiesis

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.177022

    SIX proteins interact with GATA1 and stimulate GATA1 transcriptional output. (A) Western blot analysis of total cell extracts (input) versus streptavidin pull-downs generated from biotin-labeled TF1 cells transduced with BirA* versus BirA*-SIX2 probed with anti-GATA1 or anti-BirA antibodies as indicated. (B,C) Western blot analysis of anti-MYC immunoprecipitation assays conducted on total cell lysates generated from HEK293T cells co-transfected with GATA1 and MYC-SIX1/MYC-SIX2 versus empty vector. Total cell extract (input) versus anti-MYC-IP samples probed with anti-GATA1 or anti-MYC antibodies. (D) PLA assays testing association of SIX1 and GATA1. No signal was observed in SIX1 KO compared with control cells with endogenous SIX1. Scale bar: 25 µm. Images from two independent experiments shown. (E) HEK293T cells were co-transfected with GATA1-luciferase reporter plasmid, SV40-Renilla luciferase and empty vector, SIX1, SIX2 or GATA1 expression plasmids. Firefly luciferase activity was normalized to Renilla luciferase activity. EV-transfected cultures were set to 1. Data plotted are representative set of quadruplicates with s.d. indicated (*** P
    Figure Legend Snippet: SIX proteins interact with GATA1 and stimulate GATA1 transcriptional output. (A) Western blot analysis of total cell extracts (input) versus streptavidin pull-downs generated from biotin-labeled TF1 cells transduced with BirA* versus BirA*-SIX2 probed with anti-GATA1 or anti-BirA antibodies as indicated. (B,C) Western blot analysis of anti-MYC immunoprecipitation assays conducted on total cell lysates generated from HEK293T cells co-transfected with GATA1 and MYC-SIX1/MYC-SIX2 versus empty vector. Total cell extract (input) versus anti-MYC-IP samples probed with anti-GATA1 or anti-MYC antibodies. (D) PLA assays testing association of SIX1 and GATA1. No signal was observed in SIX1 KO compared with control cells with endogenous SIX1. Scale bar: 25 µm. Images from two independent experiments shown. (E) HEK293T cells were co-transfected with GATA1-luciferase reporter plasmid, SV40-Renilla luciferase and empty vector, SIX1, SIX2 or GATA1 expression plasmids. Firefly luciferase activity was normalized to Renilla luciferase activity. EV-transfected cultures were set to 1. Data plotted are representative set of quadruplicates with s.d. indicated (*** P

    Techniques Used: Western Blot, Generated, Labeling, Transduction, Immunoprecipitation, Transfection, Plasmid Preparation, Proximity Ligation Assay, Luciferase, Expressing, Activity Assay

    Related Articles

    Construct:

    Article Title: Combined decellularisation and dehydration improves the mechanical properties of tissue-engineered sinews
    Article Snippet: An individual sinew construct after 3 weeks in culture is displayed in . .. Decellularisation After 3 weeks of culture, sinew constructs were removed from culture and decellularised using an established decellularisation protocol., Briefly, constructs were soaked in 0.1 wt% ethylenediaminetetraacetic acid (EDTA; Sigma-Aldrich) in deionised water for 4 h at room temperature. .. Next, the constructs were washed in 0.1 wt% sodium dodecyl sulphate (SDS; Sigma-Aldrich) in 0.1% EDTA for 24 h with a single change of solution at 12 h. The constructs were then washed for 1 h in phosphate-buffered saline (PBS) with a single change of PBS after 30 min. Constructs were stored in PBS until tensile testing.

    Western Blot:

    Article Title: The targetable role of herpes virus-associated ubiquitin-specific protease (HAUSP) in p190 BCR-ABL leukemia
    Article Snippet: .. Western blot and immunoprecipitation Total cell extraction was performed using co-immunoprecipitation buffer [150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 50 mM HEPES (pH, 7.5), 1% Triton and 10% glycerol] supplemented with protease inhibitor (cat no. 036K4082; Sigma-Aldrich) and a phosphatase cocktail composed of PMSF and Na3 VO4 . .. Following quantification by Bio-Rad Protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA), 30 µg protein extract was denatured, reduced, separated by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrophoretically transferred onto nitrocellulose membranes.

    Immunoprecipitation:

    Article Title: The targetable role of herpes virus-associated ubiquitin-specific protease (HAUSP) in p190 BCR-ABL leukemia
    Article Snippet: .. Western blot and immunoprecipitation Total cell extraction was performed using co-immunoprecipitation buffer [150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 50 mM HEPES (pH, 7.5), 1% Triton and 10% glycerol] supplemented with protease inhibitor (cat no. 036K4082; Sigma-Aldrich) and a phosphatase cocktail composed of PMSF and Na3 VO4 . .. Following quantification by Bio-Rad Protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA), 30 µg protein extract was denatured, reduced, separated by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrophoretically transferred onto nitrocellulose membranes.

    Protease Inhibitor:

    Article Title: The targetable role of herpes virus-associated ubiquitin-specific protease (HAUSP) in p190 BCR-ABL leukemia
    Article Snippet: .. Western blot and immunoprecipitation Total cell extraction was performed using co-immunoprecipitation buffer [150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 50 mM HEPES (pH, 7.5), 1% Triton and 10% glycerol] supplemented with protease inhibitor (cat no. 036K4082; Sigma-Aldrich) and a phosphatase cocktail composed of PMSF and Na3 VO4 . .. Following quantification by Bio-Rad Protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA), 30 µg protein extract was denatured, reduced, separated by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrophoretically transferred onto nitrocellulose membranes.

    Concentration Assay:

    Article Title: C-Reactive Protein Stimulates Nicotinic Acetylcholine Receptors to Control ATP-Mediated Monocytic Inflammasome Activation
    Article Snippet: To detect low cytokine levels in cell culture supernatants, for IL-1β the Human IL-1 beta/IL-1F2 DuoSet ELISA (R & D Systems) was used, whereas IL6 and TNF-α were measured by the Human Quantikine® Immunoassays (R & D Systems, Minneapolis, MN, USA). .. Endogenous CRP was dissolved at a concentration of 5 µg/ml in PBS devoid of Ca2+ and Mg2+ (Gibco) containing 1.1 mM ethylenediaminetetraacetic acid (EDTA; Sigma-Aldrich), incubated at 37°C for 15 min followed by ultrafiltration using Amicon® Ultra centrifugal filters. .. The high molecular weight fraction was diluted in PBS/EDTA, ultrafiltrated, and transferred to PBS, 5 mM Ca2+ , without EDTA by two additional ultrafiltration steps.

    Incubation:

    Article Title: C-Reactive Protein Stimulates Nicotinic Acetylcholine Receptors to Control ATP-Mediated Monocytic Inflammasome Activation
    Article Snippet: To detect low cytokine levels in cell culture supernatants, for IL-1β the Human IL-1 beta/IL-1F2 DuoSet ELISA (R & D Systems) was used, whereas IL6 and TNF-α were measured by the Human Quantikine® Immunoassays (R & D Systems, Minneapolis, MN, USA). .. Endogenous CRP was dissolved at a concentration of 5 µg/ml in PBS devoid of Ca2+ and Mg2+ (Gibco) containing 1.1 mM ethylenediaminetetraacetic acid (EDTA; Sigma-Aldrich), incubated at 37°C for 15 min followed by ultrafiltration using Amicon® Ultra centrifugal filters. .. The high molecular weight fraction was diluted in PBS/EDTA, ultrafiltrated, and transferred to PBS, 5 mM Ca2+ , without EDTA by two additional ultrafiltration steps.

    High Performance Liquid Chromatography:

    Article Title: An Interaction of Rhamnolipids with Cu2+ Ions
    Article Snippet: .. The rhamnolipid R-95 (the 1:1 mixture of mono- and dirhamnolipids with the HPLC purity; the mean molecular weight of 577 g mol−1 , p K a = 5.6), methylglycinediacetic acid (MGDA; analytical grade, p K a = 14.6 [ ]), ethylenediaminetetraacetic acid (EDTA; analytical grade), potassium chloride (KCl), copper (II) sulfate (CuSO4 ; analytical grade), potassium hydroxide (KOH; analytical grade), and sulfuric acid (H2 SO4 ) were purchased from Sigma Aldrich (Poznan, Poland) company. .. Distilled filtrated (0.2 µm Whatman filters; GE Healthcare UK Ltd., Little Chalfont, UK) water with the electrolytic conductivity of 0.001 ms cm–1 at 20 ± 0.1 °C) was used for the solutions preparation.

    Molecular Weight:

    Article Title: An Interaction of Rhamnolipids with Cu2+ Ions
    Article Snippet: .. The rhamnolipid R-95 (the 1:1 mixture of mono- and dirhamnolipids with the HPLC purity; the mean molecular weight of 577 g mol−1 , p K a = 5.6), methylglycinediacetic acid (MGDA; analytical grade, p K a = 14.6 [ ]), ethylenediaminetetraacetic acid (EDTA; analytical grade), potassium chloride (KCl), copper (II) sulfate (CuSO4 ; analytical grade), potassium hydroxide (KOH; analytical grade), and sulfuric acid (H2 SO4 ) were purchased from Sigma Aldrich (Poznan, Poland) company. .. Distilled filtrated (0.2 µm Whatman filters; GE Healthcare UK Ltd., Little Chalfont, UK) water with the electrolytic conductivity of 0.001 ms cm–1 at 20 ± 0.1 °C) was used for the solutions preparation.

    Flow Cytometry:

    Article Title: Monocytes mediate Salmonella Typhimurium-induced tumour growth inhibition
    Article Snippet: A portion of the cells were counted using Trypan Blue exclusion dye (Sigma, T8154). .. Cells were resuspended in flow cytometry buffer (FB: 2% FCS, 3 nM EDTA (Sigma, E9884) in PBS). .. Cells were first stained in Fixable Viability Dye eFluor® 780 (eBioscience) in PBS for 15-20 min on ice, in the dark.

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  • 97
    Millipore immunoprecipitation buffer
    Kidney-specific with no lysine kinase 1 (KS-WNK1) promotes WNK4 S335 phosphorylation through heterodimer formation. A : representative Western blot of the total lysate and KS-WNK1 lacking exon 11 (KS-WNK1-Δ11) <t>immunoprecipitation</t> (IP) from oocytes injected with WNK4 cRNA alone or together with KS-WNK1-Δ11 cRNA or KS-WNK1-Δ11 HQ/AA, as stated. IP was performed using the c-Myc tag antibody, which exclusively recognizes KS-WNK1-Δ11. Left : blot from total lysate shows total WNK4 and pWNK4 expression, as stated. Lanes were loaded as follows: lane 1 : H 2 0-injected oocytes; lane 2 : WNK4-injected oocytes; lane 3 : WNK4-injected oocytes exposed to low-chloride hypotonic stress (LCHS) conditions; lane 4 : WNK4 + KS-WNK1-Δ11-injected oocytes; lane 5 : WNK4 + KS-WNK1-Δ11 HQ/AA-injected oocytes. Right : blot from the IP assay. IP was performed against the c-Myc tag, which recognizes KS-WNK1-Δ11 and revealed against WNK4 or pWNK4, as stated. Lanes were loaded as follows: lane 1 : IP from WNK4 + KS-WNK1-Δ11-injected oocytes; lane 2 : flowthrough of WNK4 + KS-WNK1-Δ11-injected oocytes; lane 3 : IP from WNK4 + KS-WNK1-Δ11 HQ/AA-injected oocytes (the absence of WNK4 in the IP confirms the specificity of the c-Myc IP); lane 4 : flowthrough from WNK4 + KS-WNK1-Δ11 HQ/AA-injected oocytes. Identical results were observed in 3 independent experiments. B : representative Western blot of the total lysate and KS-WNK1-Δ11 IP from oocytes injected with WNK4 L322F cRNA or WNK4 S335A alone or together with KS-WNK1-Δ11 cRNA, as stated. Left : blot from total lysate shows total WNK4 and pWNK4, as stated. Right : blot from the IP assay. Lanes 1 and 3 were loaded with proteins from the IP fraction, whereas lanes 2 and 4 were loaded with proteins from the flowthrough, as stated. The blot shows pWNK4 and WNK4 expression, as stated. Identical results were observed in 3 different experiments. C : representative Western blot of the total lysate and KS-WNK1-Δ11 IP from oocytes injected with WNK4 S335A cRNA together with KS-WNK1-Δ11 or KS-WNK1-Δ11-Δ4a cRNA, as stated. Blot from the IP assay is shown. Lanes 1 , 3 , and 5 were loaded with proteins from the flowthrough, whereas lanes 2 , 4 , and 6 were loaded with proteins from the IP fraction, as stated. The blot shows total WNK4 and pWNK4 expression, as stated. Identical results were obtained from 3 independent experiments.
    Immunoprecipitation Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore denhardt s solution
    Field sample analysis with the CNV-SNP array . (a) The manufacturer-recommended starting amount is 1,000 ng of DNA to produce at least 10 μg of labeled product. However, 250 ng of parasite DNA consistently produced sufficient labeled product when using 65% AT nonamers. Error bars indicate one standard deviation. (b) Hybridizations with field samples - straight from patient blood, or whole genome amplified - produced microarray data on par with standard lab clones, even when significant human DNA contamination was present. Microarray accuracy was determined through Illumina sequencing of lab-adapted parasites. Patient blood samples were hybridized with the addition of 1× <t>Denhardt's</t> solution while WGA samples were not.
    Denhardt S Solution, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore gst lysis buffer
    (A) Endogenous MYCN and <t>p53</t> co-IP. Nuclear extracts from the neuroblastoma cell line IMR-32 treated with Nutlin-3a were co-immunoprecipitated using anti-p53 (Ab-7) antibody or IgG (negative control). Western blots of immunoprecipitated proteins were performed using anti-p53 (DO-1), anti- MYCN (B8.4.B), or anti-Max (C-17) antibodies. (B) Endogenous MYC and p53 co-IP. HeLa cells treated with Nutlin-3a were co-immunoprecipitated using anti-p53 antibody or negative control IgG. Immunoprecipitated proteins were analyzed by Western blotting, using with anti-p53 (DO-1), anti- MYC (N262), and anti-MAX (C-17) antibodies. (C) in vitro <t>GST-C-MYC</t> pull-down. Crude nuclear protein extract from transient p53 over-expressing HEK-293T cells was incubated overnight with full-length GST-MYC or GST control proteins immobilized on glutathione-agarose beads. Pull-down samples were immunoblotted with the anti-p53 antibody. Membrane Ponceau S staining is shown as a loading control. (D) MYCN and p53 in vitro pull-down. Purified recombinant MYCN-6×His, GST-p53 (full length), and GST-control proteins were loaded as input samples. Recombinant MYCN- 6×His protein was incubated with GST-p53 or GST-control proteins immobilized on glutathione-agarose beads. GST proteins were pulled down and associated MYCN was detected by Western Blotting. Stain-Free total protein staining was used as a loading control. (E) Recombinant p53 and MYCN co-immunoprecipitation. The p53-null, non-small cell lung carcinoma cell line H-1299 was transiently transfected with plasmids overexpressing p53-GFP and MYCN-3×Flag. Crude nuclear protein extract collected from cells cultured under different transfection conditions were immunoprecipitated (IP) with either anti-p53 (Ab-7) or anti-FLAG (M2) antibody, and Western blots were performed using either anti-FLAG (M2) or anti-p53 (DO-1) antibody. (F) MYCN interacts with tetrameric form of p53. Crude nuclear protein extracts from MYCN-amplified SK-N-BE2-(C) cells were incubated with GST alone and a series GST-p53 purified proteins: p53-WT (dimeric-tetrameric), p53-L344A (dimeric only) and p53-L344P (monomeric only). Input and pull-down samples were immunoblotted using anti-MYCN antibody and Ponceau staining was used as loading control.
    Gst Lysis Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Millipore co immunoprecipitation buffer
    Patch 1 and Patch 2 residues of the AP2-μ2 subunit that bind to PIP 2 lipid are not critical for the binding of the AP2 complex to CXCR2 A) Non-silencing (NS), HEK-293-μ2-KD cells and HEK-293-μ2-KD cells with transiently over-expressed AP2-μ2 mutants (P1, P1+P2, P1P2T and WT) were serum starved, stimulated with 100 ng /ml CXCL8 and cross-linked with DSP. The cells were lysed and a CXCR2 <t>co-immunoprecipitation</t> assay was performed. The CXCR2 associated proteins were eluted with 50 mM DTT and separated by 10%SDS-PAGE. The CXCR2 associated AP2 complex was probed with an anti-β2 antibody. Experiments were repeated 3 times and the mean band densities as normalized to co-immunoprecipitation with CXCR2 ±S. D. are shown. B) HA-AP2-μ2 associates with endogenous α2 subunit of AP-2. HEK-293-μ2-KD cells with transiently over-expressed HA-AP2-μ2 mutants (P1, P1+P2, T, P1P2T and WT), were lysed subjected to Western blot analysis for α2 and HA-μ2 subunits (upper panel). Each HA-tagged μ2 subunit was immunoprecipitated with anti-HA-agarose and blotted for endogenous α2 and for HA-μ2 (upper panel). A representative blot from 3 individual experiments is shown. C) Functional AP-2 complexes successfully incorporate transiently expressed HA-AP2-μ2 as shown by a reciprocal co-immunoprecipitation. A reciprocal co-immunoprecipitation of the endogenous AP2-β2 from 1.5 mg of total lysate shows that the functional AP-2 complexes contain both endogenous AP2-α2 and overexpressed HA-AP2-μ2. One thirtieth (1/30) of the total lysate input for co-immunoprecipitation was used for Western analysis of the total lysates. The top panel shows co-immunoprecipitation and the bottom panel shows the lysates. A representative blot from 2 individual experiments is shown.
    Co Immunoprecipitation Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Kidney-specific with no lysine kinase 1 (KS-WNK1) promotes WNK4 S335 phosphorylation through heterodimer formation. A : representative Western blot of the total lysate and KS-WNK1 lacking exon 11 (KS-WNK1-Δ11) immunoprecipitation (IP) from oocytes injected with WNK4 cRNA alone or together with KS-WNK1-Δ11 cRNA or KS-WNK1-Δ11 HQ/AA, as stated. IP was performed using the c-Myc tag antibody, which exclusively recognizes KS-WNK1-Δ11. Left : blot from total lysate shows total WNK4 and pWNK4 expression, as stated. Lanes were loaded as follows: lane 1 : H 2 0-injected oocytes; lane 2 : WNK4-injected oocytes; lane 3 : WNK4-injected oocytes exposed to low-chloride hypotonic stress (LCHS) conditions; lane 4 : WNK4 + KS-WNK1-Δ11-injected oocytes; lane 5 : WNK4 + KS-WNK1-Δ11 HQ/AA-injected oocytes. Right : blot from the IP assay. IP was performed against the c-Myc tag, which recognizes KS-WNK1-Δ11 and revealed against WNK4 or pWNK4, as stated. Lanes were loaded as follows: lane 1 : IP from WNK4 + KS-WNK1-Δ11-injected oocytes; lane 2 : flowthrough of WNK4 + KS-WNK1-Δ11-injected oocytes; lane 3 : IP from WNK4 + KS-WNK1-Δ11 HQ/AA-injected oocytes (the absence of WNK4 in the IP confirms the specificity of the c-Myc IP); lane 4 : flowthrough from WNK4 + KS-WNK1-Δ11 HQ/AA-injected oocytes. Identical results were observed in 3 independent experiments. B : representative Western blot of the total lysate and KS-WNK1-Δ11 IP from oocytes injected with WNK4 L322F cRNA or WNK4 S335A alone or together with KS-WNK1-Δ11 cRNA, as stated. Left : blot from total lysate shows total WNK4 and pWNK4, as stated. Right : blot from the IP assay. Lanes 1 and 3 were loaded with proteins from the IP fraction, whereas lanes 2 and 4 were loaded with proteins from the flowthrough, as stated. The blot shows pWNK4 and WNK4 expression, as stated. Identical results were observed in 3 different experiments. C : representative Western blot of the total lysate and KS-WNK1-Δ11 IP from oocytes injected with WNK4 S335A cRNA together with KS-WNK1-Δ11 or KS-WNK1-Δ11-Δ4a cRNA, as stated. Blot from the IP assay is shown. Lanes 1 , 3 , and 5 were loaded with proteins from the flowthrough, whereas lanes 2 , 4 , and 6 were loaded with proteins from the IP fraction, as stated. The blot shows total WNK4 and pWNK4 expression, as stated. Identical results were obtained from 3 independent experiments.

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Kidney-specific WNK1 isoform (KS-WNK1) is a potent activator of WNK4 and NCC

    doi: 10.1152/ajprenal.00145.2018

    Figure Lengend Snippet: Kidney-specific with no lysine kinase 1 (KS-WNK1) promotes WNK4 S335 phosphorylation through heterodimer formation. A : representative Western blot of the total lysate and KS-WNK1 lacking exon 11 (KS-WNK1-Δ11) immunoprecipitation (IP) from oocytes injected with WNK4 cRNA alone or together with KS-WNK1-Δ11 cRNA or KS-WNK1-Δ11 HQ/AA, as stated. IP was performed using the c-Myc tag antibody, which exclusively recognizes KS-WNK1-Δ11. Left : blot from total lysate shows total WNK4 and pWNK4 expression, as stated. Lanes were loaded as follows: lane 1 : H 2 0-injected oocytes; lane 2 : WNK4-injected oocytes; lane 3 : WNK4-injected oocytes exposed to low-chloride hypotonic stress (LCHS) conditions; lane 4 : WNK4 + KS-WNK1-Δ11-injected oocytes; lane 5 : WNK4 + KS-WNK1-Δ11 HQ/AA-injected oocytes. Right : blot from the IP assay. IP was performed against the c-Myc tag, which recognizes KS-WNK1-Δ11 and revealed against WNK4 or pWNK4, as stated. Lanes were loaded as follows: lane 1 : IP from WNK4 + KS-WNK1-Δ11-injected oocytes; lane 2 : flowthrough of WNK4 + KS-WNK1-Δ11-injected oocytes; lane 3 : IP from WNK4 + KS-WNK1-Δ11 HQ/AA-injected oocytes (the absence of WNK4 in the IP confirms the specificity of the c-Myc IP); lane 4 : flowthrough from WNK4 + KS-WNK1-Δ11 HQ/AA-injected oocytes. Identical results were observed in 3 independent experiments. B : representative Western blot of the total lysate and KS-WNK1-Δ11 IP from oocytes injected with WNK4 L322F cRNA or WNK4 S335A alone or together with KS-WNK1-Δ11 cRNA, as stated. Left : blot from total lysate shows total WNK4 and pWNK4, as stated. Right : blot from the IP assay. Lanes 1 and 3 were loaded with proteins from the IP fraction, whereas lanes 2 and 4 were loaded with proteins from the flowthrough, as stated. The blot shows pWNK4 and WNK4 expression, as stated. Identical results were observed in 3 different experiments. C : representative Western blot of the total lysate and KS-WNK1-Δ11 IP from oocytes injected with WNK4 S335A cRNA together with KS-WNK1-Δ11 or KS-WNK1-Δ11-Δ4a cRNA, as stated. Blot from the IP assay is shown. Lanes 1 , 3 , and 5 were loaded with proteins from the flowthrough, whereas lanes 2 , 4 , and 6 were loaded with proteins from the IP fraction, as stated. The blot shows total WNK4 and pWNK4 expression, as stated. Identical results were obtained from 3 independent experiments.

    Article Snippet: Four milligrams of protein from Xenopus laevis oocyte total protein lysate was incubated with 20 μl anti-c-Myc agarose suspension in a total volume of 700 μl attained using 1× immunoprecipitation buffer (catalog number I5779, Sigma-Aldrich).

    Techniques: Western Blot, Immunoprecipitation, Injection, Expressing

    Field sample analysis with the CNV-SNP array . (a) The manufacturer-recommended starting amount is 1,000 ng of DNA to produce at least 10 μg of labeled product. However, 250 ng of parasite DNA consistently produced sufficient labeled product when using 65% AT nonamers. Error bars indicate one standard deviation. (b) Hybridizations with field samples - straight from patient blood, or whole genome amplified - produced microarray data on par with standard lab clones, even when significant human DNA contamination was present. Microarray accuracy was determined through Illumina sequencing of lab-adapted parasites. Patient blood samples were hybridized with the addition of 1× Denhardt's solution while WGA samples were not.

    Journal: Genome Biology

    Article Title: An optimized microarray platform for assaying genomic variation in Plasmodium falciparum field populations

    doi: 10.1186/gb-2011-12-4-r35

    Figure Lengend Snippet: Field sample analysis with the CNV-SNP array . (a) The manufacturer-recommended starting amount is 1,000 ng of DNA to produce at least 10 μg of labeled product. However, 250 ng of parasite DNA consistently produced sufficient labeled product when using 65% AT nonamers. Error bars indicate one standard deviation. (b) Hybridizations with field samples - straight from patient blood, or whole genome amplified - produced microarray data on par with standard lab clones, even when significant human DNA contamination was present. Microarray accuracy was determined through Illumina sequencing of lab-adapted parasites. Patient blood samples were hybridized with the addition of 1× Denhardt's solution while WGA samples were not.

    Article Snippet: Labeled product was resuspended in water, and 10 μg of test and reference samples combined (6 μg for 5K SNP chip samples), dried down, and resuspended in hybridization buffer (Roche NimbleGen, Inc., Madison, WI, USA); hybridizations for patient blood samples included 1× Denhardt's solution (Sigma Aldrich) in the hybridization buffer.

    Techniques: Labeling, Produced, Standard Deviation, Amplification, Microarray, Clone Assay, Sequencing, Whole Genome Amplification

    (A) Endogenous MYCN and p53 co-IP. Nuclear extracts from the neuroblastoma cell line IMR-32 treated with Nutlin-3a were co-immunoprecipitated using anti-p53 (Ab-7) antibody or IgG (negative control). Western blots of immunoprecipitated proteins were performed using anti-p53 (DO-1), anti- MYCN (B8.4.B), or anti-Max (C-17) antibodies. (B) Endogenous MYC and p53 co-IP. HeLa cells treated with Nutlin-3a were co-immunoprecipitated using anti-p53 antibody or negative control IgG. Immunoprecipitated proteins were analyzed by Western blotting, using with anti-p53 (DO-1), anti- MYC (N262), and anti-MAX (C-17) antibodies. (C) in vitro GST-C-MYC pull-down. Crude nuclear protein extract from transient p53 over-expressing HEK-293T cells was incubated overnight with full-length GST-MYC or GST control proteins immobilized on glutathione-agarose beads. Pull-down samples were immunoblotted with the anti-p53 antibody. Membrane Ponceau S staining is shown as a loading control. (D) MYCN and p53 in vitro pull-down. Purified recombinant MYCN-6×His, GST-p53 (full length), and GST-control proteins were loaded as input samples. Recombinant MYCN- 6×His protein was incubated with GST-p53 or GST-control proteins immobilized on glutathione-agarose beads. GST proteins were pulled down and associated MYCN was detected by Western Blotting. Stain-Free total protein staining was used as a loading control. (E) Recombinant p53 and MYCN co-immunoprecipitation. The p53-null, non-small cell lung carcinoma cell line H-1299 was transiently transfected with plasmids overexpressing p53-GFP and MYCN-3×Flag. Crude nuclear protein extract collected from cells cultured under different transfection conditions were immunoprecipitated (IP) with either anti-p53 (Ab-7) or anti-FLAG (M2) antibody, and Western blots were performed using either anti-FLAG (M2) or anti-p53 (DO-1) antibody. (F) MYCN interacts with tetrameric form of p53. Crude nuclear protein extracts from MYCN-amplified SK-N-BE2-(C) cells were incubated with GST alone and a series GST-p53 purified proteins: p53-WT (dimeric-tetrameric), p53-L344A (dimeric only) and p53-L344P (monomeric only). Input and pull-down samples were immunoblotted using anti-MYCN antibody and Ponceau staining was used as loading control.

    Journal: Oncotarget

    Article Title: MYCN acts as a direct co-regulator of p53 in MYCN amplified neuroblastoma

    doi: 10.18632/oncotarget.24859

    Figure Lengend Snippet: (A) Endogenous MYCN and p53 co-IP. Nuclear extracts from the neuroblastoma cell line IMR-32 treated with Nutlin-3a were co-immunoprecipitated using anti-p53 (Ab-7) antibody or IgG (negative control). Western blots of immunoprecipitated proteins were performed using anti-p53 (DO-1), anti- MYCN (B8.4.B), or anti-Max (C-17) antibodies. (B) Endogenous MYC and p53 co-IP. HeLa cells treated with Nutlin-3a were co-immunoprecipitated using anti-p53 antibody or negative control IgG. Immunoprecipitated proteins were analyzed by Western blotting, using with anti-p53 (DO-1), anti- MYC (N262), and anti-MAX (C-17) antibodies. (C) in vitro GST-C-MYC pull-down. Crude nuclear protein extract from transient p53 over-expressing HEK-293T cells was incubated overnight with full-length GST-MYC or GST control proteins immobilized on glutathione-agarose beads. Pull-down samples were immunoblotted with the anti-p53 antibody. Membrane Ponceau S staining is shown as a loading control. (D) MYCN and p53 in vitro pull-down. Purified recombinant MYCN-6×His, GST-p53 (full length), and GST-control proteins were loaded as input samples. Recombinant MYCN- 6×His protein was incubated with GST-p53 or GST-control proteins immobilized on glutathione-agarose beads. GST proteins were pulled down and associated MYCN was detected by Western Blotting. Stain-Free total protein staining was used as a loading control. (E) Recombinant p53 and MYCN co-immunoprecipitation. The p53-null, non-small cell lung carcinoma cell line H-1299 was transiently transfected with plasmids overexpressing p53-GFP and MYCN-3×Flag. Crude nuclear protein extract collected from cells cultured under different transfection conditions were immunoprecipitated (IP) with either anti-p53 (Ab-7) or anti-FLAG (M2) antibody, and Western blots were performed using either anti-FLAG (M2) or anti-p53 (DO-1) antibody. (F) MYCN interacts with tetrameric form of p53. Crude nuclear protein extracts from MYCN-amplified SK-N-BE2-(C) cells were incubated with GST alone and a series GST-p53 purified proteins: p53-WT (dimeric-tetrameric), p53-L344A (dimeric only) and p53-L344P (monomeric only). Input and pull-down samples were immunoblotted using anti-MYCN antibody and Ponceau staining was used as loading control.

    Article Snippet: GST-p53 cells were lysed in GST lysis buffer (1% Triton, 1 μg/μl lysozyme, 0.5 mM EDTA, and 1 mM PMSF in phosphate buffered saline), purified and immobilized on glutathione-agarose beads (Sigma Aldrich).

    Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Negative Control, Western Blot, In Vitro, Expressing, Incubation, Staining, Purification, Recombinant, Transfection, Cell Culture, Amplification

    (A) Graphical representations of p53 and MYCN proteins. p53 (upper panel) and MYCN (lower panel) protein domains and truncation constructs. p53 protein domains: Trans Activation Domain (TAD), SRC Homology 3 domain (SH3), DNA binding domain, Nuclear Localization Signal (NLS), Tetramerization domain (TET), Regulatory domain (REG). MYCN protein domains: MYC boxes (MB), the basic region helix loop helix (BR-HLH), and the leucine zipper. The GST protein fragments are indicated with bars, and numbers refer to amino-acid positions. p53 and MYCN protein fragments were cloned in frame with the N-terminal GST in a pGEX-2T vector. GST-p53 and GST-MYCN fragments were cloned, expressed in BL-21 E.Coli strain and purified using gluthatione-agarose beads. (B) MYCN interacts with the C-terminus of p53. Crude nuclear protein extracts from MYCN-amplified SK-N-BE-(2)-c cells were incubated with the different p53 truncations or GST alone (negative control) immobilized onto glutathione-agarose beads. Input and pull-down samples were immunoblotted using anti-MYCN and anti-MAX antibodies. Stain-Free total protein staining was used as the loading control. (C) GST pull-down assay of MYCN truncations. Crude nuclear protein extract from transiently transfected p53-overexpressing HEK-293T cells was incubated with different MYCN-GST fragments immobilized on glutathione-agarose beads. GST alone was used as a negative control. Input and pull-down samples were immunoblotted using anti-p53 (DO-1) antibody. Ponceau staining was used as a loading control.

    Journal: Oncotarget

    Article Title: MYCN acts as a direct co-regulator of p53 in MYCN amplified neuroblastoma

    doi: 10.18632/oncotarget.24859

    Figure Lengend Snippet: (A) Graphical representations of p53 and MYCN proteins. p53 (upper panel) and MYCN (lower panel) protein domains and truncation constructs. p53 protein domains: Trans Activation Domain (TAD), SRC Homology 3 domain (SH3), DNA binding domain, Nuclear Localization Signal (NLS), Tetramerization domain (TET), Regulatory domain (REG). MYCN protein domains: MYC boxes (MB), the basic region helix loop helix (BR-HLH), and the leucine zipper. The GST protein fragments are indicated with bars, and numbers refer to amino-acid positions. p53 and MYCN protein fragments were cloned in frame with the N-terminal GST in a pGEX-2T vector. GST-p53 and GST-MYCN fragments were cloned, expressed in BL-21 E.Coli strain and purified using gluthatione-agarose beads. (B) MYCN interacts with the C-terminus of p53. Crude nuclear protein extracts from MYCN-amplified SK-N-BE-(2)-c cells were incubated with the different p53 truncations or GST alone (negative control) immobilized onto glutathione-agarose beads. Input and pull-down samples were immunoblotted using anti-MYCN and anti-MAX antibodies. Stain-Free total protein staining was used as the loading control. (C) GST pull-down assay of MYCN truncations. Crude nuclear protein extract from transiently transfected p53-overexpressing HEK-293T cells was incubated with different MYCN-GST fragments immobilized on glutathione-agarose beads. GST alone was used as a negative control. Input and pull-down samples were immunoblotted using anti-p53 (DO-1) antibody. Ponceau staining was used as a loading control.

    Article Snippet: GST-p53 cells were lysed in GST lysis buffer (1% Triton, 1 μg/μl lysozyme, 0.5 mM EDTA, and 1 mM PMSF in phosphate buffered saline), purified and immobilized on glutathione-agarose beads (Sigma Aldrich).

    Techniques: Construct, Activation Assay, Binding Assay, Clone Assay, Plasmid Preparation, Purification, Amplification, Incubation, Negative Control, Staining, Pull Down Assay, Transfection

    Patch 1 and Patch 2 residues of the AP2-μ2 subunit that bind to PIP 2 lipid are not critical for the binding of the AP2 complex to CXCR2 A) Non-silencing (NS), HEK-293-μ2-KD cells and HEK-293-μ2-KD cells with transiently over-expressed AP2-μ2 mutants (P1, P1+P2, P1P2T and WT) were serum starved, stimulated with 100 ng /ml CXCL8 and cross-linked with DSP. The cells were lysed and a CXCR2 co-immunoprecipitation assay was performed. The CXCR2 associated proteins were eluted with 50 mM DTT and separated by 10%SDS-PAGE. The CXCR2 associated AP2 complex was probed with an anti-β2 antibody. Experiments were repeated 3 times and the mean band densities as normalized to co-immunoprecipitation with CXCR2 ±S. D. are shown. B) HA-AP2-μ2 associates with endogenous α2 subunit of AP-2. HEK-293-μ2-KD cells with transiently over-expressed HA-AP2-μ2 mutants (P1, P1+P2, T, P1P2T and WT), were lysed subjected to Western blot analysis for α2 and HA-μ2 subunits (upper panel). Each HA-tagged μ2 subunit was immunoprecipitated with anti-HA-agarose and blotted for endogenous α2 and for HA-μ2 (upper panel). A representative blot from 3 individual experiments is shown. C) Functional AP-2 complexes successfully incorporate transiently expressed HA-AP2-μ2 as shown by a reciprocal co-immunoprecipitation. A reciprocal co-immunoprecipitation of the endogenous AP2-β2 from 1.5 mg of total lysate shows that the functional AP-2 complexes contain both endogenous AP2-α2 and overexpressed HA-AP2-μ2. One thirtieth (1/30) of the total lysate input for co-immunoprecipitation was used for Western analysis of the total lysates. The top panel shows co-immunoprecipitation and the bottom panel shows the lysates. A representative blot from 2 individual experiments is shown.

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Adaptor Protein2 (AP2) orchestrates CXCR2-mediated cell migration

    doi: 10.1111/tra.12154

    Figure Lengend Snippet: Patch 1 and Patch 2 residues of the AP2-μ2 subunit that bind to PIP 2 lipid are not critical for the binding of the AP2 complex to CXCR2 A) Non-silencing (NS), HEK-293-μ2-KD cells and HEK-293-μ2-KD cells with transiently over-expressed AP2-μ2 mutants (P1, P1+P2, P1P2T and WT) were serum starved, stimulated with 100 ng /ml CXCL8 and cross-linked with DSP. The cells were lysed and a CXCR2 co-immunoprecipitation assay was performed. The CXCR2 associated proteins were eluted with 50 mM DTT and separated by 10%SDS-PAGE. The CXCR2 associated AP2 complex was probed with an anti-β2 antibody. Experiments were repeated 3 times and the mean band densities as normalized to co-immunoprecipitation with CXCR2 ±S. D. are shown. B) HA-AP2-μ2 associates with endogenous α2 subunit of AP-2. HEK-293-μ2-KD cells with transiently over-expressed HA-AP2-μ2 mutants (P1, P1+P2, T, P1P2T and WT), were lysed subjected to Western blot analysis for α2 and HA-μ2 subunits (upper panel). Each HA-tagged μ2 subunit was immunoprecipitated with anti-HA-agarose and blotted for endogenous α2 and for HA-μ2 (upper panel). A representative blot from 3 individual experiments is shown. C) Functional AP-2 complexes successfully incorporate transiently expressed HA-AP2-μ2 as shown by a reciprocal co-immunoprecipitation. A reciprocal co-immunoprecipitation of the endogenous AP2-β2 from 1.5 mg of total lysate shows that the functional AP-2 complexes contain both endogenous AP2-α2 and overexpressed HA-AP2-μ2. One thirtieth (1/30) of the total lysate input for co-immunoprecipitation was used for Western analysis of the total lysates. The top panel shows co-immunoprecipitation and the bottom panel shows the lysates. A representative blot from 2 individual experiments is shown.

    Article Snippet: The cells were lysed in ice-cold co-immunoprecipitation buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 5 mM EDTA) containing proteinase inhibitor cocktail I and phosphatase inhibitor cocktails 3 and 2 (Sigma/Aldrich, St. Louis, MO).

    Techniques: Binding Assay, Co-Immunoprecipitation Assay, SDS Page, Immunoprecipitation, Western Blot, Functional Assay

    AP2 is essential for CXCR2-mediated chemotaxis, but β-arrestin1 is dispensable A) Top panel: LLKIL motif in CTDs of human CXC chemokine receptors is conserved. The CTDs of CXCR2 (45 residues), CXCR1 (44 residues), CXCR3 (49 residues) and CXCR4 (47 residues) were aligned with CLUSTALW (1.83) multiple sequence alignment program. The LLKIL functional motif of CXCR2 and similar putative motifs in other CXC receptor CTDs are in bold. Also, the serine residues known to be phosphorylated in CXCR2 CTD in response to CXCL8 stimulation are in bold. Bottom panel: The mutations in CXCR2 important for binding of AP2 and β-arrestin are illustrated. B) Decreased association of CXCR2 mutants with AP2 and/or β-arrestin1 after stimulation with CXCL8. dHL-60 cells stably expressing CXCR2-WT, 4A or CXCR2-4A/IL mutants were stimulated with or without CXCL8. CXCR2 was immunoprecipitated with anti-CXCR2 antibody, and blotted for AP2-β2 subunit or β-arrestin1. The blot was stripped and re-blotted for CXCR2. The relative values of fold increase in response to CXCL8 stimulation for each cell line calculated from 3 independent experiments is shown under the western blots (fold ± S.E.M.). One tenth of the total lysate input for co-immunoprecipitation was used for Western analysis of the total lysates. C) CXCL8-mediated internalization of CXCR2 is abolished in 4A/IL mutant of CXCR2, but only partially attenuated in 4A-CXCR2 mutant. The internalization of CXCR2 was performed by following the internalization of 125 I-CXCL8 in dHL60-CXCR2 cells stably expressing CXCR2-WT, 4A or 4A/IL mutant. Error bars are S.E.M and the experiments were repeated 3 times with duplicates for each treatment. ANOVA: 2 min – 4A vs. 4A/IL, p

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Adaptor Protein2 (AP2) orchestrates CXCR2-mediated cell migration

    doi: 10.1111/tra.12154

    Figure Lengend Snippet: AP2 is essential for CXCR2-mediated chemotaxis, but β-arrestin1 is dispensable A) Top panel: LLKIL motif in CTDs of human CXC chemokine receptors is conserved. The CTDs of CXCR2 (45 residues), CXCR1 (44 residues), CXCR3 (49 residues) and CXCR4 (47 residues) were aligned with CLUSTALW (1.83) multiple sequence alignment program. The LLKIL functional motif of CXCR2 and similar putative motifs in other CXC receptor CTDs are in bold. Also, the serine residues known to be phosphorylated in CXCR2 CTD in response to CXCL8 stimulation are in bold. Bottom panel: The mutations in CXCR2 important for binding of AP2 and β-arrestin are illustrated. B) Decreased association of CXCR2 mutants with AP2 and/or β-arrestin1 after stimulation with CXCL8. dHL-60 cells stably expressing CXCR2-WT, 4A or CXCR2-4A/IL mutants were stimulated with or without CXCL8. CXCR2 was immunoprecipitated with anti-CXCR2 antibody, and blotted for AP2-β2 subunit or β-arrestin1. The blot was stripped and re-blotted for CXCR2. The relative values of fold increase in response to CXCL8 stimulation for each cell line calculated from 3 independent experiments is shown under the western blots (fold ± S.E.M.). One tenth of the total lysate input for co-immunoprecipitation was used for Western analysis of the total lysates. C) CXCL8-mediated internalization of CXCR2 is abolished in 4A/IL mutant of CXCR2, but only partially attenuated in 4A-CXCR2 mutant. The internalization of CXCR2 was performed by following the internalization of 125 I-CXCL8 in dHL60-CXCR2 cells stably expressing CXCR2-WT, 4A or 4A/IL mutant. Error bars are S.E.M and the experiments were repeated 3 times with duplicates for each treatment. ANOVA: 2 min – 4A vs. 4A/IL, p

    Article Snippet: The cells were lysed in ice-cold co-immunoprecipitation buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 5 mM EDTA) containing proteinase inhibitor cocktail I and phosphatase inhibitor cocktails 3 and 2 (Sigma/Aldrich, St. Louis, MO).

    Techniques: Chemotaxis Assay, Sequencing, Functional Assay, Binding Assay, Stable Transfection, Expressing, Immunoprecipitation, Western Blot, Mutagenesis