Ecl chemiluminescence

For preparation of whole-cell lysis extracts, the cells were treated with Z-ajoene for 24 h and lysed with cell lysis buffer (Cell Signaling Technologies, Beverly, MA, USA). The cell lysates were then centrifuged at 10,000× g for 20 min at 4 °C. The supernatants were collected and protein concentrations were assessed using a BCA protein assay kit. The cytosolic and nuclear fractions were prepared as previously described with some adjustment [19 (link)]. Proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (GE Healthcare, Piscataway, NJ, USA). The membranes were probed with antibodies and visualized using the ECL chemiluminescence (GE Healthcare). The antibodies against β-catenin (BD Transduction Laboratories, San Jose, CA, USA), c-Myc, β-actin, lamin A (Cell Signaling Technology, Beverly, MA, USA) and cyclin D1 (Santa Cruz Biotechnology, Dallas, TX, USA) were used to detect the target proteins.

Protein extracts were prepared in RIPA buffer (10 mM Tris-HCl pH7.4, 150 mM NaCl, 1 mM EGTA, 0.5% Triton and complete 1% protease inhibitor mixture, Roche Diagnostics). Protein was separated on SDS-polyacrylamide gel, transferred to a nitrocellulose membrane and probed with a primary antibody against: PS6 (1:1000), TSC2 (1:1000, Cell Signaling, D93F12 Cat. 4308), calnexin (1:5000; Sigma, Cat. C4731), S6 (1:1000, Cell Signaling), KCNQ2 (1:500, Abcam, ab22897), ACTIN (1:2000, Sigma, A3853) followed by horseradish-peroxidase-conjugated secondary antibody (1:5000, Dako). KCNQ2 and ACTIN were used in reducing but not in denaturing conditions. Finally, it was visualized using ECL chemiluminescence (GE Healthcare).

The following materials were purchased for the study: Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin and streptomycin (P/S) from Gibco BRL (Grand Island, NY, USA); LPS (Escherichia coli; O111:B4); L-NMMA, indomethacin and radioimmunoprecipitation assay lysis buffer (RIPA) from Sigma-Aldrich (St. Louis, MO, USA); CCK-8 from Dojindo (Kumamoto, Japan); Griess reagent from Promega (Madison, WI, USA); 2,2′-Azino-bis-3-ethyl benzothiozoline-6-sulfonic acid (ABTS)-based antioxidant kit and PGE₂ enzyme-linked immunosorbent assay (ELISA) detection kit from Cayman (Ann Arbor, MI, USA); Primary antibodies from Cell Signaling Technology (Beverly, MA, USA) against iNOS (#13120), COX-2, NF-κB p65, IκBα, phospho-p38 (Thr180/Tyr182), p38, phospho-ERK (Thr202/Tyr204), ERK, phospho-JNK (Thr183/Tyr185), JNK, β-actin, GAPDH, Lamin B1, MyD88, Nrf2, and HO-1, and TLR4 from Santa Cruz Biotechnology (Dallas, TX, USA); horseradish peroxidase (HRP)-conjugated secondary antibodies against rabbit and mouse from Jackson ImmunoResearch (West Grove, PA, USA); Halt protease & phosphatase inhibitor cocktail and NE-PER nuclear and cytoplasmic extraction reagents from Thermo Fisher Scientific (Rockford, IL, USA); ECL chemiluminescence from GE Healthcare (Chicago, IL, USA). Polyvinylidene fluoride membrane from Millipore (Kenilworth, NJ, USA).

The ventral half of the lumbosacral spinal cords were homogenized in RIPA buffer containing protease and phosphatase inhibitors. Samples were then centrifuged to remove cell debris. Protein concentration was quantitated using BCA reagent (Pierce). Samples were separated on 4–15% SDS-PAGE and blotted to a nitrocellulose membrane. Films were scanned and analyzed using free software, ImageJ, with background correction and normalization to actin. The primary antibodies against Nav1.2 and Kv4.2 were from Alomone Labs. The blots were visualized by ECL chemiluminescence (GE Healthcare). Lysate from the ventral half of four different cords/chicks per treatment were used and blots were done in duplicate (total eight embryos per treatment).

Protein extract from 20-36 DRGOs were prepared in RIPA buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EGTA, 0.5% Triton and complete 1% protease inhibitor mixture, Roche Diagnostics). Protein was separated on SDS-polyacrylamide gel, transferred to a nitrocellulose membrane and probed with a primary antibody followed by horseradish-peroxidase-conjugated secondary antibody (1:5000, Dako). ACTIN was used as normalizer. A list of antibodies used can be found in Supplementary Table 4. Finally, bands were visualized using ECL chemiluminescence (GE Healthcare)

Twenty micrograms of total protein was resolved by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis, and transferred onto a polyvinylidene fluoride membrane (GE Healthcare, Chicago, IL, USA). The membrane was then probed with specified primary antibodies overnight as follows: HPV16 E7 (Cervimax, Vienna, Austria), β‐galactosidase (Promega, Madison, WI, USA), HA (12CA5; Roche Diagnostics, Risch‐Rotkreuz, Switzerland), glyceraldehyde‐3‐phosphate (GAPDH) (Chemicon, Billerica, MA, USA) and β‐actin (C4, Santa Cruz Biotechnology, Dallas, TX, USA). HPV58 E7 polyclonal antibody was custom synthesized from immunising rabbits with HPV58 E7 peptides (aa3‐20) using a 90‐day protocol with three boosters (ThermoFisher Scientific). Blots were visualized using ECL chemiluminescence (GE Healthcare) and images were captured using ChemiDoc™ Imagine System (Bio‐Rad, Hercules, CA, USA). Actin or GAPDH served as loading control for the blot. Band Intensities were quantitated by ImageLab and normalized with corresponding loading control level.