Rat anti gapdh

Three to five million cells were resuspended in RIPA Lysis and Extraction Buffer (ThermoFisher, Scientific, Waltham, MA, USA) supplemented with 1X Halt™ Protease and Phosphatase Inhibitor Cocktail (ThermoFisher, Scientific, Waltham, MA, USA) and incubated on ice for 20 min. The soluble fraction was recovered after 10 min of centrifugation at 10,000× g 4 °C. Protein concentration was determined by standard Bradford assay. A total of 20 µg of protein was loaded in a Mini-PROTEAN TGX gel (Bio-Rad, Hercules, CA, USA) and separated by electrophoresis. The gel was transferred to a Midi format 0.2 µm PVDF membrane using Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA, USA). The membrane was blocked with EveryBlot Blocking Buffer (Bio-Rad, Hercules, CA, USA) and incubated for 1 h at room temperature (RT) or overnight at 4 °C with rabbit anti-CBLB (Cell Signaling Technologies, Danvers, MA, USA, clone: D3C12) diluted at 1:500 and rat anti-GAPDH (Biolegend, San Diego, CA, USA, clone: W17079A) diluted at 1:1000 in blocking buffer. After washing, the membrane was incubated for 1 h at room temperature with IRDye 800CW goat anti-rat and IRDye 680RD goat anti-rabbit (LI-COR Biosciences, Lincoln, NE, USA) at 1:15,000 in a blocking buffer. The membrane was developed using LI-COR Odyssey Fc and band intensity was quantified with Image Studio 4.0 (LI-COR Biosciences, Lincoln, NE, USA).

Cells or liver tissues were lysed using a radioimmunoprecipitation assay buffer. Protein concentration was measured with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). After brief sonication and boiling at 95 °C for 5 min, the protein samples were run on a 5–20% gradient gel, e-PAGE (EHR-R520L, ATTO, Tokyo, Japan), and transferred to a PVDF membrane (Bio-Rad Laboratories). The membranes were blocked with 5% nonfat dry milk in PBS containing 0.1 % Tween-20 (PBST) and then probed overnight at 4 °C with rabbit anti-TG2 (1:200 dilution, RB-060-P0, Thermo Fisher Scientific), mouse anti-EXT1 (1:100 dilution, sc-515144, Santa Cruz Biotechnology), mouse anti-pan-cytokeratin (1:500 dilution, MA182041, Thermo Fisher Scientific), or rat anti-GAPDH (1:1000 dilution, 607902, BioLegend) antibodies. The blots were incubated with HRP-conjugated secondary antibodies (1:2000 dilution) and detected using the Amersham ECL Plus western blotting Detection System (GE Healthcare). Band intensities were quantified using ImageJ software (National Institutes of Health).