Rabbit polyclonal anti myc antibody

For immunoblotting, immunoprecipitates were transferred to nitrocellulose membrane. Membranes were incubated with 5% milk in 1× TBS-0.05% Tween-20 for 2 h at room temperature, and then incubated with HRP-conjugated Mouse anti-HA antibody (Sigma, 1:4000) or rabbit polyclonal anti-Myc antibody (Sigma, 1:10,000) in 1× TBS-0.05% Tween-20. The membranes were subsequently washed four times for 10 minutes each with 1× TBS-0.05% Tween-20. Immunoreactivity was visualized by using the ECL detection system directly or after incubation with the peroxidase-conjugated goat anti-rabbit IgG antibody (Millipore, 1:10,000) for 2 h.

Rabbit polyclonal anti-myc antibody, mouse monoclonal anti-actinin-1 antibody, and DMSO were from Sigma–Aldrich (St Louis, MO, USA). Mouse monoclonal anti-Stx6 antibody was from BD Transduction Laboratories (San Jose, CA, USA). Rabbit polyclonal anti-Stx6 and anti-Stx16 antibodies were from Synaptic Systems (Goettingen, Germany). Mouse monoclonal anti-Tubulin antibody was from Abcam (Cambridge, MA, USA). Human holo-transferrin conjugated to A488 was from Invitrogen (Grand Island, NY, USA). Mouse anti-myc (c-Myc 9E10) and rabbit anti-furin (H-220) were from Santa Cruz Biotechnology (Dallas, TX, USA). Polyclonal anti-P-Akt(308) and P-Akt(473) were obtained from Cell Signaling Technology (Danvers, MA, USA). Cy3- and A488-conjugated donkey anti-rabbit and donkey anti-mouse secondary antibodies and horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Nocodazole was purchased from EMD Biosciences Inc. (Darmstadt, Germany) (10 mM stock in DMSO) and C2-ceramide was purchased from Enzo Life Sciences (Farmingdale, NY, USA) (50 mM stock in DMSO). Pre-designed siRNA for Stx6 (siStx6: 5′-CCGAGTCATCAGAAGAACTAA-3′) and non-related (siNR: 5′-AATAAGGCTATGAAGAGATA C-3′) were from Qiagen (Valencia, CA, USA). Human insulin was purchased from Eli Lilly (Indianapolis, IN, USA).

Mouse anti-tubulin monoclonal antibody, rabbit anti-HA and anti-FLAG polyclonal antibodies were purchased from Sigma. Mouse anti-HA (12CA5) monoclonal antibody was purchased from Roche Applied Science (Indianapolis, IN). Mouse anti-phosphotyrosine monoclonal antibody (4G10) was purchased from EMD Millipore (Massachusetts, USA). Rabbit anti-Myc polyclonal antibody was purchased from Sigma and MBL (Nagoya, Japan). Rabbit anti-RBM10 polyclonal antibody was purchased from BET (Montgomery, USA). Secondary antibodies conjugated to Alexa Fluor 488 or 568, Alexa Fluor 568-phalloidin were purchased from Invitrogen. Rabbit anti-FilGAP polyclonal antibody was prepared as described previously (Ohta et al., 2006). NSC23766 (Calbiochem) was purchased from EMD Millipore (Massachusetts, USA).