Mouse anti human β actin

Western blot analysis was performed essentially as described [19 (link)]. The method used to normalize the protein levels was “Pierce BCA Protein Assay Kit” (Thermo Fisher Scientific, Waltham, MA, USA). Cell lysates (15 μg total protein per lane) were prepared in sodium dodecyl sulfate (SDS) sample buffer, separated by SDS-PAGE, and blotted on polyvinylidene difluoride membranes. The following antibodies were used: mouse anti-human β-actin (Proteintech, IL, USA), perilipin (Cell Signaling, MA, USA), phosphor-P70S6K (Cell Signaling, MA, USA), total P70S6K (Cell Signaling, MA, USA), pERK1/2 (Cell Signaling, MA, USA), total ERK1/2 (Cell Signaling, MA, USA), anti-mouse IgG HRP conjugate (Proteintech, IL, USA), and rabbit anti-rat IgG HRP (Proteintech, IL, USA). Image J software was used for densitometric analyses.

Western blot analysis was conducted following a similar procedure as described previously [21 (link)]. The protein levels were normalized using the “Pierce BCA Protein Assay Kit” from Thermo Fisher Scientific, Waltham, MA, USA. Cell lysates containing 15 μg of total protein per lane were prepared using a sodium dodecyl sulfate (SDS) sample buffer. Subsequently, the samples were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto polyvinylidene difluoride membranes. The membranes were then probed with the following antibodies: mouse anti-human β-actin (Proteintech, IL, USA), perilipin (Cell Signaling, MA, USA), and rabbit anti-rat IgG HRP (Proteintech, IL, USA). Densitometric analyses of the blots were performed using ImageJ software.

Total protein was isolated by using a RIPA lysis buffer (Beyotime Institute of Biotechnology), separated on SDS-PAGE gels, and transferred to PVDF membranes (EMD Millipore, Billerica, MA, USA). Membranes were blocked by 5% nonfat milk in Tris-buffered saline with Tween-20, then incubated with primary Abs overnight at 4 °C and with corresponding secondary Abs (1:4000; Catalogue No: SA00001-1 and SA00001-2; ProteinTech, Chicago, IL, USA). The following primary Abs were used: rabbit anti-human KAT2A (1:1000; Catalogue No: 3305 T; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-human AR (1:1000; Catalogue No: 5153 T; Cell Signaling Technology), rabbit anti-human PSA (1:1000; Catalogue No: 10679-1-AP; ProteinTech), rabbit anti-human acetylated lysine (1:1000; Catalogue No: 9441 S; Cell Signaling Technology), rabbit anti-human His tag (1:500; Catalogue No: 10001-0-AP; ProteinTech), rabbit anti-human Flag tag (1:1000; Catalogue No: 20543-1-AP; ProteinTech). Mouse anti-human β-actin (1:5000; Catalogue No: 66009-1-Ig; ProteinTech) and rabbit anti-human lamin B1 (1:2000; Catalogue No: 12987-1-AP; ProteinTech) were used as loading controls. Protein bands were visualized with ECL (Beyotime Institute of Biotechnology).