Phospho erk1 2 p erk

Immunoblotting was the same as previously described [26 (link)]. The following primary antibodies were used: TXNIP (Novus Biologicals), phospho-ERK1/2 (p-ERK; Cell Signaling, Denver, MA, USA), total ERK (t-ERK; Cell Signaling), phospho-JNK (p-JNK; Cell Signaling), total JNK (t-JNK; Cell Signaling), phospho-p38 (p-p38; Cell Signaling), total p38 (t-p38; Cell Signaling), AP-1 (Abcam), and β-actin (Cell Signaling). The relative protein expression was determined using ChemiDoc (Bio-Rad Laboratories).

Proteins were loaded in equal amounts and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Merck Millipore). Membranes were incubated with the primary antibodies directed against the following molecules: α-SMA (Sigma-Aldrich) PDGF receptor-β (Cell Signaling Technology, Danvers, MA), collagen 1α1 (R&D Systems, Minneapolis, MN), JNK, phospho-JNK (pJNK), ERK1/2, phospho-ERK1/2 (pERK), SMAD2, phospho-SMAD2 (pSMAD2), AKT, phospho-AKT (pAKT), p70S6K, phospho-p70S6K (pp70S6K) (all from Cell Signaling Technology), GAPDH (Abcam, Cambridge, United Kingdom) and β-actin (Sigma-Aldrich). The following secondary antibodies were used as needed: a goat-anti-mouse-IgG-HRP antibody (Bio-Rad, Feldkirchen, Germany), a goat-anti-rabbit-IgG-HRP (Bio-Rad), or a goat-anti-sheep-IgG-HRP antibody (R&D Systems). Visualization was performed using the ChemoCam (INTAS, Homburg, Germany) after incubation with Clarity Western ECL Substrate (Bio-Rad).

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.