Figure 5. mTORC2 association with FA elements is necessary for strain activation of Akt.

a. siRNA targeting vinculin, but not control siRNA (siCTL) blocked strain-induced activation of Akt, shown as an increase phospho-473 Akt. Densitometry confirms the failure to significantly increase pAkt when vinculin is knocked down; * = p < 0.05 different from control, n=3. b. Lysates from siCTL and siVin cells shows immunoblot for vinculin and tubulin (top figure) before pull down with antibody to Paxillin (lower figure). Rictor association with paxillin increases only when vinculin is present. c. Blebbistatin (50µM) delivered prior to strain prevented strain-induced rictor, Akt and actin association with vinculin pull down. d. Blebbistatin prevented strain induced mTORC2 activity measured by increase in pAkt. e. Strain was applied at the beginning (strain 1) and/or end (strain 2) of a 3 hour period, at which point all lysates were made. pAkt rises after strain (comparing no strain, 1^st lane, to strain 2, 2^nd lane). pAkt returns to basal levels 3 h after strain (strain 1, 3^rd lane). A 2^nd strain application (strain 1+2, 4^th lane) shows increased pAkt. Densitometry confirms pAkt return to baseline, and amplification at the second strain application; * ¹ control, * ¹ #, (n=4).