Fig. 5. Everolimus inhibited HLA I-mediated mTORC1 and mTORC2 formation in EC.

Quiescent EC were pretreated with A, F, I, M 10nM of everolimus or B, G, J, N 10nM of sirolimus for 2 or 24hr, and were stimulated with 0.1µg/ml anti-HLA I mAb for 10 min. Cells were lysed. The pre-cleared lysates were immunoprecipitated A and B with anti-mTOR Ab followed by immunoblotting with anti-Raptor, anti-Rictor, or anti-Sin1 Abs. The membrane was reprobed with anti-mTOR Ab to confirm equal loading of proteins; F and G The pre-cleared lysates were immunoprecipitated with anti-Raptor Ab followed by immunoblotting with anti-mTOR, or anti-Rictor Abs. The membrane was reprobed with anti-Raptor Ab to confirm equal loading of proteins. I and J the pre-cleared lysates were immunoprecipitated with anti-Rictor Ab followed by immunoblotting with anti-mTOR, anti-Raptor, or anti-Sin1 Abs. The membrane was reprobed with anti-Rictor Ab to confirm equal loading of proteins. M and N the pre-cleared lysates were immunoprecipitated with anti-Sin1 Ab followed by immunoblotting with anti-mTOR, anti-Rictor, or anti-Raptor. The membrane was reprobed with anti-Sin1 Ab to confirm equal loading of proteins. C, D, E, H, K, L, O, P Protein bands in the immuno complexes shown in A, B, F, G, I, J, M, N were quantified by densitometry analysis and results are expressed as the mean ± SEM percentage of maximal increase in protein expression above control values. *p<0.05, **p<0.01, and ***p<0.001 were analyzed by one way ANOVA with Fisher’s LSD. Data represent at least three independent experiments. HAEC used in these experiments include CAR, CAS, and 5555.