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. 2009 Jul 10;3(5-6):439–450. doi: 10.1016/j.molonc.2009.07.001

Figure 4.

Figure 4

Experimental validation of a subset of novel substrates identified in this study by Western blotting. NIH/3T3 cells have been grown to 80% confluence and serum‐starved for 12h followed by either no treatment, stimulation with PDGF‐BB (100ng/mL for 5min) or PDGF stimulation after preinculation with SU6656 (2μM for 1h prior to lysis or stimulation). (A) Cell lysates were subjected to immunoprecipitation using antibodies against Eps15, Cortactin, Shp2, Fyb or cPLA2 and probed by Western blotting with anti‐phosphotyrosine antibodies. (B) Cell lysates were subjected to immunoprecipitation using anti‐phosphotyrosine antibodies and probed by Western blotting with antibodies against Trim28, Ezrin, Calpain 2, respectively. The total amount of proteins in whole cell lysates across different conditions was monitored by Western blotting with specific antibodies against these proteins.