Anti gtrap3 18

Determination of the protein amount was performed using the BCA protein assay (Pierce), and the same amounts of proteins were normalized for total protein. The samples were boiled in RIPA buffer (20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and protease inhibitor cocktail (Sigma-Aldrich)), separated by SDS–polyacrylamide gel electrophoresis (PAGE), and transferred to polyvinylidene fluoride membranes (Bio-Rad). Non-specific binding was blocked with 3% skim milk in PBS-Tween20, and proteins were probed with anti-GTRAP3-18 (Novus Biologicals; NBP-84273) at 1:1000 dilution, anti-NOVA1 (Abcam; ab183024) at 1:1000 dilution, or anti-β-actin (Sigma-Aldrich; A5316) at 1:5000 dilution. After washing with PBS-Tween20, the horseradish peroxidase (HRP)-labeled secondary antibodies against rabbit (Chemicon; AP188P) or mouse IgG (Chemicon; AP181P) were probed and detected with an ECL prime HRP detection kit (GE Healthcare). Immunoblotting data was collected using a Luminograph I (ATTO) measuring emitted photons by chemiluminescence. Protein expression was then evaluated with CS analyzer4 (Ver. 2.2.3; ATTO).

We used the chloromethyl reagent 7-amino-4-chloro-methylcoumarine (CMAC) (Life Technologies), which produces a highly fluorescent adduct upon reaction with thiol groups for the evaluation of intracellular GSH in SH-SY5Y cells. Cells were incubated at 37 °C for 15 min with 5 μM CMAC and then incubated with serum-free media for 30 min. The cells were fixed with 4% paraformaldehyde (PFA) and then permeabilized with 0.05% Triton-X100 in the case of multiple staining. Non-specific staining was blocked with the reagent PBS containing 5% BSA/0.1% Tween20, and the cells were then incubated with anti-GTRAP3-18 (Novus Biologicals; NB100-1105) and anti-NOVA1 (Abcam; ab183024) at 1:1000 dilution overnight at 4 °C. After a wash with PBS-Tween20, the cells were labeled with fluorescent-labeled secondary antibodies, Alexa-Fluor 488 anti-goat IgG (Molecular Probes; A11055), and Alexa-Fluor 647 anti-rabbit IgG (Molecular Probes; A31573) at 1:1000 dilutions. Finally, the cells were mounted using Fluoromount-Plus mounting solution (Diagnostic Biosystems) and captured with a Nikon A1 confocal microscope. The fluorescence intensity was analyzed by NIS-Elements Imaging Software (NIS Elements C (Ver. 4.30); Nikon) and an average intensity value of the cells in the field was calculated for further analysis.