FIGURE 7.
Identification of ZA proteins as substrates for LPS-induced tyrosine phosphorylation. Postconfluent HMVEC-L monolayers were exposed for varying times to LPS (100 ng/ml) or medium alone. A, HMVEC-Ls were fixed, incubated with FITC-conjugated antiphosphotyrosine antibody, and analyzed by fluorescence microscopy. i, medium control (1 h); ii, LPS (1 h). The arrows indicate phosphotyrosine signal at intercellular boundaries. Magnification was ×400. These photomicrographs are representative of two experiments. B, in other studies, HMVEC-Ls were exposed to LPS (100 ng/ml) or medium alone in the presence of vanadate (200 μm) and phenylarsine oxide (1.0 μm), only during the last 0.25 h of incubation. HMVEC-L lysates were resolved by SDS-PAGE and transferred to PVDF membranes, and the blots were probed with antiphosphotyrosine antibody. To confirm equivalent protein loading and transfer, blots were stripped and reprobed for β-tubulin. Molecular masses in kDa are indicated on the left. The arrows on the right indicate bands with altered phosphotyrosine signal in response to LPS. This blot is representative of three experiments. C, lysates of LPS-treated and medium control HMVEC-Ls were immunoprecipitated with antibodies raised against VE-cadherin (lanes 1 and 2), β-catenin (lanes 3 and 4), γ-catenin (lanes 5 and 6), and p120ctn (lanes 7 and 8). The immunoprecipitates were resolved by SDS-PAGE and transferred onto PVDF, and the blots were probed with antiphosphotyrosine antibody. For normalization of phosphotyrosine signal to the immunoprecipitated protein, blots were stripped and reprobed with each immunoprecipitating antibody. Each blot is representative of ≥3 experiments. IP, immunoprecipitate; IB, immunoblot; IB*, immunoblot after stripping.