Western blot using GeneTex Affinity Purified anti-APC1 pS355 antibody (GTX10923) shows detection of a band ~215 kDa corresponding to phosphorylated human APC1 (arrowhead). Lane 1 shows lysate from asynchronous cells. Lane 2 shows lysate from cells treated with thymidine to synchronize cells at the G1/S boundary. Lane 3 shows lysate from cells treated with nocodazole to synchronize cells at the M phase. Phosphorylated APC1 is mostly present only in cell preparations arrested at cell division. Each lane contains approximately 30 ug of HeLa S3 whole cell lysates separated by 12.5% SDS-PAGE followed by transfer to nitrocellulose. After blocking with 5% non-fat dry milk in TTBS, the membrane was probed with the primary antibody diluted to 1:500 for 1 h at room temperature followed by washes and reaction with a 1:5,000 dilution of HRP Gt-a-Rabbit IgG [H&L] MX (GTX27090) for 45 min at room temperature. ECL reagent was used for detection. Other detection systems will yield similar results.