Cells were washed with cold PBS at 24e36 h after transfection and then fixed
in methanol-acetone, 1:1, for 30 min at 4 C and washed with PBS once again followed by incubation with primary antibodies at a dilution of 1:100 in PBS containing 0.5% bovine serum albumin (BSA) and secondary antibodies (1:200 dilution in PBS containing 1% BSA) both at 37 C for 1 h, respectively.