Enhanced chemiluminescence

IFN‐ω and IL‐6 luciferase fusion proteins were heated at 95°C for 4 min in reducing sample buffer [3% SDS, 10% glycerol, 0.1 M dithiothreitol, 0.02% bromophenol blue and 6.25 mM Tris–HCl, pH 6.8], run in 12% SDS–PAGE and blotted onto polyvinylidene difluoride filters. After blocking, strips of the filter were incubated with patient or control sera (1:100) or rabbit anti‐luciferase antibody (1:1000 New England Biolabs, Ipswich, MA) followed by secondary antibodies (anti‐human HRP 1:10,000 and goat anti‐rabbit‐HRP 1:5000; Jackson ImmunoResearch, West Grove, PA, USA, Inc.), and visualization by enhanced chemiluminescence using the manufacturer's protocol (GE Healthcare, Little Chalfont, UK).

Protein was isolated from nerve biopsies homogenized in lysis buffer (150 mM NaCl, 1 % Triton, 2 mM EDTA, 50 mM Tris, pH 7.4). 30 µg protein extract was electrophoresed on a 12 % SDS-PAGE gel and blotted for 40 min to PVDF membrane. The membranes were probed with rabbit anti-IGFBP5 (H-100, 1:5000, Santa Cruz Biotechnology) for human samples and goat anti-IGFBP5 antibody (GT15183, 1:5000, Neuromics) for murine samples in 5 % skim milk, and rabbit anti-phosho-IGF1 receptor beta (3918, 1:2000) in 5 % BSA, anti-IGF1 receptor beta (3027, 1:2000) in 5 % milk, for immunoprecipitation 1:1000 in combination with protein A agarose beads (11719386001, Roche), anti-phospho-Akt (9271, 1:2000 in 5 % BSA) and anti-Akt (9272, 1:2000 in 5 % milk, all from Cell Signaling Technology) for mouse extracts, in blocking buffer for 1 h. The appropriate HRP-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc) was used and visualized using enhanced chemiluminescence (GE Healthcare, Lifesciences). The blots were reprobed with mouse anti-actin antibody (Clone C4, 1:7000, Millipore). Film images were scanned and the intensity of IGFBP5 was standardized to mouse anti-actin. A minimum of n = 3 per group were tested in 3 independent experiments.

Cells were lysed in RIPA buffer (150 mM NaCl, 0.5% sodium deoxycholate, 1% NP40, 0.1% SDS, 50 mM Tris-HCl [pH 8.0]) or SDS buffer (2% SDS, 50 mM Tris-HCl [pH 7.5]) containing protease inhibitor and phosphatase inhibitor cocktail (Roche). Harvested extracts were separated by 10% SDS-PAGE and transferred to PVDF membranes. Later, the membranes were incubated with primary antibody, and then with horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratory, West Grove, PA) and were finally visualized using enhanced chemiluminescence (Thermo Fisher Scientific). Image acquisition and signal density measurements were carried out using the BioSpectrum Image System (UVP, Upland, CA). The following primary antibodies were used: anti-caspase 1 (GTX111630, GeneTex, Irvine, CA), anti-caspase 3 (#9665, Cell Signaling Technology, Danvers, MA), anti-caspase 7 (GTX1002337, GeneTex), anti-caspase 8 (#4790, Cell Signaling Technology), anti-bone morphogenetic protein 4 (BMP-4; GTX100875, GeneTex), anti-phospho-STAT1 (phospho-Tyr701, #9167 Cell signaling), anti-STAT1 (#14994, Cell Signaling), anti-phospho-STAT2 (phospho-Tyr690, GTX50721, GeneTex), anti-STAT2 (#14994, Cell Signaling), anti-DENV NS3 (GTX124252, GeneTex), and anti-GAPDH (#60004-1-Ig, Proteintech Group, Rosemont, IL).