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The RIPA kit (Beyotime, Shanghai, China) supplemented with phenylmethanesulfonyl fluoride was used to extract protein lysate from CRC tissues and cells according to the protocols. The BCA Protein Assay Kit (Beyotime, Shanghai, China) was used to quantify the protein concentration. The protein lysate was separated via 10% SDS-PAGE (Beyotime, Shanghai, China), next, it was transferred onto a polyvinylidene fluoride membrane (Millipore, USA). The membranes were blocked with 5% skim milk powder for 2 h and then incubated with the specific primary antibody overnight at 4°C, followed by a wash in TBST. Then, the membrane was incubated with the corresponding secondary antibody for 2 h at room temperature and then rinsed in TBST for three times (10 min each time). The level of protein expression was detected by ECL Plus (Millipore, USA) using a Bio-Imaging System (Bio-Rad, USA). The antibodies against SP1, IκBα, p- IκBα, p65, p-p65, E-cadherin, ZO-1, N-cadherin, Vimentin, HIF-1α, Histone H3, and β-actin were purchased from Abcam (Cambridge, MA, USA).

Whole protein was extracted from cardiac tissue (KeyGen Biotech, China) and quantified using a BCA assay (BioTeke Biotechnology, China). Total protein (30 μg per lane) from the homogenate of isolated ventricular myocytes was separated by 10% SDS-PAGE (Beyotime Biotechnology, China) and transferred to a PDVF membrane. The PVDF membrane was blocked with T-TBS containing 5% BSA (Sigma, USA) at room temperature for 1 hour and then incubated with rabbit anti-phospho-CaMKII (1 : 1000 dilution, Cat.ab182647, Abcam, USA), rabbit anti-oxidized-CaMKII (1 : 1000 dilution, Cat. # 07-1387, Millipore, USA), rabbit anti-CaMKII (1 : 1000 dilution, Cat.ab181052, Abcam, USA), mouse anti-PLN (1 : 1000 dilution, Cat.ab2865, Abcam, USA), rabbit anti-phospho-PLN (1 : 1000 dilution, Cat.ab15000, Abcam, USA), rabbit anti-phospho-RyR2 (1 : 1000 dilution, Cat.LS-C358303, LSBio, USA), mouse anti-RyR2 (1 : 1000 dilution, Cat.ab2827, Abcam, USA), and mouse anti-GAPDH (1 : 5000 dilution, Cat.ARG10112, Arigo, Taiwan) antibodies overnight at 4°C. The membranes were then washed with Tris-buffered saline-Tween (0.05%) solution and incubated with HRP-conjugated secondary antibody (1 : 5000 dilution, Lianke, China) for 1 hour. The membranes were developed using a super ECL assay kit (KeyGen Biotech, China) and a G-BOX imaging system (Syngene, UK).

The whole cell protein was extracted using RIPA buffer combined with protease inhibitor (Merck Millipore). The protein concentration was determined by the Bradford method (Beyotime, Shanghai, China). Protein was separated by 10% SDS/PAGE (Beyotime) and transferred to polyvinylidene fluoride membrane (Merck Millipore). The members were blocked in 5% skim milk with TBST for 1 h at room temperature. Then, the primary antibodies were incubated at 4 °C overnight and secondary antibodies for 1 h at room temperature. The primary antibodies used were mouse anti-GAPDH (Sigma, USA), rabbit anti-CXCR4 and rabbit anti-CXCR12 (Proteintech, Rosemont, IL, USA).

After cells were lysed by RIPA buffer (Beyotime) added with PMSF (Beyotime), protein concentration was identified by the BCA method kit (Solarbio). Protein samples were collected using 10% SDS-PAGE (Beyotime) and transferred to the polyvinylidene fluoride membranes (PVDF, Millipore). After blocked with 5% skim milk for 1 h, the membrane was incubated with primary antibodies overnight at 4°C and then with the corresponding horse radish peroxidase (HRP)-conjugated secondary antibody (1:2000 dilution) for 1 h at RT. Finally, enhanced chemiluminescence substrate (ECL kit, Millipore) were used for visualization.

Denatured proteins from PCa cells were resolved by 10% SDS-PAGE (Beyotime). The separated bands were subsequently transferred to PVDF membranes and incubated (12 h; 4°C) with PI3K (1 : 1200, ab191606), AKT (1 : 500, ab8805), p-PI3K (1 : 500, ab182651), p-AKT (1 : 500, ab38449), CDC6 (1 : 1,000, ab109315), and GAPDH (1 : 3000, ab8245) primary antibodies. They were then incubated (2 h, 25°C) with goat anti-rabbit antibody (1 : 12,000, ab205718). Antibodies were obtained from Abcam (Cambridge, UK).

The whole cell protein was extracted using RIPA buffer combined with protease inhibitor (Merck Millipore). The protein concentration was determined by the Bradford method (Beyotime, Shanghai, China), and 20 μg of protein was separated by 10% SDS/PAGE (Beyotime) and transferred to polyvinylidene fluoride membrane (Merck Millipore). Then, the primary antibodies were incubated at 4 °C overnight and secondary antibodies for 1 h at room temperature. The primary antibodies used were mouse anti‐β‐actin (Sigma, USA), rabbit anti‐SIX1 (Proteintech, Rosemont, IL, USA), PCNA, c‐myc, cyclin‐D1 and cyclin‐A1 (Cell Signaling Technology, USA).