Arg 1

Equal amounts of soluble protein extracted from cells were separated by SDS–PAGE and then transferred to a PVDF membrane (HybondTM‐P, Amershan, Piscataway, NJ). The blots were probed with specific antibodies to iNOS (BD transduction Lab., San Jose, CA), EDN1, HYAL‐1, ARG‐1 (GeneTex, Inc, Irvine, CA), Caspase‐1 (Millipore, Billerica, MA), IL‐1β (Abnova, Taipei City, Taiwan), TNFα (R&D Systems, Inc., Minneapolis, MN), p‐A‐CoA, p‐AMPKα, and (Cell Signaling, Beverly, MA), and β‐actin (Sigma‐Aldrich). The signal of electrochemical luminescence in probed blots was detected using FUJI Medical X‐ray film (FUJI Corporation, Kofu, Yamanashi, Japan). The densitometry of specific bands on the blot was measured by Image J software (NCBI).

M1 and M2 marker expression in microglia was examined using immunocytochemistry (ICC) according to previously published methods [15 (link)]. Fixed cells were probed with primary antibodies for the M1 phenotype, iNOS (1:200; BD Transduction Laboratories; Franklin Lakes, NJ), and COX-2 (1:200; Thermo Scientific, Rockford, IL) or the M2 phenotype, Arg-1 (1:200; GeneTex, Irvine, CA), RELMα/Fizz1 (1:100; PeproTech, Rocky Hill, NJ), or transglutaminase-2 (1:100; Thermo Fischer Scientific). In addition, the microglial cell markers anti-iba1 (1:5,000; Wako Pure Chemical Industries, Ltd., Tokyo, Japan) and CD68/ED1 (Macrosialin; 1:200; AbD Serotec) were used. For visualization, the secondary antibodies, goat anti-mouse Alexa-594 or anti-rabbit Alexa-594 (1:200, Life Technologies Corp.) were used along with the nuclear marker Hoechst 33342 (1:1,000; Life Technologies Corp.). The morphology of the cells was demarcated by staining with Phalloidin-488 (1:100; Life Technologies Corp). Images were acquired by sequential scanning of the immunostained cells with an Olympus Fluorescence confocal microscope (Olympus, Fluoview FV 1000). For each treatment condition, three to four randomly selected fields were imaged per well. Images obtained were representative of three independently conducted experiments.

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.