Imaging densitometer

Total RNA from stimulated cells was isolated using the Trizol reagent (Invitrogen), and cDNA was generated using M‐MLV reverse transcriptase kit (Invitrogen Life Technologies) as described by the manufacturer. All PCR reactions were performed with a MyCycler (Bio‐Rad) for 25 cycles. The primers were designed using Primer 3 software. mRNA sequence of the selected genes was obtained from NCBI website. The primer sequences are summarized in Table 1. Amplified products were electrophoresed in a 1.5% agarose gel and visualized with ethidium bromide. Quantification of mRNA was performed using an imaging densitometer (Bio‐Rad Laboratories, Inc).

Cells were lysed by the addition of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (62.5 mM Tris–HCl [pH 6.8], 2% SDS, 7.8% glycerol, 4.5% mercaptoethanol and 0.1% bromphenol blue) and subsequent boiling for 5 minutes at 100°C. Lysates were cleared by centrifugation and then supernatant protein concentrations were determined relative to a bovine serum albumin (BSA) standard. Cell lysates were subjected to SDS-PAGE on a 4% stacking gel and 10% polyacrylamide separating gel for 70 minutes at 130 V. Then, proteins were transferred onto nitrocellulose membranes with a Bio-Rad transfer unit for 120 minutes at 200 mA. Protein blots were incubated in blocking buffer (2% BSA in Tween20/Tris-buffered saline) for 1 hour at room temperature on a rotating platform. Blots were incubated overnight with primary antibodies against GFAP, CSPG, vimentin, ROCK, EphA4, TNF-α, TGF-β, phosphorylated-Smad3 (p-Smad3: a mediator of TGF-β actions), EpoR, or JAK2. Then, blots were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour, followed by 3 washes. Immuno-reactive bands were visualized by chemiluminescence reagents (PIERCE, Rockford, IL). Band optical densities were analyzed with an Imaging Densitometer (Bio-rad, GS-670). More than 3 independent experiments were performed for each experiment.

Fibroblast levels of relevant proteins were assessed by Western blot as previously described (39 (link)). Briefly, fibroblasts were lysed and homogenized with lysis buffer [10 mM Tris (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 10 mM NaF, 1 mM Na₃VO₄, 10 mg/mL leupeptin, 10 mg/mL aprotinin, and 20 mM PMSF]. Proteins from each sample were separated on 10–15% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 3% bovine serum albumin (Wisent Inc, St-Bruno, QC, Canada), the membranes were probed with primary antibodies against mouse NLRP3, caspase 1 p20 (Adipogen, San Diego, CA, USA) at 1:500 dilution; pro-IL-1β (R and D Systems, Minneapolis, MN, USA) at 1:500 dilution; nitrotyrosine (Santa Cruz, Dallas, TX, USA) at 1:1,000 dilution; and β-actin (Santa Cruz, Dallas, TX, USA) at 1:2,000 dilution. After treatment with appropriate secondary antibodies, the specific bands were detected with an enhanced chemiluminescence (ECL) system and quantified with an imaging densitometer (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Approximately 2.5 × 10⁷ cells from different cell lines were used for lipids extraction as described previously by Singh et al. [40 (link)]. Galactocerebrosides were resolved by high performance TLC (LHPK plates from Whatman) as described by Ganser et al. [41 (link)] for sphingolipids. Galactocerebroside was quantitated by densitometric scanning using Imaging Densitometer (model GS-800; Bio-Rad), and the software provided with the instrument by the manufacturer.

Immunoblotting was performed on culture supernatants, total cell lysates, or spinal cord (T7–9) tissue homogenates to quantify the expression of M1 and M2a markers according to previously published methods [15 (link)]. Nitrocellulose membranes (Bio-Rad Laboratories) were probed with specific primary antibodies; Arg-1 (1:2,000; GeneTex Inc., Irvine, CA), iNOS (1:1,000; Cell Signaling Technology, Boston, MA), TNF-α (1:500; Life Technologies Corp.), and β-actin (Sigma-Aldrich). The optical density of the bands (arbitrary units) was measured with an imaging densitometer (Bio-Rad Laboratories) and normalized to β-actin levels. The data represents values from three independent experiments.

Cells were lysed in cold RIPA buffer. Protein concentrations were determined with a bicinchoninic acid protein assay system (Pierce, Rockford, IL). Proteins were subjected to Western blots using ECL-Plus, as described previously13 . The intensity (area X density) of the individual bands on Western blots was measured by densitometry (model GS-700, Imaging Densitometer; Bio-Rad). Distribution of eNOS dimer and monomer was assayed by performed western blot under 4 °C condition.