WB using Anti-ATG13 antibody shows detection of ATG13 in 293T cells engineered to coexpress Ulk1 and Atg13 (Ulk1 + Atg13), right lane, but not in the left lane in which was loaded kinase-dead hypophosphorylated Ulk1-K46A mutant + ATG13. Detection is demonstrated at approximately 57 kDa. primary antibody was used at 1ug/mL overnight at 4°C. After washing, the membrane was probed with Goat Anti-Rabbit HRP secondary 1:5000.