Western Blot Analysis of Phosphoproteins

After the indicated treatments, cells were washed with ice-cold PBS and lysed in RIPA buffer (#sc-24948, Santa Cruz Biotechnology) or cell lysis buffer (#9803, Cell Signaling) containing protease and phosphatase inhibitors. Equal amounts of protein quantified by Bio-Rad Protein Assay Dye (#5000006) were separated by SDS-PAGE, transferred to nitrocellulose membrane and probed with the designated antibodies (1:1000 dilution). GAPDH (1:5000 dilution) was used as a loading control. The membranes were scanned using a LI-COR Odyssey IR imaging system capable of detecting antigen-antibody complexes labelled using fluorescent goat anti-rabbit (IRDye 680 RD; 1:10,000 dilution) or goat anti-mouse (IRDye 800CW; 1:10,000). Phosphoproteins were probed first, followed by stripping and re-probing the same blot with the total antibody and loading control. Quantification of bands was done using Image Studio Lite software provided with the LI-COR imaging instrument or Image J. Phosphoprotein band intensities detected by Western blotting were normalized to the levels of total protein detected by respective antibodies. All others were normalized to GAPDH. Experiments were repeated at least 3 times and a representative Western blot from one experiment is shown in the figures.