Anti ha tag

For histopathological examination, paraffin-embedded liver sections were stained with hematoxylin-eosin (HE). IHC and IF staining were described in the previous report [5 (link)]. The primary antibodies used in this study were anti-HBsAg (HBV surface antigen) (Dako, San Ramon, CA), anti-SLC2A1 (Proteintech Group, Chicago, IL), and anti-HA-tag (Santa Cruz Biotechnology, Santa Cruz, CA). Glycogen was visualized with periodic acid-Schiff (PAS) staining (Muto Pure Chemicals, Tokyo, Japan) following the manufacturer’s instructions.

Proteins for Western blotting were extracted from cells using the radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris, pH 7.4, 500 mM NaCl, 1% Na-DOC, 0,1% SDS, 1% Triton X-100) with 30 min of incubation on ice and occasional vortexing. Western blotting was performed as described previously (60 (link)). The following antibodies were used: anti-hnRNP A1 (04-1469; Millipore), anti-hnRNP A2B1 (ab227465; Abcam), anti-HPV16 E7 (GTX133411; GeneTex), anti-beta tubulin (T9026; Sigma-Aldrich), anti-actin (SC-1616; Santa Cruz), anti-HA tag (SC7392; Santa Cruz), and anti-GST (A5800; Invitrogen) antibody. Secondary antibodies conjugated with horseradish peroxidase were used, and proteins were detected using the Clarity Western ECL substrate (Bio-Rad) or the Super Signal West Femto chemiluminescence substrate (Pierce).

Cell samples and fresh tissue samples were treated using the RIPA lysis solution (supplemented with protease and phosphatase inhibitors, Vazyme Biotech Co., Ltd), and the supernatant was centrifuged, loaded and boiled. About 30 ug total proteins were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene difluoride (PVDF) membranes, blocked using 5% non-fat milk at room temperature for 2 h, and incubated in the presence of primary antibodies. The membrane was washed thrice and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1: 1000 dilution) for 1 h at room temperature. For protein half-life analysis, cells were treated with CHX (50 ug/ml) at different times. The antibodies used in this assay were: Anti-HK2 (#2867, CST), Anti-PKM2 (#4053, CST), Anti-myc (#9402, CST), Anti-p53 (#2527, CST), Anti-HIF1a (#5537, CST), Anti-PFKL (A7708, Abclonal), Anti-USP39 (23865-1-AP, Proteintech), Anti-SIX1 (10709-1-AP, Proteintech), Anti-DNAAF5 (24578-1-AP, Proteintech), Anti-myc-tag (16286-1-AP, Proteintech), Anti-Flag-tag (F1804, Sigma), Anti-HA-tag (sc-7392, Santa cruz).

Cell lysates were generated by homogenization in Cell Lysis Buffer (Cell Signaling, Boston, MA) supplemented with 1x protease inhibitor cocktail (Sigma Chemical Co., St. Louis, MO, P8340) and sonicated, twice using a Fischer Scientific sonicator FB50, then cleared by centrifugation. 40μg of protein per sample were resolved by electrophoresis on 12% tris-glycine gels from Life Technologies (Carlsbad, CA) and transferred onto a polyvinylidene difluoride membrane (Bio-Rad, Richmond, CA). Specific proteins were detected with the following antibodies, anti-XOR (Santa Cruz, Santa Cruz, CA, #sc-22006), anti-GAPDH-HRP (Cell Signaling, Boston, MA, #3683), anti-Cdk5 (Santa Cruz, Santa Cruz, CA, #sc-6247), anti-p35 C-19 (Santa Cruz, Santa Cruz, CA, #sc-820), anti-p35 N-20 (Santa Cruz, Santa Cruz, CA, #sc-821), anti-HA tag (Santa Cruz, Santa Cruz, CA, #sc-57592), anti-histone (Cell Signaling, Boston, MA, #2935), anti-phospho-H1 (Cell Signaling, Boston, MA, #2577), anti-myc antibody (Cell Signaling, Boston, MA, #2272), and anti-phospho-threonine (Cell Signaling, Boston, MA, #9391) where indicated. Immune complexes were detected using chemiluminescence (ECL; Amersham Pharmacia Biotech, Piscataway, NJ, USA).

Cell lysates were prepared by adding ice‐cold RIPA buffer (250 μl; Pierce) containing 1× cOmplete™ Mini inhibitor mixture (Roche). Lysates were mixed on a rotator at 4°C for 30 min, and the protein concentration was quantified using Bio‐Rad's DC Protein Assay. SDS–PAGE (4–12% Bis‐Tris gradient gel; Bio‐Rad) of 50–80 μg total protein per lane followed by immunoblotting was performed according to standard procedures using the following primary antibodies: anti‐HA tag (Santa Cruz Biotechnology, sc‐57592, 1:1,000), anti‐GAPDH (Santa Cruz Biotechnology, sc‐25778, 1:500), anti‐phospho‐Fak1 (Santa Cruz Biotechnology, sc‐374668, 1:1,000), anti‐total Fak1 (Santa Cruz Biotechnology, sc‐1688, 1:1,000), anti‐Nf1 (Santa Cruz Biotechnology, sc‐74444, 1:1,000), anti‐Peg10 (Abcam, ab131194, 1:1,000), anti‐Psat1 (Thermo Fisher, PA5‐22124, 1:1,000), and anti‐p53 (Santa Cruz Biotechnology, sc‐393031, 1:1,000).