Figure 2.

Btk coimmunoprecipitates with and is a substrate of Jak2. (A–D and F) COS cells were transfected with pSG5-based expression constructs encoding the indicated proteins. Expression of all proteins was verified in Western blots using specific antibodies (not depicted). After 48 h, cells were stimulated with 5 U/ml Epo (B) or 500 ng/ml SCF (C, lane 4; D and E, lane 4) for 10 min, harvested, and lysed, and Btk (A and C), EpoR (B), Jak2 (D), and c-Kit (F) were immunoprecipitated. (top panels) Immunoblots were stained with antiphosphotyrosine antibodies (PY99). The blots were restained with antibodies recognizing the immunoprecipitated proteins to check for equal loading. Arrows indicate the position of the immunoprecipitated and coimmunoprecipitating proteins. (E) wt erythroid progenitors (2B6) were factor deprived in the absence or presence of the Src kinase family inhibitor PP2 as indicated and stimulated with Epo for 10 min. Epo-induced Jak2 phosphorylation was assayed on immunoblots using PY99 (top). (bottom) Blots were restained with anti-Jak2 to confirm equal loading. (F) c-Kit immunoprecipitates were stained with antiphosphotyrosine antibodies (PY99), and blots were restained with anti–c-Kit and anti-Btk antibodies, whereas total cell lysates (TCLs) were stained with anti–c-Kit and anti-Btk to check expression levels.