Ascl1

Western blot analysis was performed as described previously
[36 (link)]. Whole cell lysates were analyzed with the following antibodies: FASN, ASCL1 (Cell signaling), SREBP1c, PLA2G3, HSulf-1 (Abcam, AB96533), CPT1A, HSL, DAGLA, β-tubulin (GeneTex) and β-actin (Sigma-Aldrich).

White adipose tissue was homogenized in homogenization buffer (150mM NaCl, 10mM Tris-HCl, 2mM EDTA, pH 7,4) and liver tissue in RIPA-buffer (Radioimmunoprecipitation assay buffer 150 mM NaCl, 2mM EDTA, 0,35% Na-deoxycholate, 0,50% Nonidet-40, 0,10% SDS both buffers were supplied with complete protease inhibitor (Sigma-Aldrich, Merk KGaA, Darmstadt, Germany) and phosphoSTOP (Sigma-Aldrich) using a Teflon douncer or a bead beater. Samples were incubated on ice for 30 min and further centrifuged at 10 000xg for 10 min at 4°C and the supernatants were collected.
Total protein concentration was determined using Bradford assay (BioRad). Total proteins were separated using 10 or 12% gradient SDS-polyacrylamide gel (BioRad) under denaturing conditions. Proteins were transferred onto nitrocellulose filters (Nitropure), and blots were probed with antibodies against phosphorylated and total HSL, GAPDH, ASCL1 (Cell Signaling Technology, Inc, Danvers, MA, USA) and FATP2 (abcam, Cambridge, UK), followed by detection by IRDye^TM 800 anti-Goat IgG and IRDye^TM 680 anti-Rabbit IgG antibodies using The Odyssey^® Imaging System (LICOR).

ASCL1 (#43666), BAP1 (#13271S), H3K27ac (#8173S), H3K4me1 (#5326S), H3K4me3 (#9751), H3K27me3 (#9733), H2AK119Ub (#8240), ASXL2 (#71257), and RING1B (#5694) were purchased from Cell Signaling Technology (CST) company. HSP90 (sc-7947) was purchased from Santa Cruz. Tubulin antibody (E7) was purchased from Developmental Studies Hybridoma Bank. FOXK1 (A301-727A), FOXK2 (A301-729A), and HCFC1 (A301-399A) antibodies were purchased from Bethyl-Laboratories. ASXL1 and ASXL3 antibodies were generated as previously described [11 (link)]. EZH2 inhibitor GSK126 was purchased from Cayman (15415).