Anti ngf

Fifty μg of protein was extracted from the ischemic myocardial tissues at 4 weeks after MI. The tissues were lysed in radioimmunoprecipitation assay (AS1004, Aspen, Wuhan, China), and lysis buffer and protein concentrations were determined using the BCA kit (AS1086, Aspen). The proteins were used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes. The membranes were blocked by incubation with 5% bovine serum albumin in a TBS-Tween buffer (10 mM Tris-HCl, 150 mM NaCl, and 0.5% Tween-20) for 1 hour at room temperature, and subsequently, incubated with different primary antibodies: rabbit anti-TH (Santa, Shanghai, China), anti-GAP43 (1:1,000; Abcam, Cambridge, UK), anti-NGF (1:500; Abcam), anti-CHAT (1:500; Bioss, Beijing, China), anti-VACHT (1:500; Abcam), or anti-GAPDH (1:10,000; Abcam) overnight at 4°C. After the membrane was washed three times, the blots were incubated with secondary HRP conjugated goat anti-rabbit antibody (1:10,000; Pierce, Rockford, IL, USA) for 30 minutes at room temperature. Membranes were detected by enhanced chemiluminescence (Beyotime Biotechnology, Jiangsu, China) and exposed to film in the dark. The optical density intensity of each band was measured using AlphaEaseFC software (Alpha Innotech Corp., San Leandro, CA, USA). Results are shown as the optical density ratio to GAPDH.

Total proteins prepared from the brain tissue were separated by 4–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for 2 h, after which resolved proteins were transferred to nitrocellulose membranes for 2 h at 40 V. Each membrane was then incubated separately at 4℃ with the following primary antibodies overnight: anti-NGF (Abcam), anti-TrkA (Cell Signaling, Danvers, MA, USA), anti-p-TrkA (Cell Signaling), anti-Akt (Cell Signaling), anti-p-Akt (Cell Signaling), anti-ERK (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p-ERK (Santa Cruz Biotechnology), anti-p-75^NTR (Abcam), anti-JNK (Cell Signaling), anti-p-JNK (Cell Signaling), anti-Bcl2 (Abcam), anti-Bax (Abcam), and anti-β-actin antibody (Sigma-Aldrich Co.). The membranes were then washed with washing buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 0.05% Tween 20) and incubated with HRP-conjugated goat anti-rabbit IgG (Invitrogen) and HRP-conjugated goat anti-mouse IgG (Invitrogen) at a 1:1,000 dilution and room temperature for 1 h. Membrane blots were developed using Amersham ECL Select Western Blotting detection reagent (GE Healthcare).