All lanes : Anti-FOXO3A (phospho S253) antibody ( ab26652 ) at 1 µg/ml Lane 1 : PC12 (Rat adrenal pheochromocytoma cell line) Cytoplasmic Lysate ( ab14883 ) Lane 2 : PC12 (Rat adrenal pheochromocytoma cell line) Cytoplasmic Lysate ( ab14883 ) with FOXO3A peptide - phospho S253 ( ab27883 ) at 1 µg/ml Lane 3 : PC12 (Rat adrenal pheochromocytoma cell line) Cytoplasmic Lysate ( ab14883 ) with Human FOXO3A (unmodified ) peptide (ab27884) at 1 µg/ml Lysates/proteins at 20 µg per lane. Secondary IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/15000 dilution Performed under reducing conditions. Predicted band size : 70 kDa Observed band size : 67 kDa ( why is the actual band size different from the predicted? )