Anti ngf

For the IL-6 sensitization of the axon-like structures, adult DRG neurons were prepared as described above (Section 2.2). On DIV 3, NGF- and GDNF-supplemented media were removed from both the soma and axonal chambers and were replaced with media containing anti-NGF (1:50, Sigma, etc.). Following 24 h of NGF deprivation, cultures were assigned to two experimental groups: axon compartments treated with 100 ng/mL IL-6 and axon compartments continued in anti-NGF for an additional 48 h (negative control). Similar times were employed for the maintenance and group assignment in the long-term cultured devices where IL-6 was added at 48 h prior to calcium imaging experiments. At either DIV 6 (short term cultures) or DIV 14 (long term cultures), axonal responses to 100 nM capsaicin were tested using calcium imaging, as previously described (Section 2.3).

Insulin, progesterone, putrescine, selenium, transferrin, anti-NGF, NGF, poly-d-lysine, NSC663284 and NSC95397 were purchased from Sigma (St Louis, MO, USA). Cell culture media DMEM, DMEM-F12, RPMI-1640, Neurobasal, B27, antibiotics, Lipofectamine 2000, Alexa Fluor, cDNA synthesis kit, real-time PCR kit and serum were purchased from Invitrogen (Life technologies, Grand Island, NY, USA). Cell culture dishes, plates and flasks were purchased from BD Falcon, Corning (Corning, NY, USA). Aβ_(1–42) and Aβ_(42-1) were purchased from American Peptide (Sunnyvale, CA, USA). ECL reagent and PVDF membrane were purchased from GE Healthcare (Buckinghamshire, UK). The PCR kits were purchased from Takara (Shiga, Japan), Fermentas (Waltham, MA, USA). Anti-Cdc25A, anti-Actin and HRP-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-phospho-Rb antibody was purchased from Cell Signaling Technologies (Denver, MA, USA). AKT inhibitor II, JNK inhibitor II and U0126 were purchased from Calbiochem (Darmstadt, Germany). Primers were purchased from IDT DNA (Gurgaon, Haryana, India). Brain tissues of AβPPswe-PS1de9 mice and control littermates were a kind gift from Dr. Anant B. Patel (Council of Scientific and Industrial Research-Centre for Cellular and Molecular Biology (CSIR- CCMB)), Hyderabad, India.

Immunocytochemistry was performed and modified according to Iida’s study. Briefly, SH-SY5Y cells were washed with PBS three times, fixed with PBS containing 4% (wt/vol) paraformaldehyde for 15 min, and then permeabilized with 0.5% (wt/vol) Triton X-100 in PBS for 20 min. Immunocytostaining was performed with anti-Akt (1:200), anti-P-Akt (1:200), anti-PI3K (1:200), anti-P-PI3K (1:200), anti-BAD (1:200), anti-Bax (1:100), anti-Bcl-2 (1:100), anti-Cytc (1:200), anti-GSK3β (1:200), anti-p53 (1:100), anti-NGF (1:200), and anti-TrkA (1:200) antibodies (Sigma). After the nonspecific reaction was blocked with PBS containing 10% (wt/vol) bovine serum albumin (BSA), the cells were incubated with the primary antibody in PBS overnight, washed with PBST, and incubated with the second antibody (1:200) in PBST for 1 h. After the samples were washed with PBS three times, they were embedded in DAPI for 5 min and then washed with PBST four times. The images were obtained using an Olympus microscope (Shanghai, China). The mean fluorescence intensity was calculated by Image-Pro software (Meyer, TX, USA).