Jip 1

POSH, JIP-1, JNK1, JNK2, Rac1, Noxa, MKK7, MLK3, MEKK1, and p-NFATc1 were purchased from Santa Cruz Biotechnology. Tak1, Puma, pSAPK/JNK, pMKK7, p-p38, pIκBα, NFATc1, JunB, Bcl2 and Bim were purchased from Cell Signaling. CD25 APC, T-bet APC, GATA3 PE, IL-4 PE, IFN-γ APC, IL-2 APC, and IL-17A APC were purchased from eBioscience. β-actin was purchased from Sigma. 4G10, Rac1 and Rac2 were purchased from Millipore. Mcl-1 was purchased from Rockland. IL-12Rβ2 PE was purchased from R&D. 7-AAD was purchased from BD.

Cells were lysed in radioimmunoprecipitation assay buffer (RIPA) containing 50 mM Tris (pH 8.0), 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40 and HALT™ protease/phosphatase inhibitor mixture (Thermo Fisher). Cellular lysates were sonicated before clarification by centrifugation. Proteins were resolved by SDS-PAGE, transferred to nitrocellulose membranes (BioRad) and probed with antibodies for JIP1 (Santa Cruz Biotechnology, sc-25267), total JNK (Santa Cruz Biotechnology, sc-474), phospho-JNK (Cell Signaling Technology, 4668), cFos (Cell Signaling Technology, 4384), acetylhistone H3 (Cell Signaling Technology, 4499), KIF5A (Abcam, ab5268), calnexin (Santa Cruz Biotechnology, sc-11397), Myc (Santa Cruz Biotechnology, sc-40), and GAPDH (Invitrogen, AM4300). Immunoblotting with an anti-LC3 antibody (Sigma, L7543) was performed using PVDF membrane (BioRad).