Differential phosphorylation of signaling components by hIL-5 in TF-1 and JYTF-1 cells.

Both TF-1 and JYTF-1 cells were cytokine-depleted for 24 hours (lanes 1 and 4) before stimulation with GM-CSF (lanes 2 and 5) and IL-5 (lanes 3 and 6) (as indicated above the picture) for 5 minutes. Cell lysates were prepared and subjected to immunoprecipitation (IP)-Western (Blot) (for βc, JAK1, JAK2, and STAT5) or direct Western (for MAPK and Akt) blot analysis. For IP-Blot analysis, cell lysates were immunoprecipitated, fractionated, and transferred by Western blotting and the membrane was probed with antiphosphotyrosine antibody (indicated as P-tyr on the right-hand side, under Blot). This membrane was stripped as described in Materials and methods and then reprobed with anti-βc, -JAK1, -JAK2, or -STAT5 antibody (as indicated on the right-hand side, under Blot). For direct Western blot analysis, 150 μg of whole cell lysates was fractionated directly in a 10% SDS-PAGE and the Western blot was then probed with antibodies against phospho-MAPK (MAPK = ERK1^p44 + ERK2^p42) and phospho-Akt. These 2 blots were then reprobed with anti-MAPK and anti-Akt antibodies. The Western blot signals were visualized by ECL reaction and exposed onto x-ray film.