Mouse anti g6pd

Cells were collected, washed with PBS, and then lysed in cell disruption buffer (Ambion) containing inhibitors of phosphatases and proteases (PhosphoSTOP and Complete, Roche). Proteins were separated by SDS/PAGE in 4–20% Tris-HEPES gradient gels (BioRad), and transferred to nitrocellulose membranes (GE Healthcare). The membranes were saturated with 3% bovine serum albumin prepared in TBS (50 mM TRIS-Cl, pH 7.5, 150 mM NaCl)-0.01% Tween-20 and then incubated overnight with the following antibodies: mouse anti-OPA1 (BD Biosciences, 1:1000), mouse anti-G6PD (Santa Cruz Biotechnology, 1:1000), rabbit anti-phospho-4EBP (Cell Signaling Technology, 1:1000), rabbit anti-non phospho-4EBP (Cell Signaling Technology, 1:1000), rabbit anti-PARP (Cell Signaling Technology, 1:1000) and rabbit anti-GAPDH (GeneTex International Corp., 1:10000). Membranes were then washed twice and incubated for 1 h with an HRP-conjugated anti-mouse or anti-rabbit antibody (Thermo Fisher Scientific, 1:5000). Chemiluminescent signals were detected using Lite Ablot Turbo (EuroClone) and a Cambridge UVITEC imaging system.

Expressions of G6PD and TLR4 in L4-L6 DRGs were determined by western blotting analysis, as previously described in detail.^{18 (link),23 (link),25 (link)} The antibodies in the present study included mouse anti-TLR4 (1:200, Santa Cruz Biotechnology, Inc.), mouse anti-G6PD (1:200, Santa Cruz Biotechnology, Inc.), rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:1000, Goodhere Biotechnology Co., Ltd.), anti-mouse horseradish peroxidase-conjugated secondary antibody (1:2000; Jackson ImmunoResearch Laboratories, Inc.), and anti-rabbit peroxidase-conjugated secondary antibody (1:2000; Jackson ImmunoResearch Laboratories, Inc.).

Immunofluorescence technology was performed as described previously.^{17 (link),18 (link)} Rabbit anti-TLR4 (1:50, Biovision), rabbit anti-G6PD (1:50, Santa Cruz Biotechnology, Inc.), mouse anti-G6PD (1:50, Santa Cruz Biotechnology, Inc.), calcitonin gene-related peptide (CGRP) (1:200, Abcam), isolectin B4 (IB4⁺) (1:200, Sigma), NeuN (1:50, Millipore Sigma), and glutamine synthetase (GS) (1:200, Abcam) antibodies were used in the present study.