FIGURE 9.

Knockdown of SFKs in HMVEC-Ls through siRNA. HMVEC-Ls were transfected with siRNAs targeting the four SFKs expressed in HMVEC-Ls, c-SRC, YES, FYN, and LYN, or control siRNAs. A, after 72 h, ECs were processed for immunoblotting with antibodies against each of these four SFK proteins. Blots were stripped and reprobed for β-tubulin. These blots are representative of three experiments. B, ECs were processed for a cell-based ELISA to detect phospho-Tyr⁴¹⁶ as a measure of SFK activation. The phospho-Tyr⁴¹⁶ was normalized to both total SFK and cellular protein and expressed as mean ± S.E. -fold increase relative to the simultaneous medium control. n, the numbers of wells studied, was 6 for each group. C, HMVEC-Ls were transfected with siRNAs specifically targeting c-SRC, YES, FYN, or control siRNAs. After 72 h, ECs were exposed for 4 h with LPS (100 ng/ml) or medium alone and lysed, and the lysates were immunoprecipitated with anti-VE-cadherin or anti-p120^ctn antibodies. The immunoprecipitates were resolved by SDS-PAGE and transferred to PVDF, and the blots were probed with anti-phosphotyrosine antibodies. To normalize phosphotyrosine signal to immunoprecipitated protein, the immunoblots were stripped and reprobed with the immunoprecipitating antibodies raised against VE-cadherin and p120^ctn. These blots are representative of four experiments. D, on each immunoblot, densitometric quantification of phosphotyrosine signal of VE-cadherin and p120^ctn immunoprecipitates each were normalized to total VE-cadherin and p120^ctn signal, respectively. Vertical bars represent mean ± S.E. -fold increase of arbitrary densitometry units of phosphotyrosine signal normalized to arbitrary densitometry units of total signal relative to the simultaneous control. n = 4. E, for the barrier assay, HMVEC-Ls cultured to 70% confluence in plastic dishes were transfected with siRNA targeting FYN, c-SRC, LYN, YES, or control siRNAs. After 24 h, transfected cells were seeded onto the filters in assay chambers and cultured for 48 h, after which base line barrier function was established. Only EC monolayers that retained ≥97% of the tracer molecule were exposed for 6 h to 100 ng/ml LPS or medium alone and again assayed for transendothelial [¹⁴C]BSA flux. Vertical bars represent mean ± S.E. transendothelial [¹⁴C]BSA flux in pmol/h immediately after the 6-h study period. n indicates the number of monolayers studied and is indicated within each bar. In A and C, IP, immunoprecipitate; IB, immunoblot; IB^*, immunoblot after stripping. In B, D, and E, ^*, significantly increased compared with the simultaneous medium with control siRNA at p < 0.05; ^**, significantly decreased compared with LPS and control siRNA at p < 0.05. In B and E, the data sets were generated from three independent experiments with 2-4 replicates/treatment/experiment. In C, the data were generated from four independent experiments, and the mean ± S.E. changes of the combined data are displayed in D.