Anti flag m2 or anti ha affinity gel

Fresh or frozen liver aliquots were suspended in RIPA buffer (Sigma) containing an EDTA-free protease inhibitor cocktail (Roche) and a Halt phosphatase inhibitor Cocktail (Thermo Scientific), and homogenized with a TissueLyser II (Qiagen). Cell debris was removed by centrifugation, and tissue lysates were resolved by SDS-PAGE and subjected to immunoblot analysis as previously described3 (link). Cell lysates were processed and analyzed as previously described3 (link). Immunoblot analysis used the following antibodies: anti-β-actin (ab8226; Abcam), anti-GAPDH (sc-25778; Santa Cruz), anti-CRTC1 (2501 or 2587; Cell Signaling), anti-NCoR (ab3482; Abcam, or sc-8994; Santa Cruz), anti-HDAC3 (ab7030; Abcam, or sc-11417; Santa Cruz), anti-Akt (9272; Cell Signaling), anti-pAkt (9271; Cell Signaling), anti-Sirt1 (07-131; EMD Millipore), anti-Flag (M2) or anti-HA Affinity Gel (Sigma), goat anti-rabbit IgG-HRP (sc-2054; Santa Cruz), IRDye 800CW–conjugated goat anti–mouse and anti–rabbit IgG (926-32210 and 926-32211, respectively; LI-COR), and IRDye 800–conjugated anti-Flag and anti-HA (600-432-383 and 600-432-384, respectively; Rockland). Immunocomplexes were visualized with an Odyssey Infrared Imaging System (LI-COR).

HEK293T cells transfected FLAG-tagged CHCHD10 WT or S59L and TDP-43-tomato-HA or HA-tagged CHCHD10 WT or S59L and TDP-43-FLAG were solubilized with NP-40 lysis buffer (20 mM Tris, 137 mM NaCl, 1% NP-40, 2 mM EDTA with protein inhibitor cocktail). After sonication on ice, lysates were collected by centrifuge. An equal amount of protein lysates were incubated with anti-FLAG M2 or anti-HA affinity gel (Sigma) overnight at 4 °C, washed three times with phosphate-buffered saline (PBS) containing 1% Tween-20, and then resuspended the pellet with 2× LDS sample buffer. The precipitates were subjected SDS-PAGE and analyzed with the Odyssey FC System (LI-COR).

All cells and tissues were processed and analyzed as previously described^{5 (link)}. For RT-PCR, total RNA was extracted using a TRIzol extraction method. The sequences of primers for RT-PCR and ChIP assays are listed in Supplementary Tables S2 and S3^{5 (link)}. Flag-tagged MafB was purified with M2 Affinity Gel as previously described^{5 (link)}, and Myc/DDK-tagged LXRα was obtained from OriGene. Immunoblot and ChIP analyses used the following antibodies: anti-β-actin (ab8226; Abcam), anti-GAPDH (sc-25778; Santa Cruz), anti-MafB (MAB3810; R&D Systems), anti-Stat6 (sc-621; Santa Cruz), anti-pStat6 (sc-11762; Santa Cruz), anti-LXRα (ab41902; abcam), anti-Flag (M2) or anti-HA Affinity Gel (Sigma), goat anti-rabbit IgG-HRP (sc-2054; Santa Cruz), IRDye 800CW–conjugated goat anti–mouse and anti–rabbit IgG (926-32210 and 926-32211, respectively; LI-COR), and IRDye 800–conjugated anti-Flag and anti-HA (600-432-383 and 600-432-384, respectively; Rockland). Immunocomplexes were visualized with an Odyssey Infrared Imaging System (LI-COR).