Western Blot Protein Quantification and Analysis

As previously described [22 (link)], cells were washed twice with PBS and solubilized with radio immunoprecipitation assay (RIPA) buffer (Vazyme Biotec Co., LTD, Nanjing, China) supplemented with protease inhibitor (Sigma). Lysates were quantitated with bicinchoninic acid (BCA) kit (Vazyme Biotec Co., LTD); equivalent amounts of protein were subjected to electrophoresis, and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). Blocking with 5% dried skimmed milk in TBST (Tris Buffered Saline and 0.1% Tween 20) for 2 h at room temperature, the membranes were incubated with different primary antibodies, including KLF13 (#41724, SAB, Maryland, MD, USA), FASN (sc-20140 AC, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), FABP4 (sc-18661, Santa Cruz Biotechnology, Inc.), PPARγ (#2435, CST, Cell Signaling Technology, Inc., Danvers, MA, USA), and β-tubulin (KM9003, Sungene, Tianjin, China), at 4 °C overnight. After washing, the membrane was incubated with HRP-conjugated secondary antibodies for 1 h at room temperature and developed with ChemiDoc^TM XRS + Chem iluminescence detection system (Bio-Rad, Hercules, CA, USA). Image Lab5.2 was used for densitometric analysis of the expressed protein bands.