Phenyl methyl sulfonyl fluoride (pmsf)

Porcine GCs were cultured in 6-well plates. RIPA buffer containing 1% PMSF (Bioworld, Nanjing, Jiangsu, China) was added to lyse the cells after 72 h treatment. A BCA determination kit (Beyotime, Shanghai, China) was applied to measure protein concentrations. Briefly, the BCA working solution and protein sample were mixed and reacted at 37 °C, then the complex was measured by Multiskan Go (Thermo, Boston, MA, USA) at 562 nm wavelength. Proteins (final concentration of 1–2 μg/uL,10–20 μg/lane) were separated on 15% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The antibodies used in this study were polyclonal anti-SRSF1 (1:1000 dilution, 12929-2-AP, Proteintech Group, Rosemont, IL, USA), polyclonal anti-tubulin (1:1000 dilution, #6181S, Cell Signaling Technology, Boston, MA, USA), polyclonal anti-Cleaved Caspase-3 (C-CASP3) (1:1000 dilution, #9664S, Cell Signaling Technology, Boston, MA, USA), and the secondary antibody (1:2000 dilution, SA00E1–2, Proteintech Group, Rosemont, IL, USA). Protein levels were detected by an imaging system (Image LAS-4000, Bio-Rad, Hercules, CA, USA) with ECL Plus reagent (Promega, Madison, WI, USA) and analyzed using ImageJ. The results were normalized with Tubulin. Each experiment was performed three times.

GCs were collected 48 h after transfection, and RIPA buffer (Bioworld, Nanjing, China) containing 1% PMSF (Bioworld, Nanjing, China) was added to lyse the cells. Protein concentration as determined by a BCA determination kit (Beyotime, Shanghai, China). Protein samples were separated by SDS-PAGE according to our previous description [33 (link)]. The antibodies used in this study were anti-tubulin (TUBB) (diluted 1:1000, #6181S, Cell Signaling Technology, Boston, MA, USA) and anti-Cleaved Caspase-3 (C-CASP3) (diluted 1:1000, #9664S, Cell Signaling Technology, Boston, MA, USA), and the secondary antibody (diluted 1:2000, SA00001-2, Proteintech Group, Rosemont, IL, USA). Protein levels were detected with an ECL Plus reagent (Promega, Madison, WI, USA) and analyzed using ImageJ software (v1.8.0). Each experiment was performed three times.