Anti phospho gsk 3β tyr216

Cells were washed with ice-cold PBS 3 times and lysed with NET lysis buffer. The buffer was composed of 150 mM NaCl, 50 mM Tris, 1 mM EDTA, and 1% Nonidet P-40, had a pH of 7.4, and was supplemented with a protease inhibitor cocktail (Roche). Lysates were boiled in the SDS sample buffer for 5 min and then subjected to Western blot analysis. The following antibodies were used:
Anti-O-GlcNAc (RL2: Thermo Fisher Scientific, #MA1-072), Anti-SMAD4 (Santa Cruz, #sc-7966), Anti-SMAD2/3 (Cell Signaling Technology, #3102S), Anti-OGT (DM-17: Sigma, #O6264), Anti-OGA (Abcam, #ab124807), Anti-GAPDH (Millipore, #M171-3 or Santa Cruz, #sc-32233), Anti-FLAG (MBL, #M185-3L), Anti-Ubiquitin (Santa Cruz, #sc-8017), Anti-HA (Santa Cruz, #sc-805 or Cell Signaling Technology, #C29F4), Anti-GSK-3β (Cell Signaling Technology, #27C10), Anti-Phospho-GSK-3β (Ser9) (Cell Signaling Technology, #D3A4), Anti-Phospho-GSK-3β (Tyr216) (Abcam, #ab75745), Anti-Phospho-threonine (Santa Cruz, #sc-5267), Anti-c-myc (Santa Cruz, #sc-47694).
For immunoprecipitation and sWGA purification, total cell lysates were incubated with antibody-bound beads or agarose-conjugated succinylated wheat germ agglutinin (sWGA, Vector Laboratories) overnight at 4 °C. Beads were then washed 5 times with lysis buffer and bead-bound proteins were eluted by boiling the beads for 5 min in the SDS sample buffer.