Pan kcr

Total protein (30 μg) was extracted from iWAT or adipocytes, separated by SDS-PAGE and transferred to PVDF nitrocellulose membrane (Millipore, Boston, MA, USA). After being blocked with 5% nonfat milk for 2 h at room temperature, the primary antibody was incubated for 2 h at room temperature. The antibodies used in the WB process are: Pan-KCr (PTM BIO, Hangzhou, China), FLAG (Abways, Shanghai, China), UCP1 (Beyotime, Shanghai, China), PRDM16 (Beyotime, Shanghai, China), ATLGL (Beyotime, Shanghai, China), FASN (Abways, Shanghai, China), FABP4 (Beyotime, Shanghai, China), GPD1 (Abcam, Shanghai, China), NDUFA8 (Abways, Shanghai, China), TPI1 (Abways, Shanghai, China), AK2 (Abways, Shanghai, China), GAPDH (Abways, Shanghai, China). Mouse or rabbit HRP-conjugated secondary antibodies (Baoshen, Beijing, China) were added and incubated for 1 h at room temperature. Proteins were visualized using chemiluminescent peroxidase substrate (Millipore, Boston, MA, USA) and the ChemiDoc XRS system (Bio-Rad, Hercules, CA, USA), and the blots were quantitatively analyzed using ImageJ (ij142-jdk6-setup) software.

HeLa S3 and 293T cells were cultured in Dulbecco’s Modified Eagle’s medium (GIBCO, Thermo Fisher, Waltham, MA, USA) with 10% fetal bovine serum (GIBCO) at 37 °C in a 5% CO₂ in air atmosphere. The following antibodies were used in this study: pan-Kac (PTM-Biolabs 101, Shanghai, China), pan-Kcr (PTM-Biolabs 501), pan-Kpr (PTM-Biolabs 201), H3K9cr (PTM-Biolabs 516), H3K18cr (PTM-Biolabs 517), p300 (Carlsbad, CA, USA) (Active Motif 61401).
H3 (Epitomics M1309-1, Hangzhou, China), Flag (Sigma 7425/1804, St Louis, MO, USA), and Myc (Abmart 20002 mouse, Shanghai, China). CBP rabbit polyclone antibody was homemade. CBP/p300 inhibitor C646 was from Sigma (SML0002) and was used in final concentration 20 or 40 μm. TGF-β1 was added to a final concentration of 2 ng ml⁻¹ overnight after cultured HEK293T cells 16 h in no fetal bovine serum Dulbecco’s Modified Eagle’s medium (GIBCO). The pCMV-Flag-CBP, pCMV- p300-Myc, pCMV-Flag-TIP60, pCMV-Flag-PCAF, pcDNA3.1-Flag-HBO1, pcDNA3.1-Flag-GCN5 and pcDNA3.1-MOF were either as described or constructed by our lab [45 (link)]. All mutants were generated by PCR-based point mutagenesis strategy and verified by DNA sequencing. Lipofectamine 2000 (Thermo Fisher) was used for the transfection of nucleic acids (DNA and RNA) into eukaryotic cells according to manufacturer’s instructions.