Quantitative Western Blot Analysis of Neurotrophin Receptors

Protein lysates were prepared as previously described^{49 (link)}. Protein content was quantified by Bradford assay, and samples were separated in 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were electrophoretically transferred onto Hybond ECL nitrocellulose membrane (GE Healthcare Bio-Sciences Corporation, Piscataway, NJ, USA), and incubated with primary antibodies raised against human TrkA (sc-118, 1:1000), TrkB (sc-20542, sc-12, 1:1000), TrkC (sc-14025, sc-117, 1:1000), p75NTR (sc-8317, 1:1000), phospho-Trk (Tyr680/Tyr681, sc-7996-R, 1:1000), Erk1 (sc-94, 1:1000), phospho-Erk (sc-7383, 1:1000), α-tubulin (sc-8035, 1:1000; all from Santa Cruz Biotechnologies Inc., Santa Cruz, CA, USA), Akt (9272, 1:2000), and phospho-Akt (4060, 1:2000, all from Cell Signaling Technology, Danvers, MA, USA). Species-specific infrared secondary antibodies (Li-Cor Biosciences, Lincoln, NE) were used to detect bound antibodies, followed by visualization using Li-Cor Odyssey Infrared Imaging System. Densitometry was performed using Image Studio Lite software (Li-Cor Biosciences).