Benchmark pre stained protein standard

Western blot analysis was performed as previously described [46 (link)]. Cells were collected and lysed in RIPA buffer (Solarbio, China) plus phenylmethysulfonyl fluoride (PMSF) on ice for 30 min, followed by centrifugation at 12000 g for 20 min. The protein concentration in the extract was measured with a BCA Protein Assay Kit (Pierce, USA). After boiling for 10 min to denature the protein, the samples were diluted in 4× loading buffer (Beyotime, China) and loaded onto SDS-PAGE (10%) gels for electrophoresis and transferred to PVDF membranes (Millipore, USA). BenchMark Pre-Stained Protein Standard (Invitrogen, USA) was used as the size marker. The membranes were blocked with TBST containing 5% non-fat milk for 1 hour, followed by immunoblotting with primary antibodies at 4°C overnight. After incubation with HRP-conjugated secondary antibody (Zhongshan Goldenbridge Biotechnology Company, China) for 1 h, the protein bands were evaluated using enhanced chemiluminescence (Millipore, USA) and detected with a LAS-4000 MINI System (GE, USA). β-Actin or β-tubulin was used as an endogenous control. All the experiments were repeated three times. Antibodies for VRK1 (1:100000), c-Jun (1:1000), phospho-c-Jun (Ser63) (1:5000), c-MYC (1:10000), β-actin (1:1000) and β-tubulin (1:1000) were purchased from Abcam.

Hind paw extracts were prepared by homogenization in RIPA lysis buffer (0.1% SDS, 1% Igepal CA-630, 1% sodium deoxycholate, 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 μg/mL aprotinin, 1 μg/mL leupeptin, 100 μg/mL PMSF, 0.5 mM EDTA) containing a cocktail of protease and phosphatase inhibitors. Protein concentration was determined by Bradford assay, and 30 μg of protein was loaded for each sample in SDS-page gel 10%. Proteins were transferred to a nitrocellulose membrane and blocked with 5% BSA for 1 h at 4 °C. Following washes in TBS-T, membranes were incubated overnight at 4 °C with antibodies for AKT1 and AKT2 (total/phospho 1:1000, rabbit; Cell Signaling cat# 2964S and 8599S, respectively). To detect the bands of interest, we used the Benchmark Pre-Stained Protein Standard (Invitrogen cat # 10748-010). Normalization was carried out with β-actin (1:5000, mouse; Abcam cat# ab6276). Quantification of gels was performed using the Image-J software. The bands that are presented in the results proceeded from cropping and merging bands from the same original images.