Stromal contact prevents TPA-induced recruitment of pY-p100 to c-Cbl but enhances recruitment of Crk to c-Cbl.

Immunoprecipitation of c-Cbl followed by immunoblotting for phosphotyrosine showed TPA-induced recruitment of an about 100 kDa tyrosine-phosphorylated protein (indicated by arrow as pY-p100). Stromal contact completely blocked TPA-induced recruitment of pY-p100. Levels of tyrosine-phosphorylated c-Cbl (arrow: pY-Cbl) did not vary significantly with any of the treatments. Reprobing for Crk showed that stromal contact, by contrast, enhanced recruitment of Crk (p28) to c-Cbl. IgL indicates immunoglobulin light chain. Immunoblotting of the whole cell extracts (WCE) showed no significant variation in total levels of c-Cbl or Crk (p28) in any of the treatments. The results shown are representative of experiments performed 3 times. K562 cells with or without TPA for 48 hours and with or without stromal contact were subjected to whole cell extraction as described in “Materials and methods.” Whole cell extracts were then used both for direct immunoblotting and for immunoprecipitation of c-Cbl followed by immunoblotting.