2.4. Western Blot Analysis of Phospho-TrkA, ERK, Phospho-ERK, CREB and Phospho-CREB Proteins

PC12 cells (passage number < 13) were seeded in collagen type IV-coated 24-well culture plates at a density of 50,000 cells/mL per well with the complete growth medium for 24 h, before being shifted to a low serum (1% v/v HS) medium for 24 h before exposure to NGF (25 ng/mL) and the test compounds (reference control, ILA or tryptophan at a final concentration of 100 nM) for 24 h to examine the sustainability of ERK signaling. Nontreated control (cells without NGF and any test compound) and NGF control (cells treated with NGF only) were also grown under the same conditions. Following treatment, cells were washed with ice-cold PBS, scraped in ice-cold NP-40 cell lysis buffer containing 150 mM NaCl, 50 mM Tris (pH 8.0), 2 mM EDTA (pH 8.0) and 1% (v/v) NP-40, and incubated on ice for 15 min. The cell lysate was collected by centrifugation (8,000 g for 15 min) at 4 °C, and the protein concentration was determined using a BCA kit with bovine serum albumin as a standard.

After boiling for 5 min, the cell lysate (20 µg) was separated on 10% SDS-PAGE and then transferred onto the polyvinylidene difluoride (PVDF) membrane on an iBlot dry blotting system (Invitrogen, Paisley, UK). Nonspecific reactivity was blocked by Bullet Blocking One solution (Nacalai Tesque Inc., Kyoto, Japan) at room temperature for 5 min with shaking. Blots were incubated with the appropriate antibodies: anti-phospho-TrkA (Tyr490) (1:2000), anti-phospho-p44/p42 MAPK (ERK1/2) (Thr202/Tyr204) (1:1000) (Cell Signalling Technology, Danvers, MA, USA), anti-β-actin (1:5000), anti-CREB (1:1000), anti-phospho-CREB (Ser133) (1:1000) (Abcam, Cambridge, UK), and anti-ERK (pan-ERK) (1:5000) (BD, Franklin Lakes, NJ, USA) overnight at 4 °C. After three washes with Tris Buffered Saline containing 0.1% Tween-20 (TBST), the blots were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (1:5000) (Abcam, Cambridge, UK) for 1 h. The blots were washed with TBST, and the proteins were detected by Amersham ECL Select Western Blotting Detection Reagent (GE Healthcare, Chicago, Illinois, USA) according to the manufacturer’s instructions, and then the chemiluminescence signal was visualized using ChemiDoc Imager (Bio-Rad Laboratories, Hercules, CA, USA)and quantified using Image Lab software, version 6.0.1 (Bio-Rad Laboratories, Hercules, CA, USA).