Hrp linked anti rabbit igg

After 36 h of transfection, GFP-positive and -negative cells were sorted using the FACSAria™ apparatus (Becton Dickinson, San Jose, CA, USA). Total extracted proteins from cultured cells were quantified using the Lowry method. Equal amounts of total protein from each sample were loaded onto a 12.5% (w/v) SDS–polyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Membranes were then incubated with the primary antibodies at 4°C overnight, followed by incubation with the HRP-linked anti-rabbit IgG (DAKO), anti-goat IgG (DAKO), or anti-mouse IgG (DAKO) antibodies for 1 h. Signals were detected using Immobilon Western Chemiluminescent HRP (Millipore). Intensity of specifically amplified products was quantified by densitometric scans of the films using computer software for a Macintosh “CS analyzer” (ATTO, Tokyo, Japan).