Hrp labeled goat anti rabbit secondary antibody

After the experiment, the rats were sacrificed immediately, the heart was taken, and the left ventricle was separated. After an appropriate amount of RIPA lysis buffer, 1% phosphatase inhibitor and 1% protease inhibitor were added, and the tissues were smashed using the ultrasound homogenizer and centrifuged at 12,000 rpm and 4°C for 10 min. The supernatant was taken as the total protein, and its concentration was determined. After inactivation via boiling, the protein was subjected to electrophoresis and transferred onto a PVDF membrane. Then, the target band was cut according to the molecular weight of protein, sealed with freshly-prepared 5% skim milk powder for 2 h, and incubated with the B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), cleaved Caspase-3, phosphorylated ERK (p-ERK), ERK, NF-κB, and GAPDH antibodies (1 : 1000, Cell Signaling Technology, USA) at 4°C overnight. After the band was washed with TBST for 3 times, it was incubated again with the HRP-labeled goat anti-rabbit secondary antibodies (1 : 5000, Boster Biological Technology Co., Ltd.) at room temperature for 1 h and washed again with TBST for 3 times. Then, the mixed ECL solution was added for image development and exposure. After scanning, the protein expression level in each group was analyzed using the ImageJ software.

Total protein in MC3T3-E1 cells was extracted, and the concentrations of protein were detected by a BCA kit (Beyotime). 30 µg of each sample was subjected to 10–12% SDS-PAGE for separation, and the blots were then transferred onto PVDF membranes. After blocking with 5% skim milk in TBS for 1 h, the primary antibodies, including anti-Bcl-2 (1:1000 dilution; Affinity), anti-cleaved caspase 3 (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), anti-p65 (1:1000 dilution; Affinity), anti-p-p65 (1:1000 dilution; Affinity), anti-NRF2 (1:1000 dilution; Affinity), anti-HO-1 (1:1000 dilution; Beyotime), and anti-β-actin (1:1000 dilution; Solarbio), were incubated with the membranes at 4 °C overnight. HRP-labeled goat anti-rabbit secondary antibodies (1:5000 dilution; Boster, Wuhan, China) were added at room temperature for 1 h. Finally, protein bands were measured by the enhanced chemiluminescence detection system and evaluated by ImageJ.

Western blotting analysis was performed with standard techniques, as described previously [3 (link)]. Cell proteins were extracted by a modified RIPA buffer containing 0.5% sodium dodecyl sulfate (SDS) in the presence of a proteinase inhibitor cocktail (Roche, IN, USA). Polyacrylamide gel electrophoresis (PAGE) was performed to separate cell lysate proteins and then fractionated proteins were transferred onto a PVDF membrane (Amersham Biosciences, NJ, USA). Immonodetection was performed using antibodies including rabbit anti-FOXM1 polyclonal antibody, anti-cyclinD1, anti-p21, anti-LEF1, anti-c-myc, anti-Rb, anti- phosphorylated –Rb, and β-actin antibodies (Cell Signaling Technology, Danvers, MA, USA) at the dilution ratio of 1:1000. The membrane was then incubated with HRP labeled goat anti-rabbit secondary antibody (BosterBio, CA, USA) at the dilution ratio of 1:6000. Anti-β-actin (Cell Signaling Technology, Danvers, MA, USA) served as an internal control. Signals were detected by exposure to films with SuperSignal West Pico Chemoluminescent substrate (Thermo Fisher Scientific, MA, USA).