QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

Help viewing high resolution images Return to article

Fig. 1. TACE phosphorylation at Thr735 in intact COS-7 cells is ERK activation-dependent. (A) COS-7 cells were transfected with pcDNA3.1+ (1 µg), pWT.TACE (0.5 µg), pCA.MEK-1.HA (0.25 µg) and/or pWT.ERK-HA (0.25 µg) for 24 hours. Amounts of transfected DNA were kept equivalent to 1 µg using pcDNA3.1+/Zeo. Cells were incubated in the presence of 2 mCi [³³P]orthophosphate for 6 hours before the cells were lysed and TACE immunoprecipitated with the anti-TACE ICD antibody. Immune complexes were washed, resolved using SDS-PAGE and western transfer followed by autoradiography (top panel). The same membrane was probed with the anti-TACE ICD antibody to reveal immunoprecipitated TACE protein (-TACE IP panel). Whole cell lysates (WCL) were also analysed by western blotting to determine the expression levels of TACE, CA.MEK-1.HA and WT.ERK2-HA (lower three panels). (B) Cells were transfected with expression plasmids for WT- and T735A-TACE and TACE protein as in A, and isolated 24 hours later by immunoprecipitation. Immune complexes were washed and probed with an anti-phosphothreonine monoclonal antibody (-pThr, top panel) and anti-TACE (second panel). WCL were also analysed for expression of TACE, MEK-1 and ERK2 (lower three panels). Arrows highlight the detected TACE species.

Return to article