Analysis of requirement for MYPT1 expression in TNF-induced MLC phosphorylation and leak.

<p>A) Immunoblot analysis of MLC phosphorylation in lysates of MYPT1 KD HDMEC. In the absence of TNF, phospho-MLC expression levels were higher in lysates from MYPT1 siRNA-transfected HDMEC than in lysates of siRNA control. TNF treatment for 6 hours at 0.8 ng/ml further increased levels of phospho-MLC expression in MYPT1 KD HDMEC, but MYPT1 knockdown did not limit inhibition of TNF-induced MLC phosphorylation by 10 μM Y-27632. Con, siRNA control lysate; MP, MYPT1 siRNA lysate. B) ECIS analysis of TNF leak in MYPT1 KD HDMEC. Compared to control-transfected HDMEC, MYPT1 KD did not inhibit leak induced by TNF at 0.8 ng/ml. Basal levels of TEER in MYPT1 KD HDMEC were decreased to 69 ± 0.3% that of control siRNA (data not shown). The corrected basal TEER for this experiment was 65.3 ± 1.9 Ω·cm² for MYPT siRNA 5-transfected HDMEC, 41.6 ± 1.5 Ω·cm² for MYPT siRNA 7-transfected HDMEC, and 42.1 ± 0.8 Ω·cm² for negative control siRNA-transfected HDMEC. n = 3,3,3. Representative of 2 (A, B) independent experiments with similar results.