Bio 1d advanced software

Livers were homogenized in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, and 50 mM Tris-HCl pH 7.4) containing protease and phosphatase inhibitors (Roche, Rotkreuz, Switzerland). Protein concentration was measured with the Pierce^TM BCA assay (Thermo Fisher Scientific, Rockford, IL, USA). Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes, blocked for 1 h with 5% nonfat milk or BSA, then incubated overnight at 4 °C with primary antibodies. After incubation with peroxidase-conjugated secondary antibody (Thermo Fisher Scientific, Rockford, IL, USA), signals were revealed with enhanced chemiluminescence (Amersham ECL Prime, GE Healthcare, Glattburg, Switzerland) and a Fusion CCD camera coupled to a computer equipped with Fusion Capt Fx Software (Vilber-Lourmat, Marne-la-Vallée, France). Signals were quantified with the Bio-1D Advanced software (Vilber-Lourmat). Uncropped and unprocessed scans of the blots are supplied as Supplementary Figs. in the Supplementary Information.

Frozen liver tissues were thawed and washed twice with ice-cold PBS. Livers were homogenized with 0.5 mm zirconium oxide beads (Next Advance, Troy, NY, USA) in a bullet blender (Next Advance, Troy, NY, USA) in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, and 50 mM Tris-HCl, pH 7.4) containing Halt™ protease and phosphatase inhibitor (Thermo Fisher Scientific, Rockford, IL, USA). Protein concentration was measured with the Pierce^TM BCA assay (Thermo Fisher Scientific, Rockford, IL, USA). 15 µg of total lysate was loaded into 10–12% sodium dodecyl sulfate polyacrylamide gel and proteins were resolved by electrophoresis. The proteins were transferred to nitrocellulose membranes, blocked for 1 h with 5% nonfat milk, and then incubated overnight at 4°C with primary antibodies (Supplementary table 1S). Next, the membranes were incubated with peroxidase-conjugated secondary antibody (Thermo Fisher Scientific, Rockford, IL, USA) for 1 h and signals were revealed with enhanced chemiluminescence (Amersham ECL Prime, GE Healthcare, Glattburg, Switzerland) and a Fusion CCD camera coupled to a computer equipped with Fusion Capt Fx Software (Vilber-Lourmat, Marne-la-Vallée, France). Band densitometry was measured with the Bio-1D Advanced software (Vilber-Lourmat).

Cells were lysed in sample buffer (2 % sodium dodecyl sulfate (SDS) in 125 mM Tris HCl, pH 6.8). An equivalent protein amount, extracted from 1 × 10⁷ to 8 × 10⁷ cells, was separated by electrophoresis on 12 % SDS–polyacrylamide gels and transferred to Polyvinylidene difluoride (PDVF) membranes (GE Healthcare).
Blots were then blocked for 1 h in 6 % milk before incubation with specific antibodies as follows: rabbit anti-human CD20 specific to the COOH-terminal region [22 (link)] (Thermo Scientific) and rabbit anti-actin (#8457L, Cell Signaling). Blotted proteins were detected and quantified on a bioluminescence imager and BIO-1D advanced software (Vilber-Lourmat) after blots were incubated with a horseradish peroxidase–conjugated appropriate secondary antibody (Beckman Coulter).