Figure 2

Downregulation and dissociation of VE-PTP and SHP2 from VE-cadherin in HIV-1 Tat C treated BMVECs. (A) Downregulation of VE-PTP expression in HIV-1 Tat C treated hBMVECs. HIV-1 Tat C treatment has been given for 12 hours on hBMVECs and western blot analysis was done by using anti VE-PTP antibody to show decrease in expression level of phosphatase VE-PTP. (B) Immnunoblot images of co-immunoprecipitaion of VE-PTP along with VE-cadherin complex showing the decreased association of VE-PTP with VE-cadherin. Same blots have been re-probed with VE-cadherin antibody to exhibit equal loading of pulled down cadherin complex. Monoclonal VE-cadherin antibody has been used for pull down of VE-cadherin complex by using NP-40 mild lysis buffer in cold conditions. (C) Compared to control hBMVECs (buffer treated), HIV-1 Tat C treated hBMVECs are showing a dose-dependent decrease in SHP2 expression levels. (D) SHP2 dissociation from cadherin complex has been shown by immunoblotting against SHP2 in pulled down cadherin complex by using anti VE-cadherin antibody. (E) The densitometry graph is showing almost 60–70% decrease in VE-PTP expression at higher doses of HIV-1 Tat C treatment. (F) Quantitation of image density by ImageJ software normalized by VE-cadherin image density. Pull-down experiment has been repeated three times and results are shown as mean ± S.E. (*p value ≤0.05). (G) Quantitation of image density of SHP2 showing a significant dissociation (**p value ≤0.005) from cadherin complex, normalized with amount of VE-cadherin pull down. All the results shown here are representative of three independently repeated experiments and shown as mean ± S.E.