Anti traf6 h274

Nuclear and cytoplasmic extracts from naive T cells cultured for 3 days were prepared with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce), as previously described^{21 (link)}. The purity of the nuclear and cytoplasmic fractions was verified by probing with antibodies against Lamin B1 (D4Q4Z; 1,000×; Cell Signaling Technology). Whole-cell extracts were immunoprecipitated with an anti-Raptor (24C12; 100×; Cell Signaling Technology), anti-Def6 (Rabbit polyclonal; 100×^{21 (link)}), anti-p62 (H-290; 50×; Santa Cruz), anti-TRAF6 (H274; 50×; Santa Cruz), or anti-HA (3F10; 50×; Roche Applied Science) antibodies. Antibodies to p-STAT3 (Y705; 1,000×), p-4E-BP (T37/46; 1,000×), 4E-BP (1,000×), p-S6K1 (S371; 1,000×), S6K1 (1,000×), p-AKT (S473; 1,000×), AKT (1,000×), p-AKT (T308; 1,000×), p-PRAS40 (T246; 1,000×), PRAS-40 (1,000×), p-AMPK (T172; 1,000×), AMPK (1,000×), p-Raptor (S792; 1,000×) and p62 (5114; 1,000×) were obtained from Cell Signaling Technology. Antibodies to IRF4 (M-17; 1,000×), TRAF6 (H274; 500×), and c-Myc (9E10; 500×) were obtained from Santa Cruz. Anti-Bcl6 antibody was obtained from BD (K112-91; 1,000×). Anti-Flag monoclonal antibody M2 (horseradish peroxidase (HRP)) was obtained from Sigma (1,000×).

pCMV/Myc-TRAF6, TRAF2, and TRAF5 was provided by Dr. Lingqiang Zhang (State Key Laboratory of Proteomics, Beijing, China). pEFNeo/HA-CHIP, pRKIM/Myc-CHIP, and pRKIM/Myc-CHIP (H260Q) were generated in our lab as described previously.^{23 (link)} I-κBα, phosphor-I-κBα (Ser32) and anti-α-tubulin (3873p) were purchased from Cell Signaling Technology (Beverly, MA). Anti-TRAF6 (H-274) and anti-p65 (C-20) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-CHIP antibody was developed in our lab.^{16 (link)} MG132 was purchased from Millipore (Billerica, MA). Cyclohexanone (CHX) was purchased from Amresco (AMRESCO Inc., OH).