Gfp 3e6 a11120

The unbiased screen for htt protein interactors was performed using a Panomics transcription factor (TF) immunoprecipitation array, according to the manufacturer’s instructions: http://www.veritastk.co.jp/attached/2209/TFInteractionArrayKitcombined.pdf. Htt IP for the Panomics TF IP array utilized anti-htt antibody EM48. Protein lysates were prepared as previously described^{55 (link)}. For co-immunoprecipitations, homogenized protein was incubated with indicated co-immunoprecipitation antibody (GFP: 3E6 A11120, Life Technologies (1:100); Htt: MCA2050, Abcam (1:100); PPARδ: SC-7197, Santa Cruz; (1:100)). Proteins were run on 10% Bis-Tris gels (Invitrogen) and transferred to PVDF membranes (Millipore) prior to blocking in Odyssey Blocking Buffer (LI-COR biosciences). Membranes were incubated with antibodies as indicated: Flag (F7425, Sigma; 1:1000), Htt (MAB2166, Millipore; 1:1000), GFP (A11121, Life Technologies, 1:1000), PPARδ (PA1-823A, Pierce; 1:1000), PPARα (PA1-822A, Pierce; 1:1000), or PPARγ (PA3-821A, Pierce; 1:1000), or β-actin (ab8226, Abcam; 1:10,000), and imaged on the Odyssey instrument (Licor). To obtain proteins in vitro for the direct interaction co-immunoprecipitation, the Promega TnT T7 Quick Coupled Transcription/ Translation System (Promega, #L1170) was used prior to co-immunoprecipitation.