Western blot using Rocklands affinity purified anti-ATG13 pS318 antibody shows detection of phosphorylated ATG13 in 293T cells engineered to coexpress Ulk1 and Atg13 (Ulk1 + Atg13). In the left lane was loaded kinase-dead hypophosphorylated Ulk1-K46A mutant + ATG13. The right lane contains the 293T Ulk1 + ATG13 lysate and shows detection at approximately 57 kDa. The antibody was purified and resolved by SDS-PAGE, then transferred to nitrocellulose membrane. The membrane was blocked with 5% Blotto (p/n B501-0500) and probed with the primary antibody at 1microg/mL overnight at 4°C. After washing, the membrane was probed with Goat Anti-Rabbit HRP secondary 1:5000 in detection buffer (p/n MB-070) for 45 minutes at room temperature. In collaboration with Charles Dorsey at Eli Lilly, Indianapolis, IN and John Cleveland at Scripps, Jupiter, FL.