Gel dol ez imager

Total proteins from cells and tissues were extracted, and the concentrations were determined using a bicinchoninic acid kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s protocol. The extracted proteins were run on sodium dodecyl sulfate polyacrylamide gel electrophoresis with the voltage increasing from 80 V to 120 V. Then the proteins were transferred onto polyvinylidene fluoride membranes using the semi-dry method at 80 mV for 30–45 min. The membranes were incubated with 5% bovine serum albumin at room temperature for 1 h, and then incubated with the primary antibodies against OPG (1:1000, ab2302), receptor activator of nuclear factor kappa B (RANK, 1:1000, ab32370), RANK ligand (RANKL, 1:5000, ab32064) and β-actin (1:5000, ab227387) (all purchased from Abcam) at 4 °C overnight. Afterwards, the membranes were washed with tris-buffered saline tween (TBST) (3 × 5 min), and incubated with the corresponding secondary antibody horseradish peroxidase-labeled immunoglobulin G (ab6747, 1:10000, Abcam) at room temperature for 1 h, After 3 times of TBST washes (5 min for each), the bands were visualized using chemiluminescence reagent on a Bio-Rad Gel Dol EZ imager (Bio-Rad Laboratories, California, USA). The target band was analyzed by calculating the gray value using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA).

The cells from the three groups were digested with cell lysis buffer (0.1
mol/l NaCl, 0.01 mol/l Tris/HCl (pH 7.6), 0.001 mol/l
EDTA (pH 8.0), 1 μg/l aprotinin and 100 μg/l
phenylmethylsulphonyl fluoride) for 30 min and then centrifuged at 1200 rpm for 5 min
to collect the supernatant. The amount of proteins were determined by the Coomassie
Brilliant Blue method and an equal volume of sampling buffer was added. The resultant
was boiled for 5 min and then sampled at 100 μg per strip. Subsequently,
SDS/PAGE and Western blotting assay were performed. The Bio–Rad Gel Dol
EZ imager (Bio–Rad Laboratories, Hercules, CA, U.S.A.) was used for developing
and the ImageJ software was used to detect the grey value of the BMP-2 band.
β-actin served as the internal reference. The concentration of spacer gel was
4.5% and the concentration of separation gel was 10%. The protein
separated by electrophoresis was transformed into the PVDF membrane and 1:600 rhBMP-2
rabbit anti-human monoclonal antibody (Wuhan Boster Biological Technology Ltd.,
Wuhan, Hubei, China) was added into the blocked PVDF membrane. This was then stored
overnight at 4°C. Subsequently, the protein was incubated with 1:1000 goat
anti rabbit IgG (Wuhan Boster Biological Technology Ltd., Wuhan, Hubei, China),
labelled with horseradish peroxidase for 3 h, washed and developed in DAB
solution.

The concentration of the extracted protein was measured with the bicinchoninic acid (BCA) reagent kit (Beyotime Biotechnology Co., Shanghai, China). Equal amounts (30 μg) of total protein was mixed with SDS loading buffer and boiled for 10 min at 100°C and resolved on 10% SDS-PAGE for 30 min at 80V and then at 120V for 1h. Then, the proteins were wet transferred onto a PVDF membrane for 90 min at 100V. The membrane was blocked in 5% BSA (Beyotime Biotechnology Co., Shanghai, China) at room temperature for 1h, and then probed with primary antibodies against GSTM1 (ab113432, 1: 1000), 78 kDa glucose-regulated protein (GRP78) (ab173613, 1: 100), p-PERK (CST, #3179, 1: 1000), p-IRE1α (NB100-2323, 1: 1000) and C/EBP homology protein (CHOP) (ab10444, 1: 250), GAPDH (CST, #2118, 1: 1000) and superoxide dismutase (SOD) (ab13533, 1: 5000) overnight at 4°C. The membranes were then washed by TBST thrice for 5 mins and incubated with the corresponding secondary antibodies for 1h. The blots were then washed in TBST thrice for 5 min and developed with ECL chemiluminescence reagent (Nanjing Vazyme Biotech Co. Ltd, Nanjing, China) and imaged by the Bio-Rad Gel Dol EZ imager (GEL DOC EZ IMAGER, Bio-Rad, CA, USA). GAPDH was used as internal reference and the relative levels of different proteins were quantified.

Proteins from cells in each group were extracted to identify the protein concentration using a BCA kit (Boster, Wuhan). Loading buffer was then added to the proteins, which were boiled at 95 °C for 10 min. Then, 30 μg of loading buffer was added to each well, and the proteins were separated by a 10% polyacrylamide gel (Boster, Wuhan). The voltage for electrophoresis was increased from 80 V to 120 V, and the proteins were transferred to a membrane at 100 mV for 45–70 min. The membrane was transferred to PVDF, followed by blocking with 5% BSA at ambient temperature for 1 h. β-actin was considered as a control. Primary antibodies for BMP-2, Smad1, p-Smad1, Runx2, osteocalcin (OC), and osteopontin (OPN) (1: 1000, Abcam, America) and primary antibody for β-actin (1:3000, Abcam, America) were added and incubated overnight at 4 °C. The membrane was washed three times with TBST for 5 min each. Incubation with secondary goat anti-rat antibodies (Jackson, America) at ambient temperature was conducted. Then, the membrane was washed three times for 5 min each. The color of the membrane was developed using chemiluminescence reagents. A Bio-Rad Gel Dol EZ imager (GEL DOC EZ IMAGER, Bio-Rad, California, USA) was used for the color observation. ImageJ software was used to analyze the grayscale value of the target band. Experiments were conducted three times to obtain the average value.