Ecl western blotting detection kit

The purified proteins were detected with western blot method (Das et al., 2018 (link)). The purified proteins were cut off acrylamide gels (SDS-PAGE) and transferred on the nitrocellulose membrane after electrophoresis. The membrane was then incubated with rabbit anti-6 ×His tag antibody (Genscript, Nanjing, China) at 37 °C for 2 h. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin (BoShiDe Biotechnology, Wuhan, China) was used as the secondary antibody. The hybrid membrane was washed with TBST buffer (pH 7.4), and detection was carried out with the ECL Western Blotting Detection kit (SW2010, Solarbio, Beijing).

Total proteins were extracted from 85–90% confluent BMSCs using a Total Protein Extraction Kit (Solarbio, Beijing, China), and the protein concentration was determined with a BCA protein assay kit (Solarbio, Beijing, China) according to the manufacturer's instructions. All protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Polyvinylidene fluoride (PVDF) membranes (Bio-Rad). All membranes were blocked by 5% non-fat milk for 1 h and incubated overnight at 4°C with the following primary antibodies (all from Abcam): β-actin (#ab8227, 1:2000 dilution), BMAL1 (#ab3350, 1:1500 dilution), p53 (#ab26, 1:1500 dilution), p21 (#ab80633, 1:2000 dilution), Runx2 (#ab23981, 1:1000 dilution), Osx (#ab22552, 1:1000 dilution), Ocn (#ab134220, 1:1000 dilution) and Alp (#ab83259, 1:1000 dilution). After washing with TBST three times, the membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody (1:2500, Solarbio, Beijing, China) and anti-mouse IgG antibody (1:2500, Solarbio, Beijing, China) for 1 h. β-actin was used as an internal control. Finally, the ECL Western Blotting Detection Kit (Solarbio, Beijing, China) and Western-Light Chemiluminescent Detection System (GE Healthcare, NY, USA) were used to acquire the protein bands.

Cells or fresh tumor samples were homogenized in RIPA Buffer (R0010, China) as per the manufacturer’s specification to extract total protein which then was quantified by BCA Protein Assay Kit (PC0020, China). Equal amounts of protein samples (15 μg/lane) were separated using 10% SDS-PAGE and loaded onto polyvinylidene difluoride membranes (0.45 μm, 88585, Thermo Fisher), followed by blocking with TBST-diluted 5% non-fat milk (GC310001, Servicebio, China). Primary antibodies against CES2 (ab184957, 62 kDa), IMPDH1 (ab33039, 55 kDa), IMPDH2 (ab129165, 56 kDa) and GAPDH (ab181603, 36 kDa) were incubated with the membranes at 4°C overnight. Next, the membranes were hybridized with HRP-conjugated secondary antibody (ab6721) at RT for 1 h. All antibodies used in Western blot were provided by Abcam (UK). After that, the stripped membranes were treated with ECL Western Blotting Detection Kit (SW2040, Solarbio, China), and immunoblots were detected by ChemiDoc XRS+ imaging system (Bio-Rad). Bandscan software (Bio-Rad) was exploited to analyze relative protein expression (represented as the ratio of the gray-scale value of the target band to that of the GAPDH band).

Proteins were extracted from HGFs using ice-cold radioimmunoprecipitation (RIPA) lysis buffer (Solarbio). After being quantified by BCA (Thermo Fisher Scientific), the protein samples were mixed with loading buffer (Solarbio), separated by electrophoresis on SDS-PAGE. The proteins in the gel were transferred on a polyvinylidene fluoride (PVDF) membrane (Beyotime). The membranes were blocked with 5% skimmed milk (Solarbio) and incubated overnight at 4°C with primary antibody. The membranes were washed with Tris-buffered saline and incubated with secondary antibody for 90 min at room temperature. The PVDF membranes were subjected to chemiluminescence detection using an ECL Western Blotting Detection Kit (Solarbio).