BMP4-induced activation of ITGA4 expression is mediated via SMAD independent p38 MAPK phosphorylation. KLS cells were cultured with SCF+TPO in addition to BMP4 alone or combined with various inhibitors, for 5 days (ST = SCF/TPO; STC = SCF/TPO/CHD; STT = SCF/TPO/TSG; STB = SCF/TPO/BMP4; STBD = SCF/TPO/BMP4/Dorsomorphin; STBS = SCF/TPO/BMP4/SB203580), and analyzed by qRT-PCR, Western blot and flow cytometry. (A) Fold change in Itga4 transcript levels in KLS progeny from cultures stimulated with SCF+TPO and either BMP4, CHD, or TSG compared with KLS progeny from SCF+TPO cultures (n = 6; *P < .05). (B) Immunoblotting with antibodies against ITGA4 and β-actin (n = 3). (C) qRT-PCR analysis for Itga4 using KLS progeny cultured with ST, STC, STB, STBD, and STBS. (n = 3; *P < .05). (D). FACS analysis of KLS progeny cultured with ST, STB, STBD, and STBS using antibodies against ITGA4 (n = 4). (A) Western blot analysis using antibodies against ITGA4 and β-actin in KLS progeny cultured with ST, STC, STB, STBD, and STBS. (E) Western blot analysis of KLS progeny cultured with ST or STB using phospho-specific antibodies against JNK/SAPK, Erk1/2, and S6 kinase. (F) Western blot analysis of KLS progeny cultured with ST, STB using antibodies against phospho-p38 MAPK and total p38 MAPK. (G) Western blot analysis of KLS progeny cultured with ST or STB using antibodies against phospho-TAK1 and phospho-MKK3/6. β-actin was used as internal control (representative for panels D through F). All western blots are representative examples of 3 independent experiments.