Vegf antibody

The protein expression levels of VEGF, FLK1, and GAPDH in the femoral head tissues obtained from rats in different groups were detected by western blot analysis. The protocol and semiquantitative analysis were carried out following the protocol of our previous study [26 (link)]. The following antibodies were used: VEGF antibody (rabbit antibody, dilution 1 : 50, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), FLK1 (rabbit antibody, dilution 1 : 50, Santa Cruz), and GAPDH antibody (internal control, rabbit polyclonal antibody, dilution 1 : 200, Santa Cruz).

VEGF and KDR were detected by immunocytochemistry in HUVECs treated with TFA (0 ug/ml, 5 μg/ml, 10 μg/ml, 20 μg/ml). Briefly, HUVECs were seeded on sterile coverslips in 6-well culture plates at a density of 3×10⁵ cells/ml to allow attachment. When cells reached 60% confluence, the medium was removed. Then, HUVECs were treated with TFA at different concentrations (0 μg/ml, 5 μg/ml, 10 μg/ml, 20 μg/ml) and incubated for 72h. Each group was performed in triplicate. Afterwards, the cells were fixed with 10% formalin for 10 min. Each coverslip was treated with 3% (v/v) H₂O₂ for 15 min to block the activity of endogenous peroxidase and blocked with 5% (w/v) bovine serum albumin for 10 min. The medium was discarded and each coverslip was stained with VEGF antibody (1:200, Santa Cruz, Dallas, TX, USA), or KDR antibody (1:200, Santa Cruz, Dallas, TX, USA) at 4°C overnight prior to incubation with HRP-conjugated secondary antibody for 30 min. The antibody binding sites were visualized by incubation with 3-Amino-9-ethylcarbazole (AEC) solution and counterstained with hematoxylin. One hundred cells were counted in three randomly selected fields for each coverslip with 400× magnification. A score from 0 to 3 was conferred to identify the staining intensity (3 = strongest expression).

G-Rh2 was obtained from National Standard Material Center (Beijing, China). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and trypsin were bought from GIBCO/BRL (Grand Island, NY, USA). VEGF-ELISA kit was purchased from R&D Systems (Minneapolis, MN, USA). VEGF antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA).MMP9 and MMP2antibodies were purchased from Abcam (Cambridge, UK). The flow cytometry antibodies CD206, CD16/32were purchased from Peprotech (New Jersey, NJ, USA). Lipopolysaccharide (LPS) was from Sigma-Aldrich (St. Louis, MO, USA).Interferon-γ (IFN-γ) and interleukin-4 (IL-4) were produced by BioLegend (San Diego, CA, USA).

Vascular endothelial growth factor (VEGF) antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phospho-specific anti-p65 (Ser 536) was purchased from Cell Signaling (Beverly, MA, USA). β-actin antibody were obtained from Sigma Chemicals Co (St. Louis, MO, USA). To determine the levels of various proteins, skin wound tissue extracts were prepared and fractionated by SDS-polyacrylamide gel electrophoresis (PAGE) as described previously [37] (link). After electrophoresis, the proteins were electro-transferred to a nitrocellulose membrane, blocked with 5% nonfat milk to minimize non-specific binding, and probed with various antibodies (1∶1000) overnight at 4°C. The blot was washed, exposed to HRP-conjugated secondary antibodies for 2 h, and the expression of various proteins was detected by chemiluminescence emission (ECL; GE Healthcare, Little Chalfont, Buckinghamshire, UK).

Cells were seeded into 6‐well plates. Following overnight incubation, the cells were treated with LY294002, RAD001 or DMSO and harvested at 48 hours. Western blot analysis was performed as previously described.³² The membranes were exposed with an enhanced chemiluminescence reaction (ECL) kit, and the grey levels of the protein bands were analysed using the ImageJ 1.47v software. The sources of the primary antibodies applied were as follows: GAPDH (#2118), β‐tubulin(#2146), p‐PI3K S1981 (#4228), PI3K (#4249), p‐AKT S473(#4060), AKT(#4685), p‐mTOR S2448 (#5536), mTOR (#2983), p‐P70S6KT421/S424 (#9204), P70S6K(#2708), p‐S6 S235/236 (#4858) and S6 (#2217) were purchased from Cell Signaling Technologies; polyclonal rabbit anti‐12‐LOX was from Novus Biologicals (NBP2‐29941; Novus Biologicals; Centennial, CO, USA); VEGF antibody was obtained from Santa Cruz (sc‐7269; Santa Cruz; Dallas, TX, USA).