Phosstop inhibitor tablet

Cells were plated to be confluent two days later. If cells were treated, media was changed to media containing the appropriate treatment for the indicated time. Each plate was rinsed twice with PBS, and then scraped into Kischkel buffer supplemented with Roche cOmplete Protease Inhibitor Cocktail, 1 mM PMSF, and Roche PhosSTOP inhibitor tablet. For suspension cells, cells were pelleted, washed twice in PBS, and then resuspended in lysis buffer. Lysates were sonicated for 15 s and cleared by centrifugation for 10 minutes at 4°C. Protein concentrations were measured by Bio-Rad Protein Assay Dye Reagent. For each IP 1–5 mg of lysate was used as total input. Antibodies used for IPs were 6–10 μg anti-WDR5 (Bethyl A302–429A), 4–6 μg anti-PDPK1 (Bethyl A302–130A), and an equivalent amount of Normal Rabbit IgG (Cell Signaling Technologies #2729S). Antibodies and lysates were rotated at 4°C overnight, and the next day a 20 μl bed volume of Roche Protein A agarose, blocked for at least 20 minutes with 1 mg/ml BSA in Kischkel buffer, was added to each sample. IPs were incubated with protein A agarose for 2–6 hours and then washed four times for five minutes with 1 mL cold Kischkel buffer, transferring to new tubes before last wash. Samples were eluted with SDS sample buffer supplemented with β-mercaptoethanol and taken forward for western blotting analysis.