Erk1 2

Protein samples were extracted with lysis buffer. 15 μg protein samples from each group were loaded onto the SDS polyacrylamide gel for electrophoresis and transferred to NC membranes (Millipore, Billerica, MA, USA). After blocking in 5% non-fat milk at room temperature for 2 h, primary antibodies: NPRA (1:1000; Santa Cruz Biotechnology), NPRC (1:1000; Santa Cruz Biotechnology), PGRMC1 (1:1000; Cell Signaling), PGRMC2 (1:1000), Bax (1:2000; Proteintech), Bcl-2 (1:2000; Proteintech), Caspase 8 (1:1000; Proteintech), Caspase 9 (1:1000; Proteintech), PCNA (1:2000; Proteintech), EGFR (1:500; Proteintech), p-EGFR (1:500; Cell Signaling), ERK 1/2 (1:500; Beyotime Biotechnology), p-ERK 1/2 (1:500; Beyotime Biotechnology), p-c-Fos (1:500; Cell Signaling), c-Fos (1:500; Cell Signaling), p-c-Jun (1:500; Cell Signaling), c-Jun (1:500; Cell Signaling) and GAPDH (1:2000; Proteintech) were incubated at 4 °C overnight. Next day, secondary antibodies (1:2000; Cell Signaling) were incubated for 45 min at room temperature. ECL detection system was used to visualize the bands. All experiments were repeated at least three separate times.

Cell climbing sheets were prepared and fixed in 4% paraformaldehyde for 10 min at room temperature. Subsequently, permeabilization was performed with 0.5% Triton X-100 for 10 min at room temperature. Additionally, three washes were performed with PBS for 5 min each, and the solution blocked with 5% BSA for 2 h. Primary antibodies against Flag (Beyotime, Shanghai, China, 1:200), Nesprin2 (Abcam, Cambridge, UK, 1:200), ERK1/2 (Beyotime, 1:200), and pERK1/2 (Cell Signaling Technology, Danvers, MA, USA, 1:100) were incubated at 4 ℃ overnight. After washing with PBS three times, a fluorescent secondary antibody was used and incubated at room temperature for 1 h. The nuclei were stained with DAPI, blocked with glycerol, and finally observed under fluorescence microscope (Zeiss, Jena, Germany). Each process was repeated three times.

MC3T3-E1 cells were harvested and washed 3 times with ice-cold PBS and lysed with TEN-T buffer (150 mM NaCl, 10 mM Tris- HCl, pH 7.4, 5 mM EDTA, pH 8.0, 1% Triton X-100, 1 mM PMSF, and 2 μg/mL of aprotinin). Cell debris was discarded by centrifuging at 12,000 × g for 15 minutes at 4 °C. Using 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels, 30 μg of total protein was fractioned. Proteins were transferred to polyvinylidene fluoride (PVDF) membranes for blotting. Proteins were blocked with 5% skim milk for 2 hours at room temperature. The antibody against p-Erk1/2 was used at a 1:1000 dilution (Cell Signaling Technology, USA) and Erk1/2 at 1:1000 (Beyotime Biotech, China) for blotting overnight at 4 °C. Secondary antibodies were used at 1:10,000 dilutions. An enhanced chemiluminescent substrate for the detection of horseradish peroxidase (HRP) (Pierce, USA) was used to visualize the immunoreactivity. Molecular analyst software (Quantity One, Bio-Rad, USA), was used to detect the density of the areas of interest in the western blotting experiment. Erk1/2 was used as an internal control. The density of p-Erk1/2/Erk1/2 was computed and represented.

BMSCs were planted on the MBG-NH₂, MBG-NH₂/IGF and MBG-NH₂/IGF@SF/VEGF scaffolds in a 6-well plate at a density of 1 × 10⁵ cells/mL. After 5 days of culture, the collected cells were washed with PBS and then lysed (lysate: EDTA: phosphatase inhibitor: protease inhibitor = 50:1:1:1) on ice. The protein was obtained after centrifuging at 12,000× g for 15 min at 4 °C. After determining the protein concentration with a BCA protein assay kit (Thermo, Waltham, MA, USA), 30 μg of protein was mixed with a ×6 loading buffer and lysate before being heated in a water bath to denature it. Then, 10% sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis was performed on the denatured protein, and the protein bands were transferred to a polyvinylidene fluoride (PVDF) membrane. The bands were stained with reed red and blocked with 5% (w/v) skim milk. After incubation for 2 h with Erk1/2 (Beyotime Biotech, Haimen, China, 1:1000), phospho-Erk1/2 (p-Erk, Cell Signaling Technology, Danvers, MA, USA, 1:2000), mTOR (Abcam, Cambridge, UK, 1:2000), and phospho-mTOR (p-mTOR, Abcam, Cambridge, UK, 1:2000), the PVDF membrane was treated with the corresponding species source of the second antibody at a 1:5000 dilution. Immunoreactivity was determined with enhanced chemiluminescent substrate (Pierce, MO, USA) for HRP detection.