Anti actin

Western blotting was performed as previously described (33 (link)). Briefly, total cell lysates (30 μg) were subjected to SDS-PAGE and western blotting analysis using the following primary antibodies: anti-Blimp-1 (1:1,000 dilution, Abcam), anti-STAT3 (1:500 dilution, Cell Signaling), anti-phospho-STAT3 at Tyr705 (1:500 dilution, Cell Signaling), anti-phospho-STAT3 at Ser727 (1:500 dilution; Cell Signaling), anti-actin (1:2,000 dilution, GenScript), and anti-Flag (1:1,000 dilution, Sigma) antibodies. Secondary antibodies were as previously described (33 (link)). Representative blots from at least two independent experiments are shown.

All the antibodies used in the study were purchased commercially, including Anti-Flag tag (Sigma, catalog F7425), anti-Myc (HuaBio, catalog EM31105), anti-BAP1 (Santa Cruz Biotechnology, catalog sc-28383 for IP and WB; GST, catalog 78105 for ChIP), anti-ASXL1 (Abcam, catalog ab228009; Abcam, catalog ab221455), anti-FOXK1 (Abcam, catalog ab18196), anti-FOXK2 (Abcam, ab5298), anti-HCF-1 (GST, catalog 50708), anti-H2AK119 mono-ubiquitination (Abcam, catalog ab193203), anti-H3K27me3 (Abcam, catalog ab6002), anti-Histone H2A (Abcam, catalog ab18255), anti-Histone H3 (CST, catalog 9715), anti-TXNIP (Abcam, catalog ab188865), anti-HIF-1α (Abcam, catalog ab216842), anti-STAT3 (CST, catalog 12640), anti-p-STAT3 (CST, catalog 9145), anti-JAK2 (Abcam, catalog ab108596), anti-p-JAK2 (Abcam, catalog ab32101), anti-GAL4 (Santa Cruz Biotechnology, catalog sc-510), anti-ACTIN (Genscript, catalog A00702).

Liver tissue from mice was homogenized in RIPA buffer supplemented with Protease Inhibitor Cocktail from Sigma (St. Louis, MO) using a QIA Tissue Lyser (Qiagen, Valencia, CA). The homogenates were centrifuged at 14,000 rpm for 15 min at 4 °C and the supernatants collected. Protein concentration was measured using a BCA kit from Pierce (Rockford, IL). Supernatant (50 μg protein) was combined with 4 × concentrated SDS loading buffer and reducing agent and heated at 80 °C for 10 min. The proteins were separated on 4–20 % SDS PAGE gels from Pierce (Rockford, IL), transferred electrophoretically to nitrocellulose membranes, blocked with 5 % bovine serum albumin and probed with the following primary antibodies (Abs): anti-LDLR from R&D Biosciences (Minneapolis, MN); anti-GAPDH from Epitope Biotech Inc (Burnaby, B.C., Canada); anti-S1P from Santa Cruz Biotech Inc (Santa Cruz, CA); anti-actin from Genscript (Piscataway, NJ). Horse radish peroxidase conjugated secondary Abs were obtained from Jackson Immunoresearch (West Grove, PA) and the visualized using the ECL Super Signal West Pico or Femto reagent from Pierce (Rockford, IL). Bands were scanned using a Microtek ScanMaker 5950 (Microtek, Santa Fe Springs, CA) and quantified using Image-Pro Plus 6.0 (Media Cybernetics, Warrendale, PA).

All DNA constructs were made in the corresponding author’s laboratory and verified by DNA sequencing and/or restriction enzyme analysis. Chemicals were purchased at molecular biology or analytical grade purity from established vendors (e.g., Sigma-Aldrich, VWR). Enzymes were obtained from Promega or New England Biolabs. The following antibodies were used for western blotting or immunoprecipitation: anti-Flag M2 (Sigma-Aldrich F1804), anti-Myc 9E10 (Sigma-Aldrich M4439), anti-cyclin D1 DCS6 (Cell Signaling #2926), anti-SMAD4 B-8 (Santa Cruz sc-7966) and anti-actin (GenScript A00730) mouse monoclonal antibodies; anti-ETV1 (Abcam ab81086 or our previously described #959 antibody^{9 (link)}), anti-p21 H-164 (Santa Cruz sc-756), anti-PMEPA1 2A12 (Abnova H00056937-M01) and anti-SMAD3 (Zymed 51–1500) rabbit polyclonal antibodies; and anti-p-SMAD3-Ser423/425 C25A9 (Cell Signaling #9520) rabbit monoclonal antibodies.