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5 protocols using anti hla g 4h84

1

Quantitative Gene Expression Analysis

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To quantify specific gene expression, samples were lysed in lysis buffer (Sigma-Aldrich). Samples containing equal quantities of protein were run on 10% to 12% SDS polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes, which were then blocked in 5% BSA and incubated overnight at 4°C with anti-catalase and anti-human PRL-1 (1:1,000, both from Abcam, Cambridge, UK); anti-GPX3, anti-HLAG (4h84), and anti-PGC1A (1:1,000, all from NovusBio, Littleton, CO, USA); anti-POU5F1 (1:1,000, AbFrontier, Seoul, Republic of Korea); antibodies from a Mitochondrial Marker Antibody Sampler Kit (1:1,000, Cell Signaling Technology, Danvers, MA, US); anti-HO-1 (1:500, Cell Signaling Technology); anti-ATP5B (1:500, Santa Cruz Biotechnology, Dallas, TX, USA); anti-NRF2 (1:1,000, Bioss, Woburn, MA, USA); anti-TFAM (1:1,000, Thermo Fisher Scientific, Waltham, MA, USA); and anti-GAPDH (1:3,000, AbFrontier). The membranes were then incubated with peroxidase-conjugated secondary anti-rabbit IgG and anti-mouse IgG (1:8,000, Bio-Rad). Bands were detected using enhanced chemiluminescence reagent (Bio-Rad Laboratories).
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2

Evaluation of PD-MSCs and Liver Tissue Gene Expression

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To assess the specific gene expression of PD-MSCsPRL-1 and cirrhotic liver tissues, samples were lysed in protein lysis buffer (Sigma-Aldrich). The protein lysates were loaded on sodium dodecyl sulfate polyacrylamide gels, and separated proteins were transferred to PVDF membranes. The following primary antibodies were used: anti-human PRL-1, anti-Oct4 (1:1000; all from Abcam, Cambridge, UK), anti-albumin, anti-HLA-G (4 h84) (1:1000; all from Novus Biologicals, Littleton, CO, USA), anti-ATP5B (1:200; Santa Cruz Biotechnology, Dallas, TX, USA), anti-ROCK1, anti-RhoA, anti-CDK4, anti-cyclin D1, and mitochondrial marker antibody sampler kit (1:1000; all from Cell Signaling Technology, Denvers, MA, USA). The loading control was anti-GAPDH (1:3000; AbFrontier, Seoul, Korea). The following secondary antibodies were used: anti-mouse IgG (1:5000; Bio-Rad, Hercules, CA, USA) and anti-rabbit IgG (1:10,000; Bio-Rad). The bands were detected using ECL reagent (Bio-Rad).
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3

Gene Expression Analysis of PD-MSCs and Cirrhotic Liver

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To assess specific gene expression of PD-MSCs PRL-1 and cirrhotic liver tissues, samples were lysed in protein lysis buffer (Sigma-Aldrich). The protein lysates were loaded on sodium dodecyl sulfate polyacrylamide gels, and separated proteins were transferred to PVDF membrane. The following primary antibodies were used: antihuman PRL-1, anti-Oct4 (1:1,000; all from Abcam, Cambridge, UK), anti-albumin, anti-HLA-G (4h84) (1:1,000; all from Novus Biologicals, Littleton, CO, USA), anti-ATP5B (1:200; Santa Cruz Biotechnology, Dallas, TX, USA), anti-ROCK1, anti-RhoA, anti-CDK4, anti-cyclin D1, and mitochondrial marker antibody sampler kit (1:1000; all from Cell Signaling Technology, Denvers, MA, USA). The loading control was anti-GAPDH (1:3,000; AbFrontier, Seoul, Korea). The following secondary antibodies were used: anti-mouse IgG (1:5,000; Bio-Rad, Hercules, CA, USA) and anti-rabbit IgG (1:10,000; Bio-Rad). The bands were detected using ECL reagent (Bio-Rad).
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4

Molecular Profiling of Cirrhotic Liver

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To assess the speci c gene expression of PD-MSCs PRL-1 and cirrhotic liver tissues, samples were lysed in protein lysis buffer (Sigma-Aldrich). The protein lysates were loaded on sodium dodecyl sulfate polyacrylamide gels, and separated proteins were transferred to PVDF membranes. The following primary antibodies were used: anti-human PRL-1, anti-Oct4 (1:1,000; all from Abcam, Cambridge, UK), antialbumin, anti-HLA-G (4h84) (1:1,000; all from Novus Biologicals, Littleton, CO, USA), anti-ATP5B (1:200; Santa Cruz Biotechnology, Dallas, TX, USA), anti-ROCK1, anti-RhoA, anti-CDK4, anti-cyclin D1, and mitochondrial marker antibody sampler kit (1:1000; all from Cell Signaling Technology, Denvers, MA, USA). The loading control was anti-GAPDH (1:3,000; AbFrontier, Seoul, Korea). The following secondary antibodies were used: anti-mouse IgG (1:5,000; Bio-Rad, Hercules, CA, USA) and anti-rabbit IgG (1:10,000; Bio-Rad). The bands were detected using ECL reagent (Bio-Rad).
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5

Molecular Profiling of Cirrhotic Liver

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To assess the speci c gene expression of PD-MSCs PRL-1 and cirrhotic liver tissues, samples were lysed in protein lysis buffer (Sigma-Aldrich). The protein lysates were loaded on sodium dodecyl sulfate polyacrylamide gels, and separated proteins were transferred to PVDF membranes. The following primary antibodies were used: anti-human PRL-1, anti-Oct4 (1:1,000; all from Abcam, Cambridge, UK), antialbumin, anti-HLA-G (4h84) (1:1,000; all from Novus Biologicals, Littleton, CO, USA), anti-ATP5B (1:200; Santa Cruz Biotechnology, Dallas, TX, USA), anti-ROCK1, anti-RhoA, anti-CDK4, anti-cyclin D1, and mitochondrial marker antibody sampler kit (1:1000; all from Cell Signaling Technology, Denvers, MA, USA). The loading control was anti-GAPDH (1:3,000; AbFrontier, Seoul, Korea). The following secondary antibodies were used: anti-mouse IgG (1:5,000; Bio-Rad, Hercules, CA, USA) and anti-rabbit IgG (1:10,000; Bio-Rad). The bands were detected using ECL reagent (Bio-Rad).
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