Anti phospho plcγ

DNCB, anti-DNP-IgE and DNP-HSA were purchased from Sigma-Aldrich (St. Louis, MO, USA), Eucalyptus oil, procured from Nippon Terpene Chemicals (Tokyo, Japan), conforms with the Japanese Pharmacopoeia, and its main component, 1,8-cineole, accounts for 82.5% of the oil. 1,8-cineole was obtained from Nacalai Tesque (Kyoto, Japan), and dimethyl sulfoxide (DMSO) was acquired from Wako (Osaka, Japan). The following antibodies for immunoblotting were purchased from Cell Signalling Technology (Beverly, MA, USA): anti-Syk, anti-phospho-Syk, anti-Lyn, anti-phospho-Lyn, anti-PLA2, anti-phospho-PLA2, anti-PLCγ, anti-phospho-PLCγ, anti-p38 and anti-phospho-p38. Goat anti-rabbit IgG horseradish peroxidase was purchased from Cell Signaling Technology (Danvers, MA, USA).

Commercial antibodies included: anti-TrkB (BD Biosciences, New Jersey, NJ, USA; #cat 610102), anti-TrkB (Millipore, Burlington, MA, USA; #cat 07-225), anti-phospho-TrkB (Tyr 515) (Sigma, St Louis, MO, USA; #cat SAB4503785), c-Abl (Sigma #cat A5844), anti-c-Abl (Santa Cruz Biotechnology, Dallas, TX, USA; #cat K-12), anti-c-Abl (Santa Cruz Biotechnology, #cat 24-11), anti-phospho-c-Abl (Tyr 412) (Sigma, #cat C5240), anti-PLC-γ (Cell Signaling, Danvers, MA, USA; #cat 2822), anti-phospho-PLC-γ (Tyr 783) (Cell Signaling, #cat 2821), anti-AKT (Cell Signaling, #cat 9272), anti-phospho-AKT (Ser 473) (Cell Signaling, #cat 9291), anti-ERK (Cell Signaling, #cat 9102), anti-phospho-MAPK (Erk1/2; p42/44; Thr202/Tyr204) (Cell Signaling, #cat 9101), anti-Flag (Sigma, #cat F3040), anti-GAPDH (Santa Cruz, #cat 6C5), β-III Tubulin (Sigma, #cat T8578), anti-MAP2 (Sigma, #cat M3696), anti-phospho-CrkII (Tyr221) (Cell Signaling, #cat 3491), anti-CrkII (Cell Signaling, #cat 3492), Secondary Antibody: Goat anti-Mouse IgG (H + L) and Goat anti-Rabbit IgG (H + L), HRP (Invitrogen, Carlsbad, CA, USA, #cat 31430 and #cat 31460) and secondary antibody Alexa-555, Alexa-488, Alexa-633 and Alexa 547 (1:1000) (Invitrogen, Carlsbad, CA, USA).

The protein concentrations were measured using a BCA protein assay kit (Beyotime, Beijing, China). The proteins were then separated using 10% SDS–PAGE and transferred to PVDF membranes. After blocking in 5% skim milk for 2 h, the membranes were incubated with primary antibodies at 4 °C overnight and then incubated with secondary antibodies for 1 h at RT. Enhanced chemiluminescence reagent was used for signal visualization. Anti-PLCγ (D9H10, PLCγ, 1:1000), anti-phospho-PLCγ (D25A9, P-PLCγ, 1:1000), anti-IP3R (D53A5, IP3R, 1:1000), anti-phospho-IP3R (D10E3, P-IP3R, 1:1000), and anti-GAPDH (D16H11, GAPDH, 1:2000) antibodies were purchased from Cell Signaling Technology (Boston, MA, USA). Goat anti-mouse IgG (32160702, 1:4000) and goat anti-rabbit IgG (32160702, 1:4000) secondary antibodies were purchased from Life Science (Santa Cruz, CA, USA).