Figure 6. The effect of EGCG treatment and PKA silencing on the level of MYPT1^pThr696, eNOS^pThr497 and ppMLC20^Thr18/Ser19.

(a) BPAECs were pretreated with 100 nM PMA for 30 minutes, then challenged with EGCG in the indicated concentrations for 1 hour. Changes in the level of MYPT1^pThr696, eNOS^pThr497 and ppMLC20 were assessed by Western blotting with phospho-specific antibodies upon the different challenges (upper panel). Cropped images of representative Western blots are shown. Uncropped, full-length blots are presented in Supplementary Information in Fig. S8. Bar graphs represent the changes in the level of MYPT1^pThr696 (upper graph), eNOS^pThr497 (middle graph) and ppMLC20 (lower graph) determined by densitometric analysis of blots from 3–4 independent experiments (means ± SEM, n.s.: not significant, *p < 0.05, **p < 0.01, ***p < 0.001 compared to control, i.e. without EGCG, One-way ANOVA, Newman-Keuls post-hoc testing). (b) BPAECs cells were treated with 20 μM EGCG and the phosphatase activity in the lysates of untreated (control) or EGCG treated cells was determined using ³²P-MLC20 as substrate. Data represent means ± SEM (n = 3), two-tailed Student’s t-test. (c) BPAECs were transfected with scrambled or PKA catalytic subunit (PKAc) specific siRNA then treated with PMA and EGCG and the changes in the level of MYPT1^pThr696, eNOS^pThr497 and ppMLC20 were assessed as described in (a).