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. 2017 Mar 31;7:45545. doi: 10.1038/srep45545

Figure 3. RhoA induces bleb formation in PCTK3-knockdown HEK293T cells.

Figure 3

(a) HEK293T cells plated on poly-L-lysine-coated coverslips were transfected with NC ((i), (iii), (v), (vii)) or PCTK3 ((ii), (iv), (vi), (viii)) siRNA. After 24 hours, cells were transfected with empty FLAG vector ((i), (ii)), FLAG-tagged RhoA wild type (WT) ((iii), (iv)), T19N mutant ((v), (vi)), or Rac1 ((vii), (viii)) for 12 hours, and then were serum starved for 12 hours. Cells were fixed and stained with anti-FLAG or anti-phospho-MLC (Thr-18/Ser-19) antibodies. Fluorescence for FLAG and phospho-MLC is shown in green and red, respectively. Hoechst nuclear staining is represented in blue. Confocal images were performed using a 63× objective lens. (b) The cell lysates were subjected to immunoblot analysis using anti-PCTK3 and anti-FLAG antibodies. (c) Total cells and cells with blebs were counted in 6-7 fields randomly selected. At least 30 cells were counted, and the number of cells with blebs was expressed as a percentage of the total number of counted.