Mouse anti β actin a1978

The endometriotic epithelial cell line (11Z) was established by Professor Anna Strazinski-Powitz [24 (link)]. The human endometrial stromal cell line (ESC) was established by Dr. Krikun [25 (link)]. All cell lines were cultured in Dulbecco’s Modified Eagle Medium/Ham’s F-12 50/50 Mix (DMEM/F-12) supplemented with 10% FBS (Gibco, Carlsbad, CA, USA) with 100 μg/mL penicilin and 100 μg/mL streptomycin at 37 °C and 5% CO₂.
Mouse anti-β-actin (A1978) was from Sigma-Aldrich, and dilution: 1:5000. Mouse anti-HSF1 (sc-17,757) was from Santa Cruz, and dilution: 1:1000. Rabbit anti-PFKFB3 (ab181861) was purchase from Abcam, and dilution: 1:2000. KRIBB11 were obtained from Med Chem Express (MCE), 50 mg/kg.

Spinal cord tissue samples were homogenized using cell lysis buffer (Cell Signaling, Danvers, MA, USA) and the Bullet Blender (Next Advance, Averill Park, NY, USA). Total protein concentrations were quantified using Quick Start Bradford 1x Dye (Bio-Rad). Tissue homogenates (30 μg) were subjected to electrophoresis using 4–12% SDS-PAGE gels and specific protein signals were detected using the following antibodies: mouse anti-β-actin (A1978, 1:4000, Sigma); rabbit anti-RAGE (GTX23611, 1:1000, GeneTex); rabbit anti-S100B (ab52642, 1:5000, Abcam); and rabbit anti-HMGB1 (GTX101277, 1:1000, GeneTex). After incubation with indicated antibodies, protein signals were visualized with IRDye 680RD Goat anti-mouse (1:25,000; LI-COR, Lincoln, NE, USA) and IRDye 800CW Goat anti-rabbit (1:10,000; LI-COR) and protein signals were visualized using the Odyssey Infrared Imaging System Model 9120 (LI-COR). Quantification was carried out using Image J open source software as described previously (Juranek et al., 2013a (link)).

Protein extraction from rat brains was performed at P13 using an ULTRARIPA kit for lipid rafts (F015; BioDynamics Laboratory Inc.); the reagent solution contained 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40 alternative, 0.1% SDS, and 0.5% sodium deoxycholate. Proteins were separated using SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore). The membranes were blocked with 5% nonfat skim milk overnight at 4°C and incubated in primary antibody solution containing rabbit anti- voltage-gated sodium channel alpha subunit 1 (Na_V1.1) (ASC-001, 1:200; Alomone Labs) and mouse anti-β-actin (A1978, 1:1000; Sigma-Aldrich) for 1 h at room temperature and sequentially incubated with secondary antibodies containing anti-Rb IgG HRP (AP307P, 1:2000; Millipore) and anti-mouse IgG HRP (AP308P, 1:2000; Millipore) for 1 h at room temperature. The signal was detected using Chemiluminescence HRP Substrate (Takara Bio) on the LAS3000 Mini apparatus (FUJI FILM). Semiquantitative analysis of the signal was performed using ImageJ software (National Institute of Health).