Super ecl plus

The brain tissue of mice was lysed with lysis buffer (Solarbio, whole protein extraction kit, cat. no. BC3710) to extract total protein. The protein concentration was determined by using a BCA assay (NCM biotech, BCA protein assay kit, cat. no. WB6501). After treatment with protein loading buffer, a 10% PAGE precast gel was used for protein electrophoresis. Subsequently, the protein was transferred to a PVDF membrane (Millipore, IPVH00010). After blocking with 7% fat-free milk at room temperature for 1 h, the membrane was incubated with primary antibody overnight at 4°C. The primary antibodies used in this study were OPN (1:1,000, abcam, cat. no. ab8448) and β-actin (1:1,000, Bioss, cat. no. bs-0061R). After washing 5 times with TBST containing 0.01% Tween-20 at room temperature (3 times for 5 min, 2 times for 10 min), the blot was incubated with goat anti-rabbit IgG horseradish peroxidase (1:10,000, ZSGB-BIO, cat. no. ZB-2301) at room temperature for 1 h. After washing again with TBST, the immunoblots was visualized with a chemiluminescence reagent (APPLYGEN, Super ECL Plus, cat. no. P1050), and the gray value of the immunoblots was semi-quantified using ImageJ software.

Protein samples (50 µg) were solubilized in 2×SDS gel-loading buffer. The proteins were separated using 10% SDS-PAGE and electrotransferred to a PVDF membrane (Millipore, Billerica, MA, USA). The membranes were blocked in TBST buffer (TBS plus 0.1% Tween-20) with 5% w/v skimmed milk and incubated with primary antibodies (rabbit polyclonal antibodies) to PPARγ, DNMT1 (Abcam), Akt, phospho-Akt (Ser473) (Cell Signaling Technology, Beverly, MA, USA) and mouse polyclonal antibodies to β-actin (Santa Cruz Biotechnology Inc., California, USA). This was followed by an incubation with a specific HRP-conjugated secondary antibody (1∶2000, Zhongshan-Bio). Super ECL plus (ApplyGEN, Beijing, China) was used to measure protein bands. β-actin was used as a reference protein, and the results were analyzed with Bandscan version 4.3 software.

The total protein was extracted from transfected HEK 293 T cells, blank HEK 293 T cells and skin sample from mouse using RIPA cell lysis solution (Applygen Technologies Inc., China), while blank cells and the mouse skin sample were used as a positive control. The samples were separated on a 10% SDS-PAGE gel (30 μg protein per sample) and transferred onto nitrocellulose membranes (Bio-Rad, USA). After blocking with 5% skimmed milk powder solution for 1 h at 37 °C, the membranes were incubated overnight at 4 °C with a murine polyclonal rabbit-antibody against HOXC13 and GAPDH (Signalway Antibody, USA), then incubated with a HRP conjugated secondary goat-anti-rabbit IgG (Zhongshan-Bio, China). The protein bands were visualized with Super ECL Plus (ApplyGEN, China). And GAPDH was taken as a reference control.

Heart tissues were homogenized on ice in RIPA buffer with a protease inhibitor (Complete, Roche). After centrifugation for 20 min at 4°C, the supernatant was used for Western blot analysis. Protein concentration was measured by BCA method (Bradford, 1976). 40–100 μg proteins were loaded for Western blot analysis. The protein extract was transferred in sample buffer, loaded on 10% SDS-polyacrylamide gel, and blotted onto a nitrocellulose membrane with a wet blotting system. After being blocked for 1 h in Tris-buffered saline/Tween 20 (TBST) with 5% nonfat milk, the membranes were incubated with primary antibodies in blocking buffer (1 : 1000) at 4°C overnight. Then the membranes were incubated with peroxidase conjugated secondary antibody at a 1 : 6000 dilution at room temperature for 1 h. After being rinsed with TBST for 3 times, the proteins were detected by autoradiography on enhanced chemiluminescence (Super ECL Plus; Applygen Technologies, Beijing, China). Semiquantifications were performed with densitometric analysis using NIH ImageJ software.

The liver and ileal samples were ground and lysed using a high-efficiency RIPA lysis buffer (Beijing Solarbio Science & Technology Co Ltd., Beijing, China) followed by high-speed centrifugation to extract total tissue proteins. The BCA protein assay kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) was used to detect protein content and made the sample solution (50 μg/20 μL) for subsequent tests. To separate the protein, 8–15% SDS-PAGE was used (10–50 μg per well), which was then transferred to a PVDF membrane. Later, the membranes were blocked using the 1% BSA blocking buffer (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) for 2 h, followed by 2 h of incubation using the respective primary antibodies and another 1 h incubation using HR-conjugated secondary antibodies (Table S2). Besides, the ECL luminescence reagent was used in the measurement. The expression of target proteins was quantitatively analyzed using the image analysis system (Super ECL Plus, Applygen, Beijing, China). β-actin or VDAC were the reference proteins used to normalize the result of target proteins. At last, the digital quantification of protein signals and normalization was conducted based on β-actin or VDAC [25 (link)]. Each densitometry value was normalized according to VDAC or β-actin and expressed as the level relative to the CON value.

After scrapping, the lung tissue was lysed in RIPA lysis buffer for 30 min to isolate total protein. The lysate was transferred into an EP tube and centrifuged for 5 min at 12,000 rpm/min. Total protein content was assessed by BCA assay kit (Thermo Fisher Scientific). The protein samples were separated on 12% sodium dodecyl sulfate Tris-HCl gels according to their molecular weight, and then immobilized onto PVDF membranes via electroblotting. After blocking with 5% skimmed milk in TBS containing 0.1% Tween (TBST) for 1 h, the membrane was exposed to the primary antibody against p53 (Immunoway, YT3528), Bcl-2 (Biorbyt, orb10173), Bax (Proteintech, 50599-2-Ig), cytochrome C (immunoway, YM3402), caspase-3 (Proteintech, 19677-1-AP), caspase-9 (Proteintech, 66169-1-AP), VDAC (Abcepta, AP6627A) or GAPDH (GOOD HERE, AB-P-R 001) at 4°C for 24 h. After rinsing in TBST for five times (each for 5 min), the membrane was exposed to the corresponding secondary antibody for 2 h at 37°C. Super ECL Plus (Applygen Technologies, P1050) was added onto the membranes to visualize the protein bands. The intensity of each band was measured by ImageJ software.

Whole cell lysates were generated conventionally using RIPA lysis buffer (cat. no. P0013B, Beyotime Biotechnology, Co., Ltd.). Next, 40 μg protein samples were separated by 12.5% SDS‐PAGE gels and blotted to polyvinylidene difluoride membranes (PVDF; Millipore; Merck KGaA). After blocked with skim milk (5%), PVDF membranes were incubated with primary antibodies at 4°C overnight and with secondary antibodies at room temperature for 1 h. Protein bands were visualized with Super ECL Plus (Applygen Technologies, Inc.) and scanned with a camera (Leica ICC50W, Leica Biosystems Co., Ltd.). Finally, the Image J software (version 1.51j8; National Institutes of Health) was utilized to conduct the densitometric analysis. The antibodies used in this study are listed in Table S3.