miR-1306 Mediates the Feedback Regulation of the TGF-ß/SMAD Signaling Pathway in Granulosa Cells

Yang, Liu; Du, Xing; Liu, Lu; Cao, Qiuyu; Pan, Zengxiang; Li, Qifa

Porcine GCs were lysed in radioimmunoprecipitation assay (RIPA) (Bioworld, Nanjing, China) containing 1% phosphatase inhibitor (v/v). The protein concentration was determined by the BCA Protein Assay Kit (Beyotime, Shanghai, China) and diluted to the same concentration using the 5 × Protein Loading Dye (Sangon, Shanghai, China). Total protein extracts were separated on using SDS-PAGE on 12% gels and electrophoresed for 1 h. Following that, the total protein was transferred onto a PVDF membrane (Millipore, Billerica, MA, USA) and the membrane was blocked with 5% non-fat milk for 2 h. After washing with tris buffered saline tween (TBST) for 15 s, the membrane was incubated with primary antibodies (1:1000 dilution) at 4 • C for 12 h. After that, the membrane was washed thrice for 10 min each time using TBST and incubated with the appropriate secondary antibodies (1:2000 dilution). Chemiluminescence was detected by WesternBright TM BCL (Advansta, Menlo Park, CA USA).