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. 2011 Aug 23;105(7):1002–1011. doi: 10.1038/bjc.2011.303

Figure 4.

Figure 4

GS-168AT2 co-precipitates with both CD9 and CD15, but poorly with CD81. Human EC were incubated with vehicle or GS-168AT2 for 2 h, washed twice with vehicle and hEC were lysed in 1% Brij 97 lysis buffer, and the indicated tetraspanins were immunoprecipitated (CD9, CD81 and CD151) by incubating the cell lysate (the protein contents of cell lysates were adjusted by Bradford protein assay) with the indicated antibody at 4 °C. The immunocomplexes were pulled down with agarose beads coated with G-protein, and the beads were washed twice with lysis buffer. The immunoprecipitates were resolved in SDS–PAGE and western blotted with the indicated antibody. GS-168AT2 was used as control. (A) Upper panel: WB with 229T mAb of the immunoprecipitates with anti-CD81 mAb showing that GS-168AT2 poorly co-precipitated with CD81. Lower panel: WB with anti-CD81 of the immunoprecipitated CD81 showing equivalent quantities of CD8 deposited in the upper panel. (B) Upper panel: WB with 229T mAb of the immunoprecipitates with anti-CD9 mAb showing that GS-168AT2 co-precipitated with CD9. Lower panel: WB with anti-CD9 of the immunoprecipitated CD9 showing equivalent quantities of CD9 deposited in the upper panel. (C) Upper panel: WB with 229T mAb of the immunoprecipitates with anti-CD151 mAb showing GS-168AT2 co-precipitated with CD151. Lower panel: WB with anti-CD151 of the immunoprecipitated CD151 showing equivalent quantities of CD151 deposited in the upper panel. (D) Upper panel: WB with 229T mAb of the immunoprecipitates with anti-β1 mAb showing GS-168AT2 do not co-precipitated with β1 integrins. Lower panel: WB with anti-β1 mAb of the immunoprecipitated integrin β1 showing equivalent quantities of integrin β1 deposited in the upper panel. Western blots presented in this figure are representative images of at least three independent experiments.