Supplementary methods

NF-κB double immunolabeling with glial markers

In order to identify the CNS cell types where NF-κB expression is expressed following EAE induction, we performed the double immunolabeling for NF-κB p65, Iba1 (a marker for activated microglia), and GFAP (a marker for reactive astrocyte) using combined method of DAB exposure and immunofluorescent labeling. Cryostat sections (20 mm) of EAE spinal cord were processed for antigen-retrieval by incubation with 1X Tris-EDTA at 100°C for 30 min, followed by blocking with 1% fetal bovine serum (FBS). Sections were then incubated with rabbit NF-κB p65 primary antibody (1:50; Santa Cruz Biotechnology, USA) for 16 h at 4°C and subsequently incubated with a biotinylated secondary antibody (1:200) for 2 h at room temperature. Sections were incubated with avidin/biotin complex (ABC, 1:100) and NF-κB p65 signals were visualized with DAB exposure. These NF-κB labelled sections were used for the subsequent immunolabeling with antibodies against Iba1 and GFAP, respectively. For this, sections were washed with PBS, exposed to avidin-biotin solution, blocked with 1% FBS, and incubated with primary antibodies against Iba1 (1:500, Wako Chemicals) or GFAP (Invitrogen, 1:500). Sections were further labeled with secondary antibodies conjugated with Cy3 (1:1000 Jackson Immuno Research, PA, USA) and mounted with VECTASHIELD® mounting media. Colored and fluorescent images were obtained with the BX53 microscope equipped with a DP72 camera (Olympus).

Supplementary figures

Fig. S1. Exendin-4 administration reverses the downregulation of GLP-1 and GLP-1R mRNA expression in EAE spinal cords.

Spinal cord samples from naïve and EAE mice exposed to vehicle (veh) or exendin-4 (Ex4, 5 mg/kg) were used to determine changes in GLP-1 and GLP-1R mRNA expression levels. The quantitative real time-PCR (qPCR) analysis showing that exendin-4 administration upregulates the mRNA level of both GLP-1 (A) and GLP-1R (B). n = 4 per group. *P<0.05 and #P<0.05 vs. naïve and vehicle-treated EAE, respectively. Newman-Keuls multiple range test.

Fig. S2. NF-κB p65 is abundantly expressed in activated microglia in EAE spinal cords.

Spinal cord sections were processed for double immunolabeling to determine a localization of NF-κB p65 in activated microglia or reactive astrocytes. Upper panels: representative images of NF-κB p65-positive cells and double-immunopositive cells (NF-kB p65, brown color; Iba1, red fluorescence). Lower panels: representative images of NF-κB p65-positive cells and double-immunopositive cells (NF-kB p65, brown color; GFAP, red fluorescence). Arrowheads are inserted to indicate the representative double-immunopositive cells. Scale bar, 20 µm.

Table S1-Primers used for qPCR analysis

Target / Direction / Sequence / Gene Accession #
β-actin
(mouse) / Forward / 5’-AGCCTTCCTTCTTGGGTATG-3’ / NM_007393
Reverse / 5’-CTTCTGCATCCTGTCAGCAA-3’
GAPDH
(rat) / Forward / 5’-GAACATCATCCCTGCATCCA-3’ / NM_017008
Reverse / 5’-GCCAGTGAGCTTCCCGTTCA-3’
GLP-1
(mouse) / Forward / 5’-CTCTGATGAGATGAGCACCATTCT-3’ / NM_008100
Reverse / 5’-GGTTTGAATCAGCCAGTTGATG-3’
GLP-1
(rat) / Forward / 5’-GCTTTGCATGCGGACGAATA-3’ / NM_012707
Reverse / 5’-CAGGGCGGTAACTTCAAAACG-3’
GLP-1R
(mouse) / Forward / 5’-GGCTCCTCTCCTATCAGGACTCT-3’ / NM_021332
Reverse / 5’-GTTGGCTGCCACGCAGTACT-3’
GLP-1R
(rat) / Forward / 5’-TCTGCCCTGCATGGGTACTA-3’ / NM_012728
Reverse / 5’-TTTCAGGTCAACAGCACCCC-3’
TNF-a
(mouse) / Forward / 5’-CATCTTCTCAAAATTCGAGTGACAA-3’ / NM_013693
Reverse / 5’-TGGGAGTAGACAAGGTACAACCC-3’
TNF-a
(rat) / Forward / 5’-GAAACACACGAGACGCTGAA-3’ / NM_012675
Reverse / 5’-AGGGAGGCCTGAGACATCTT-3’
IL-1b
(mouse) / Forward / 5’-CAACCAACAAGTGATATTCTCCATG-3’ / NM_008361
Reverse / 5’-GATCCACACTCTCCAGCTGCA-3’
IL-1b
(rat) / Forward / 5’-CTAATGCCTTCCCCAGGACAT-3’ / NM_031512
Reverse / 5’-AGGGAAACACACGTTTTCTTTGAC-3’
IL-6
(mouse) / Forward / 5’-GAGGATACCACTCCCAACAGACC-3’ / NM_031168
Reverse / 5’-AAGTGCATCATCGTTGTTCATACA-3’
IL-6
(rat) / Forward / 5’-TCGGCAAACCTAGTGTGCTA-3’ / NM_012589
Reverse / 5’-ACATTCATATTGCCAGTTCTTCGT-3’
IL-17
(mouse) / Forward / 5’-TCCAGAAGGCCCTCAGACTA-3’ / NM_010552
Reverse / 5’-AGCATCTTCTCGACCCTGAA-3’