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. 2017 Jul 31;8(47):82506–82530. doi: 10.18632/oncotarget.19714

Figure 7. Assessment of RhoA activity and ROCK inhibition in EOC cells.

Figure 7

(A) GTP-Rhotekin RhoA pull-down samples from cell lysates of SKOV3 mock-transfected control (Ctrl) and shRNA-Hic-5 knockdown clones (KD) (sh-S1 and sh-S2) were immunoblotted with a Rho specific antibody for assessment of the active protein levels using Western blot. Cell lysates were also immunoblotted with a Rho specific antibody for total Rho protein levels. (B) GTP-Rhotekin RhoA pull-down samples from cell lysates of A2780s control clone (pCMV-Ctrl) and Hic-5 pCMV clone (pCMV-Hic-5) were immunoblotted with a Rho specific antibody for assessment of the active protein levels using Western Blot. Cell lysates were also immunoblotted with a Rho specific antibody for total Rho protein levels. (C) SKOV3 mock-transfected control (Ctrl) and shRNA-Hic-5 knockdown (KD) (sh-S1 and sh-S2) cells were either untreated (-) or treated (+) with 10mM of the ROCK inhibitor (Y27632) over a period of 48 hr. Western blot analysis were performed on cell lysates to examine protein expression of Hic-5, E-cadherin, N-cadherin, FAK-pY397, and FAK. (D) SKOV3 mock-transfected control (Ctrl) and shRNA-Hic-5 knockdown (KD) (sh-S1 and sh-S2) clones were either untreated (-) or treated (+) with 10mM of the ROCK inhibitor (Y27632) followed by stimulation without (-) or with (+) 5ng/ml of TGFβ1 for 48 hr. Western blot analysis were performed on cell lysates to examine protein expression of Hic-5, E-cadherin, N-cadherin, FAK-pY397, and FAK. β-actin was used as the loading control.