Flag-Ppp1r11 Pulldown in HEK293T

Pull-down of Ppp1r11 was performed in HEK293T cells 48 h after reverse transfection of Flag-Ppp1r11 ± GSK3β. BIO treatment (1 µM) was performed for 2 h at 37 °C prior to harvest. Cultured cells were resuspended in Triton lysis buffer (20 mM Tris HCl, 150 M NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 1 mM b-glycerolphosphate, 2.5 mM sodium pyrophosphate and 1 mM sodium orthovanadate). After cell lysis, 30 min incubation on ice, centrifugation and concentration measurement, the samples were incubated with Protein A/G PLUS-Agarose (Santa Cruz; sc-2003) and Flag antibody (Mouse monoclonal anti-FLAG M2, Sigma Aldrich, Cat#F3165) 1 :250 for 3 h. As a negative control for IP, mouse γ-globulin was used 1 :250. Lysates were washed 3x in lysis buffer then heated at 70 °C in NuPAGE LDS buffer (Thermo Scientific; Cat#NP0007) supplemented with 50 mM DTT for subsequent immunoblotting.