Alexa flouro 568 phalloidin

RNAi treated cells were placed on poly-L-Lysine coated coverslips, fixed with 4% paraformaldehyde, blocked for 1 hour with standard PBS containing 10% normal goat serum (NGS) at room temperature (RT), then stained with Alexa Flouro 568 phalloidin (1:1000; #A12380, Molecular Probes, Inc., Oregon, USA) in PBS with 10% NGS for 45 min at room temperature (RT). A minimum of eight frames of fluorescence micrographs was taken containing on average 30–40 cells counted and categorized for cell shape for each treatment. For the transfected cells, the appropriate primary antibody: mouse anti-GFP (1:200; #MS-1315 Thermo Fisher Scientific, USA), rabbit anti-FLAG (1:200; #F7425 Sigma-Aldrich, Missouri, USA), mouse anti-HA (1:200; #H9658 Sigma-Aldrich, Missouri, USA) was added overnight in PBS with 10% NGS at 4°C. Cells were next incubated in PBS containing 10% NGS and secondary anti-mouse or anti-rabbit FITC (1:200) antibody. After two hours, cells were washed and stained with Alexa Flouro 568 phalloidin (1:1000; #A12380, Molecular Probes, Inc., Oregon, USA) in PBS with 10% NGS for 45 min at RT before mounting. Cells that were FITC-positive were counted and characterized.