The method was adapted from previous studies as described (49, 52, 53). Cells were grown on glass coverslips for 24 hours and fixed with 3.7% formaldehyde in 1× phosphate-buffered saline (PBS) for 15 min at room temperature and permeabilized with 0.1% Triton X-100 in 1× PBS for 5 min. Samples were rinsed three times in 1× PBS with 5 min each wash, blocked for 30 min with 5% BSA, and incubated with primary antibodies as indicated for 2 hours. After three 5-min washes in 1× PBS with Tween-20 (PBST) (0.1% Tween 20), the coverslips were incubated with Alexa 488–conjugated goat anti-rabbit secondary antibody or Alexa 594–conjugated goat anti-mouse secondary antibody (Invitrogen) for 60 min and washed three times with 1× PBST. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole for 10 min. Coverslips were washed twice for 3 min each with 1× PBS and mounted onto slides using ProLong Gold anti-fade reagent (Invitrogen). Images were acquired by Nikon NIS Elements 4.0 software using Nikon Eclips Ti.

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