Chemidoc eq system

Total proteins were extracted from tissues or cells using RIPA lysis buffer (Solarbio, China) containing protease inhibitor and then denatured at 100 °C for 10 min. Equal amounts of proteins in each group were separated by 10% SDS-PAGE. After PVDF membrane transfer, blocking in 5% skim milk and incubation with primary antibodies (MMP3, CST, USA; MMP9, CST, USA; Wnt2, Abcam, USA; β-actin, Santa Cruz, USA) and secondary antibody, respectively, the immunoreactive bands on the membrane were incubated with ECL kit and detected by using the Chemidoc EQ system (BioRad, USA).

Total proteins were extracted from homogenate of olfactory epithelium (n=8), as well as from arm, optic lobe, sub-supraoesophageal masses, and quantified by Bradford Protein Assay, using a BSA standard, according to manufacturer's instructions (Bio-Rad Laboratories, Inc., Hercules, CA). After 10% sodium dodecylsulphate (SDS)-polyacrylamide gel electrophoresis, proteins were transferred on nitrocellulose membrane (Whatman) and incubated for 30 min in a blocking solution (non-fat milk 5% in PBS). Membranes were incubated in antibody solution (1:1000 anti-OMP in non-fat milk 5%) at 4°C overnight. After several rinses with PBS-T (PBS with 0.1% of Tween 20), membranes were incubated with secondary antibodies (1:5000) for 1 h at room temperature. Immunopositive band was visualized using the SuperSignal West Pico Chemiluminescent Substrate in accordance with the manufacturer's instructions (Pierce Biotechnology, Inc., Rockford, IL, USA) using a Chemidoc EQ System (Bio-Rad).

To determine the protein expression levels, the ES2 and OV90 cells were treated with 0, 0.25, 0.5, or 1 mM 4-MU and lysed with lysis buffer containing Triton X-100 (catalog number: X100; Sigma-Aldrich). The Bradford protein assay (Bio-Rad, Hercules, CA, USA) was performed to measure the protein concentration in whole-cell lysates. After denaturation, proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Each membrane was, then, incubated with primary antibodies (1:1000). An enhanced chemiluminescence detection solution (Super Signal West Pico; Pierce, Rockford, IL, USA) was used to develop the blots and a ChemiDoc EQ system and Quantity One software (Bio-Rad) were used to detect and quantify the chemiluminescence intensity of the protein bands. The intensity of the target proteins in the blots was normalized using the intensity of total proteins or that of α-tubulin (TUBA). Data from three independent experiments were analyzed according to the statistical analysis methods described below.

The protein concentrations in whole-cell extracts were determined using the Bradford Protein Assay (Bio-Rad, Hercules, CA, USA) with BSA as the standard. Proteins were denatured, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membranes. Blots were developed using enhanced chemiluminescence detection and quantified by measuring the intensity of light emitted from correctly sized bands under ultraviolet light using a ChemiDoc EQ system and Quantity One software (Bio-Rad). Immunoreactive proteins were detected using goat anti-rabbit polyclonal antibodies against phosphorylated and total proteins at a 1:1000 dilution and separated by 10% SDS-PAGE. As a loading control, total protein and α-tubulin (TUBA) were used to normalize results for the detection of target proteins. Multiple exposures of each western blot were used to ensure the linearity of chemiluminescent signals.

The protein was extracted from each whole cell and the concentration determined via a Bradford assay (Bio-Red, Hercules, CA, USA) with BSA as the standard. The proteins were denatured and isolated via 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a nitrocellulose membrane. Primary and secondary antibodies for each protein were dispensed in serial order and measured using a ChemiDoc EQ system and Quantity One software (Bio-Rad). The results were measured using three cell culture plates, and changes in phosphorylation were indicated in a bar graph. The assay was performed as described in a previous study [18 (link)].

Huh7 and Hep3B cells were treated with increasing doses of brassinin (0, 20, 50 and 100 µM). To measure the protein concentration, a Bradford protein assay (Bio-Rad, Hercules, CA, USA) was performed. The denatured proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Light intensity of the whole blots was measured using a ChemiDoc EQ system and Quantity One software (Bio-Rad) as previously described [16 (link)].