Erk1 2 monoclonal antibody

Cells extracts were prepared and analyzed as previously described [16 (link)]. Western blotting was performed by anti-ZKSCAN1 (Bioss, P17029), FBXW7 Rabbit Polyclonal Antibody (ABclonal, A5872), mTOR Mouse Monoclonal antibody (Proteintech, 66,888–1-lg), β-Actin Rabbit mAb (ABclonal, AC026), anti-His Mouse Monoclonal antibody (ABclonal, AE003), HA-tag antibody (ABclonal, AE008), anti DDDDK-Tag Mouse Monoclonal antibody (ABclonal, AE005), anti-GAPDH (Sangon Biotech, D110016), AKT Polyclonal Antibody (Proteintech, 51,077–1-AP), Phospho-Akt (Ser473) (Cell Signaling Technology, 4060), Phospho-Akt (Thr308) (Cell Signaling Technology, 13,038), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling Technology, 4370), ERK1/2 Monoclonal Antibody (67170–1-Ig), Phospho-mTOR (Ser2448) (Cell Signaling Technology, 5536), Phospho-PDK1 (Ser241) (Cell Signaling Technology, 3438), Phospho-MDM2 (Ser166) (Cell Signaling Technology, 3521), Phospho-p70 (S6) Kinase (Thr389) (Cell Signaling Technology, 97,596), MDM2 (Proteintech, 66,511–1-Ig), PDK1 (Proteintech, 18,262–1-AP), P70 (S6K) (Proteintech, 14,485–1-AP), Biotinylated Rabbit anti-Goat IgG (ZSGB-BIO, ZB-2050) and Biotinylated Mouse anti-Goat IgG (ZSGB-BIO, ZB-2020) antibodies. The antigen-antibody complexes were visualized by chemiluminescence.

Assay kits for measuring superoxide dismutase (SOD), malondialdehyde (MDA), total glutathione/oxidized glutathione (T-GSH/GSSG), nitric oxide (NO), and total antioxidant capacity (T-AOC) were purchased from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). Anti-phospho-FGFR1 (Tyr653+Tyr654) polyclonal antibody (rabbit) was purchased from Bioss (Beijing, China). BDNF polyclonal antibody (rabbit) was purchased from Cloud-Clone Corp (Wuhan, Beijing). Goat-anti mouse IgG, goat-anti rabbit IgG, β-actin monoclonal antibody (rabbit), AKT (pan) monoclonal antibody (mouse), phospho-AKT (Ser473) monoclonal antibody (rabbit), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) monoclonal antibody (rabbit), Erk1/2 monoclonal antibody (mouse), Bcl-2 monoclonal antibody (rabbit), and caspase-3 polyclonal antibody (rabbit) were purchased from Cell Signaling Technology (Danvers, United States). FGF-2 was produced in the EK’s lab, and the purification data, western blot data and activity data were presented in Supplementary Figure 1.

Aliquots containing 30 μg of protein were blotted as previously described7 (link)8 9 (link). Briefly, protein samples were analyzed on 10% SDS–PAGE gels and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA) in a wet transfer unit (Bio-Rad, Hercules, CA, USA). Membranes were with 5% non-fat dry milk at room temperature for 1 h and incubated overnight at 4 °C with the following primary antibodies: GPR91 polyclonal antibody (1:1000), p-ERK1/2 monoclonal antibody (1:3000, Cell Signaling Technology, Boston, MA, USA), ERK1/2 monoclonal antibody (1:3000, Cell Signaling Technology, Boston, MA, USA), c-Fos monoclonal antibody (1:1000), C/EBP β monoclonal antibody (1:1000), Hypoxia inducible factor-1α (HIF-1α) monoclonal antibody (1:200, abcam, Cambridge, MA, USA), VEGF monoclonal antibody (1:1000, abcam, Cambridge, MA, USA), β-actin monoclonal antibody (1:5000, Sigma Aldrich, Saint Louis, MO, USA). β-Tublin monoclonal antibody (1: 1:1000, abcam, Cambridge, MA, USA) and Histone H3 monoclonal antibody (1:1000, ProteinTech Group, Chicago, IL, USA). Membranes were washed with TBS-Tween 20 and incubated with the appropriate HRP-conjugated secondary antibodies (1:1000, ProteinTech Group, Chicago, IL, USA) for 1 h at room temperature. Bands were visualized using an enhanced ECL detection kit (Bio-Rad, Hercules, CA).