Western blot analysis of extracts from HT-29 cells, untreated or synchronized in G1 phase, using Phospho-CDK4 (Thr172) Antibody (upper) or CDK4 (DCS156) Mouse mAb #2906 (lower). After western transfer, the membrane was either left untreated (-CIP, left) or treated with Calf Intestinal Phosphatase (+CIP, right). G1 phase synchronization was performed by thymidine block (2 mM, 16 hours), release into nocodazole (100 ng/mL, 24 hours), selective detachment of mitotic cells, and release into G1 phase (4 hours).
Western blot analysis of extracts from U-2 OS cells, untreated or synchronized in G1 phase, using Phospho-CDK4 (Thr172) Antibody. The antibody was preincubated with a phospho-CDK4 (Thr172) peptide or a nonphosphorylated CDK4 (Thr172) peptide.