Polyacrylamide gel electrophoresis

The proteins from the renal tissues of mice were extracted and analyzed with a bicinchoninic acid kit (Boster Biological Technology Co., Ltd., Wuhan, Hubei, China). The extracted protein samples were separated using 10% polyacrylamide gel electrophoresis (Boster Biological Technology Co., Ltd., Wuhan, Hubei, China), followed by transferring onto a polyvinylidene fluoride membrane. The membrane was blocked using 5% bovine serum albumin (BSA) for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies to p-Skp2 (1: 1000, 14,865, Cell Signaling Technology, Beverly, MA, USA), Skp2 (1: 1000, ab183039, Abcam, Cambridge, UK), p-p27 (1: 1000, sc-12,939, Santa Cruz Biotech, Santa Cruz, CA, USA), p27 (1: 1000, ab193379, Abcam, Cambridge, UK) and β-actin (1: 1000, ab6276, Abcam, Cambridge, UK). Membranes were then washed and incubated with the secondary antibody for 1 h at room temperature and developed using chemiluminescent reagents. β-actin was regarded as the internal reference. The gray value of the target band was analyzed by Image J. Data are mean values from three independent experiments.

The cells were rinsed twice with phosphate-buffered saline (PBS) and then added with loading buffer for heating at 95°C for 10 min. The proteins were separated by 10% polyacrylamide gel electrophoresis (Boster Biological Technology Co., Ltd., Wuhan, Hubei, China) (40 μg/well) and then transferred onto polyvinylidene fluoride (PVDF) membranes using the wet transfer method. The membranes were blocked with 5% bovine serum albumin (BSA) at room temperature for 1 h and then incubated with primary antibodies diluted at 1: 500–1: 1000: Osteocalcin, (AM0911, Millipore, Billerica, MA, USA), Osterix, (ab22552, Abcam Inc., Cambridge, MA, USA) and ATF4 (abl05383, Abcam Inc., Cambridge, MA, USA) at 4°C overnight. After being rinsed three times (5 min/time) with Tris-buffered saline Tween-20 (TBST), the membranes were added with the corresponding secondary antibody (Abcam Inc., Cambridge, MA, USA) (Shanghai Precision & Scientific Instrument Co., Ltd., Shanghai, China) and incubated at room temperature for 1 h, followed by three washes with TBST (5 min/time). The immunocomplexes on the membrane were visualized using enhanced chemiluminescence (ECL) reagent (Shanghai Genmed Gene Pharmaceutical Technology Co., Ltd., Shanghai, China) and band intensities were quantified using a multifunctional imaging system.