In vitro translation and detection of Jak2. Approximately 1 μg Jak3 and Jak2 cDNAs were translated in a combined transcription and translation reticulocyte lysate system (Invitrogen) including (35S) methionine. Aliquots of each product were resolved on SDS-PAGE gel, followed by transfer to nitrocellulose and visualization by autoradiography. The reactions yielded proteins of 120 and 130 kD, corresponding to Jak3 and Jak2, respectively (lanes 1 and 2). The remaining lysate of each reaction was precleared with Protein A–Sepharose CL-4B and subsequently immunoprecipitated with anti-human Jak2 antibody, resolved by SDS-PAGE gel, electrotransferred to nitrocellulose membrane, and visualized by autoradiography. Only Jak2 was immunoprecipitated by the anti-human Jak2 antibody with no cross-reactivity to Jak3 (lanes 3 and 4). Only the immunoprecipitated Jak2 in lane 4 was detected by ECL (lane 6) after the same membrane was probed with an anti-mouse Jak2 antibody (PharMingen).
