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ajt12669-sm-0001-SuppData-S1.pdf183KFigure S1: Anti-HLA I Abs stimulate activation of mTOR signal pathway, Akt and ERK in endothelial cells (EC). (A) Quiescent human aortic EC were treated with anti-HLA I mAb W6/32 for various time points or mIgG for 10 min as control. (D) Quiescent EC were treated with different concentration of anti-HLA I mAb W6/32 for 10 min. Proteins in the precleared cell lysates were separated by SDS–PAGE followed by immunoblotting with anti-phospho-mTOR Ser2448, anti-phospho-p70S6K Thr389, Thr421/Ser424, anti-phospho-S6RP Ser235/236, anti-phospho-Akt Ser473 or anti-phospho-ERK Thr202/Tyr204 Abs. The membrane was reprobed with anti-mTOR, anti-S6K, anti-S6RP, anti-Akt or anti-ERK total Ab to confirm equal loading of proteins. (B, C, E, F, G, H, I, J and K) Phosphorylated protein bands shown in A and D were quantified by densitometry scan analysis and results are expressed as the mean ± SEM percentage of maximal increase in phosphorylation 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 and CAS.

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