Ripa buffer

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.

Animals were collected at indicated age and frozen in liquid nitrogen. Animals were lysed in protease inhibitor cocktail (Abcam, #ab65621) and 3× phosphatase inhibitor (Thermo Scientific, #78420)-supplemented RIPA buffer (bioWORLD) with a micropestle in 1.5-ml Eppendorf tubes. Lysates were resolved on 4–15% precast polyacrylamide gels (Bio-Rad). Phospho-p38 MAPK (used with 1:1000 dilution) was purchased from Cell Signaling Technology (#4511S) and α-tubulin antibody (used with 1:5000 dilution) from Merck (T5168). Secondary antibodies Goat Anti-Rabbit IgG H&L (HRP) (ab97051) and Goat Anti-Mouse IgG H&L (HRP) (ab97023) (used with 1:10,000 dilution) were purchased from Abcam. SuperSignal™ West Pico PLUS Chemiluminescent Substrate (#34577, Thermo Fisher Scientific) was used for protein detection. Western blots were quantified by using Fiji (see Supplementary Table S6 for Western blot quantifications).

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.

Twenty-four hours after treatments, cells were lysed with RIPA buffer (BioWorld, Dublin, OH, USA) containing phosphatase and protease inhibitor cocktails (Roche, Risch-Rotkreuz, Switzerland). The samples were adjusted to the same protein concentration (BCA protein assay kit, Pierce, Rockford, IL, USA) and denatured by boiling in Laemmli sample buffer with 5% β-mercaptoethanol. Then were subjected to electrophoresis separation in SDS-PAGE. Gels were transferred to a PVDF membrane using a Trans-Blot turbo transfer system (BioRad, Hercules, CA, USA) and membranes were blocked with 5% nonfat milk in Tris-buffered saline (TBS) with 0.1% Tween 20, for 2 h. After blocking, membranes were incubated overnight with specific primary antibodies and then with HRP-conjugated secondary antibodies (Supplementary Table 1) and developed by chemiluminiscence (ECL, Thermo Fisher Scientific Inc., Walthan MA USA) using a ChemiDoc system (BioRad, Hercules, CA, USA). Bands corresponding to the different proteins were quantified, digitalized and analyzed with Image Lab 2.0.1. software (BioRad, Hercules, CA, USA) and Adobe PhotoShop CS5 12.0 (Adobe Systems Inc., USA).