Radio immunoprecipitation assay cell lysis buffer

Western blotting was performed as described following the previous method [21 (link)]. Cells (1 × 10⁶ cells/well) were harvested and homogenized in the radioimmunoprecipitation assay cell lysis buffer (Cell Signaling Technology, Beverly, MA, USA). Protein concentrations were measured using a BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). Proteins (80 μg) were resolved using 10% sodium dodecyl sulfate-polyacrylamide gel. Subsequently, resolved proteins were transferred to nitrocellulose membranes. Membranes were blocked in 5% BSA for one hour. The blots were washed with Tris-buffered saline with Tween 20 buffer (TBST) and incubated with primary antibodies: phosphorylated-Syk (Cell Signaling Technology), phosphorylated-Gab (Cell Signaling Technology), phosphorylated c-Jun (Cell Signaling Technology), and β-actin (Cell Signaling Technology). The immunoblots were washed in TBST(Bio-Rad Laboratories, Hercules, CA, USA) and incubated with horseradish peroxidase-labeled secondary antibody (Cell Signaling Technology). The immuno blots were developed using EZ west Lumi plus (Atto, Tokyo, Japan). Finally, developed immuno blots were visualized, and a densitometry analysis of the bands was performed using LI-COR Odyssey (LI-COR Biosciences, Lincoln, NE, USA).

Total protein from the cells was extracted using radio-immunoprecipitation assay cell lysis buffer (Cell Signaling Technology) supplemented with the protease inhibitor (Roche Ltd, Basel, Switzerland). The protein concentration was determined using a bicinchoninic acid kit (Thermo Fisher Scientific). Equal amounts of protein samples (30 μg) were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Following a 1-h block in 5% non-fat milk at room temperature, the membranes were incubated with the primary antibodies against KDM5C (1:1,000, ab264168, Abcam), PFDN5 (1:1,000, ab129116, Abcam), ATG7 (1:1,000, ab52472, Abcam), P62 (1:1,000; NBP1-48320, Novus Biologicals, Littleton, CO, USA), Beclin1 (1:1,000, #3495, Cell Signaling Technology), LC3 (1:1,000; #3868, Cell Signaling Technology), Vimentin (1:1,000, NBP1-31327, Novus Biologicals), N-cadherin (1:1,000, NBP2-19457, Novus Biologicals), GAPDH (1:1,000; ab181602, Abcam) at 4 °C overnight. Subsequently, the membranes were incubated with the HRP-conjugated IgG (1:20,000, ab6721, Abcam) at room temperature for 2 h. Protein blots were developed using enhanced chemiluminescence reagent and analyzed using Image J.