Pgc1α h 300

Nuclear lysates were incubated with specific antibodies overnight at 4°C, followed by precipitation with protein G Dynabeads (Invitrogen) at 4°C for 2 h. For Figure 3h, nuclear lysates from V5-ID2 stably-expressing A375P cells were subjected to co-IP with 1 μg ID2 antibody (C-20, Santa Cruz Biotechnology), followed by Western blot with TCF4 (M03, Abnova) and ID2 (4E12G5, Thermo Scientific); for Figure 4d, 10 mg of nuclear lysates from A375P cells treated with DMSO or 1 μM PLX4032 for 16 h were subjected to co-IP with 4 μg ID2 antibody (C-20). ChIP was performed with the MilliPore ChIP Kit with slight modification. Following sonication, nuclear lysates were precleared with protein A/GDynabeads (Invirogen) for 1 h. Equal amounts of precleared lysates were incubated with IgG or gene specific antibodies (PGC1α 4C1.C from Millipore, or PGC1α H-300, and TCF4 K-15 from Santa Cruz Biotechnology) overnight, followed by precipitation with protein A/G-Dynabeads for 2 h. qPCR with SYBR Green was performed to quantify the promoter occupancy. For Figure 4e, A375P cells stably expressing V5-TCF4 were cultured with PLX4032 at 5 μM for 16 hours and followed by ChIP and qPCR.