Anti normal mouse igg

ChIP experiments were performed using a Simple ChIP Enzymatic Chromatin IP kit (no.9003; Cell Signaling Technologies) according to manufacturer instructions. The fresh GAS from 8‐week‐old C57BL/6J mice were cut with scissors and then cross‐linked with 37% formaldehyde at a final concentration of 1% at room temperature for 10 min. Fragmented chromatin was treated with nuclease and subjected to sonication. Samples were then precleared with protein‐A/G sepharose beads and immunoprecipitated with anti‐p‐CREB‐1 (10E9) antibody (sc‐81,486; Santa Cruz Biotechnology), anti‐normal mouse IgG (sc‐2025; Santa Cruz Biotechnology) as a negative control overnight at 4°C. Chromatin protein/DNA complexes were eluted from the agarose beads by adding 100 μL of elution buffer at room temperature and heated to 65°C for 4 h. After reverse cross‐linking and DNA purification, immunoprecipitated DNA was quantified by real time PCR using power SYBR green (Catalog no. 4367659; Applied Biosystems) with primers for CREB binding sites in the Pgc1α4 promoter. Fold enrichment was calculated based on the threshold cycle (CT) value of the IgG control using the comparative CT method. Primers used in this assay are listed in Table S2.

Following antibodies were used at a dilution of 1:1000 except where otherwise stated: Anti-Actin (sc-1616 HRP; 1:10,000), anti-ATP7A (sc-376467; 1:100), anti-normal mouse IgG (sc-2025; 1.4 µg for IP), anti-ERK (sc-514302) from Santa Cruz; anti-GFP (2555), anti-OTULIN (14127; 1.4 µg for IP), anti-IκBα (4814), anti-phospho-IκBα (Ser32/36) (9246), anti-LAMP1 (15665; 1:100), anti-phospho-ERK (Thr202/Tyr204) (9101), anti-normal rabbit IgG (2729; 1.4 µg for IP), anti-EEA1 (3288, 1:100) all CST; anti-SNX27 (ab77799; 1.4 µg for IP and 1:50 for IF), anti-VPS26 (ab23892), anti-VPS35 (ab157220; 1:10,000), anti-MRP4 (ab15602; 1:2500), anti-DGKζ (ab105195; 1:100), anti-GLUT1 (ab15309; 1:100 and ab115730) all Abcam; anti-Beta PIX (07-1450-I; 1:5000), anti-Met1-Ub (MABS199) all Millipore; anti-STEAP3 (17186-1-AP), anti-KIDINS220 (21856-1-AP) all Proteintech; anti-SNX27 (for murine SNX27) gift from W. Hong; anti-Beta-catenin (610153, 1:2000) BD; anti-FLAG M2 (F3165; 1:10000 and 1 µg for IP) Sigma; anti-HOIP (MAB8039; 1:5000) R&D; anti-HA (Core facility monoclonal antibodies HMGU; 25 µl for IP); anti-CD2-APC (17-0029-41; 1:200) eBioscience.