After treatment, the adherent cells were washed twice with cold
phosphate buffered saline (PBS) and fixed in paraformaldehyde solution
(4% in PBS, pH 7.4) for 30 min at 4 °C. The fixed cells were then
washed twice with PBS, treated with permeabilization solution (0.1%
Triton X-100 in 0.1% sodium citrate) on ice for 2 min, and incubated in
blocking buffer (3% BSA in TBST) for 1 h.