2.9. Western blot phosphorylation analyses of TrkC downstream kinases in osteoclast lineage cells

As a step to demonstrate direct action of NT-3 and examine potential activation of TrkC downstream kinases in osteoclast lineage cells, RAW_264.7 osteoclast precursor cells (at 80% confluence) were firstly serum-starved for 2h and then cultured in α-MEM with or without rhNT-3 (100 ng/ml) for 5, 15, or 30 min, or with 100 ng/ml rhBDNF for 15 min as a comparison, and in the absence or presence of RANKL (100 ng/ml). After 3 washes with cold PBS, cells were lysed for 30 min at 4 °C in RIPA buffer containing protease inhibitor and phosphatase inhibitor cocktails (Roche, Basel, Switzerland). Supernatants were harvested after centrifuging for 15 min at 13000 rpm and protein concentrations quantified. After being denatured, proteins were separated through a 4–15% Criterion™ SDS-PAGE gel (Bio-Rad, NSW, Australia), transferred onto nitrocellulose membrane, incubated with rabbit antibodies against phosphorylated (p-) or total (t-) Erk½ (extracellular signal-regulated kinases-½, Cell signaling, Beverly, MA), p-Akt or t-Akt (also known as protein kinase B, Santa Cruz, Dallas, Texas), or sheep anti-mouse GAPDH (as a loading control, Osenses, Adelaide, Australia), and then incubated with HRP-conjugated secondary antibodies (Dako) and finally processed for signal development using an ECL kit (Thermo Fisher Scientific, Melbourne, Australia) as described [18].