Figure 6. mTOR regulates HLA class I Ab-induced ERM phosphorylation through the Rho pathway.

ECs were pretreated with Rapa or RAD for 24 h, or Ro-31-7549 (PKC inhibitor), Y-27632 (ROCK inhibitor), or U0126 (C3 transferase; Rho inhibitor) for 4 h, then stimulated with HLA I Ab (anti-HLA I; clone W6/32) for 10 min. (A) Cells were lysed and proteins were separated by SDS–PAGE followed by immunoblotting to detect phosphorylated MYPT1^Thr696 (p-MYPT1), p-ERM, and phosho-MLC^Ser19 (p-MLC), as well as Vinculin to confirm equal loading of proteins. Images from different parts of the same gel are separated by boxes. (B) Protein bands were quantified by densitometry scan analysis using ImageJ and results are expressed as the mean fold increase in phosphorylation above untreated with and without the indicated inhibitor ± SEM over 3 independent experiments. *P<0.05; **P<0.01; ****P<0.0001 by two-way ANOVA with Tukey’s multiple comparisons test.