Chemidoc touch

The immunoprecipitation of NCL protein and Bcl‐2 mRNA complexes was undertaken according to the instructions provided by the manufacturer (EZ‐Magna RIP Kit, Millipore, Billerica, MA). Briefly, CA46 cells were lysed in RIPA lysis buffer. A total of 5 µg of anti‐NCL antibody (Santa Cruz) was cross‐linked with protein A/G magnetic beads, for which mouse IgG was used as a control antibody. Then, the cell lysates were incubated with the cross‐linked magnetic beads overnight at 4°C in a RNA‐immunoprecipitation (RIP) buffer. The magnetic beads were eventually washed off from any unbound material. Proteinase K buffer was used to digest the proteins in magnetic beads. The RNA was extracted and purified with a phenol:chloroform:isoamyl alcohol mixture. The cDNA was synthesized using random hexamer primers and reverse transcriptase, as described above. RT‐PCR was conducted under the following conditions: initial denaturation for 10 min at 95°C, followed by 40 cycles of denaturation at 95°C for 15 s and amplification at 60°C for 60 s. The PCR products were separated on 1.5% agarose gels and stained with GoldView, and the results were quantified by determining the band intensity using Bio‐Rad ChemiDoc™ Touch (Bio‐Rad Laboratories, Hercules, CA).

Following protein harvest using Laemmli lysis buffer, protein concentrations of whole cell extracts were measured using DC protein Assay (Bio-Rad). Next, equal amounts of protein were loaded onto a Bio-Rad Mini-Protean^® TGX™ 4-20% gel. Proteins were subsequently transferred onto polyvinylidene difluoride (PVDF) membranes, after which membranes were blocked using ECL advance blocking agent (GE Healthcare) in TBS-Tween 0.1%. Proteins of interest were detected using the following antibodies: anti-N-myc (Cell Signaling, Cat: 9405), anti-CHK1 (Cell Signaling, Cat: 2360), anti-phospho-CHK1 S296 (Cell Signaling, Cat: 2349), anti-WEE1 (Cell Signaling, Cat: 13084), anti-CDC2 (Cell Signaling, Cat: 9116), anti-phospho-CDC2 Y15 (Cell Signaling, Cat: 4539), anti-γH2AX (Abcam, Cat: ab26350), anti-alpha-tubulin (Cell Signaling, Cat: 3873), anti-beta-actin (Cell Signaling, Cat: 4967). Following treatment with HRP-link secondary antibodies (Invitrogen), detection was performed using Bio-Rad Chemidoc™ Touch (BioRad).

GBS cell wall extracts, prepared as described previously (41 (link)), were separated using 8 to 12% Bis-Tris gels (Invitrogen) and transferred to nitrocellulose membranes. The membranes were then incubated with biotin-conjugated wheat germ agglutinin (WGA; Vector Labs, Burlingame, CA, USA), followed by incubation with horseradish peroxidase (HRP)-conjugated streptavidin (0.2 μg/ml; Sigma-Aldrich). Signals were visualized using a chemiluminescent Western blotting substrate (Thermo Scientific, Waltham, MA, USA), and data were acquired using the Bio-Rad ChemiDoc Touch imaging system (Bio-Rad Laboratories, Hercules, CA, USA).

The medulla dorsal horn was cut into small pieces, and the tissues were washed three times by adding an appropriate amount of cold PBS after weighing. Add 100 μL of lysis buffer and corresponding protease inhibitors per 50 mg of tissue. Protein concentration was measured by using the BCA protein assay kit (servicebio). After heating the mixture with loading buffer at 100°C for 5 min, the collected proteins were loaded and separated on a 10% SDS PAGE gel (Epizyme) and electrically transferred to PVDF membranes. Membranes were incubated with the primary antibody overnight at 4°C following 5% nonfat milk blocking for 2 h at room temperature. The primary antibody was diluted as follows: PFKP (1:1,000; servicebio), HK1 (1:1,000; servicebio), G6PD (1:1,000; servicebio), COXIV (1:1,000; proteintech), CGRP (1:1,000; Santa Cruz), c-Fos (1:1,000; Santa Cruz), Iba-1 (1:1,000; woko), IL-6 (1:1,000; proteintech), IL-1β (1:1,000; proteintech), β-actin (1:2,000; servicebio), pro-BDNF (1:2,000; abcam), BDNF (1:2,000; abcam). β-actin served as respective controls. Incubation with the secondary antibodies followed for 2 h at room temperature. Enhanced chemiluminescence (ECL; biosharp) luminescent solution was used to visualize bands. Images were acquired using a chemiluminescence imaging system (BIO-RAD ChemiDoc Touch). The acquired images were analyzed by using Image J software (Image J 1.48v).