Journal: The Journal of Cell Biology
Article Title: EWI-2 regulates ?3?1 integrin-dependent cell functions on laminin-5
Figure Lengend Snippet: Tetraspanins support EWI-2- α 3 β 1 integrin association. (A) ∼10 7 A431 EWI-2 cells were labeled with sulfo-NHS biotin and lysed in 1% Brij 96. Equal portions of lysate were depleted three times with protein G alone (mock) or with protein G plus anti- tetraspanin or α2 integrin antibodies. Depletions are indicated at the top of the figure. Depleted lysates were further divided and immunoprecipitated with anti-CD81, anti-α3 integrin, or anti-α2 integrin antibodies conjugated to agarose followed by SDS-PAGE and blotting with HRP-ExtrAvidin ® . EWI-2* is a 50-kD EWI-2 cleavage fragment. Densitometry revealed that CD9 and CD81 depletion removed only ∼10% of total α3 integrin, but ∼85% of the α3-associated EWI-2. Densitometry of an independent experiment confirmed that only ∼10% of α3 is CD81-associated in A431 cells (not depicted). (B) U937 cells lacking CD9 and CD81 were transduced with CD81 and EWI-2 retroviral expression vectors and selected to obtain stable (CD81−, EWI-2+) or (CD81+, EWI-2+) cell lines. Then, both cell types were super-infected with an α3 integrin retroviral expression vector, to yield equivalent α3 expression levels, as confirmed by flow cytometry (not depicted). EWI-2 (M2 anti-FLAG mAb), CD81 (M38 mAb), or α3 integrin (A3X8 mAb) were immunoprecipitated from 1% Brij 96 lysates of CD81 − or CD81 + cells. Immunoprecipitates were blotted for EWI-2 (biotinylated M2 anti-FLAG mAb), α3 integrin (D23 pAb), or CD81 (M38 mAb). Apparent mature and immature forms of EWI-2 and α3 integrin are indicated with filled and open arrows, respectively. 6 × 10 6 cell equivalents were analyzed in EWI-2 and CD81 immunoprecipitations, and 4.8 × 10 7 cell equivalents in the α3 immunoprecipitations. (C) Biotinylated CD81 − or CD81 + cells were lysed in 1% Brij 96, and EWI-2 (FLAG), α3 integrin, or CD81 were immunoprecipitated as above. Cell surface–labeled proteins were revealed by HRP-ExtrAvidin ® blot. 1.7 × 10 6 cell equivalents were analyzed in each immunoprecipitation.
Article Snippet: The M2 anti-FLAG epitope mAb, either biotinylated or conjugated to agarose, and a rabbit anti-FLAG pAb were purchased from Sigma-Aldrich.
Techniques: Labeling, Immunoprecipitation, SDS Page, Transduction, Expressing, Infection, Plasmid Preparation, Flow Cytometry, Cytometry