Iblot 2 gel transfer stacks

Proteins in keratinocytes were collected using blue loading buffer with DTT (Cell Signaling Technology, Inc., Danvers, MA, USA). The samples were analyzed by gel electrophoresis using 10 % SDS-polyacrylamide gel (Mini-protein TGX Gel, Bio-Rad Laboratories, Inc., Tokyo, Japan) and were transferred onto polyvinylidene difluoride membranes (iBlot^TM 2 Gel Transfer Stacks, Invitrogen Corporation, Carlsbad, CA, USA). The membranes were blocked for 1 h with PBS containing 5 % nonfat dried milk and were incubated overnight at 4 °C with primary antibodies diluted in the blocking solution. The primary antibodies used were against phospho-Akt (Ser473, #4060S, 1:1000 dilution, Cell Signaling Technology, Inc.), total-Akt (#2920S, 1:1000 dilution, Cell Signaling Technology, Inc.) and GAPDH (#2118S, 1:1000 dilution, Cell Signaling Technology, Inc.). Subsequently, the membranes were washed in PBS and incubated for 2 h at room temperature with anti-rabbit IgG, horseradish peroxidase (HRP)-linked secondary antibody (#7074S, 1:1000 dilution, Cell Signaling Technology, Inc.), diluted in the blocking solution. The immunoreactive bands were detected by Amersham ECL Prime Western Blotting Detection Reagents (RPM2232, GE Healthcare, Tokyo, Japan) and analyzed using luminescent image analyzer (ImageQuant LAS 4000 mini, FUJIFILM Corporation, Tokyo, Japan) with Image Reader LAS-4000 software.