Hrp conjugated species specific secondary antibodies

Iodoacetamide was purchased from GE Healthcare. RPMI-1640 and DMEM media were from Gibco. Microcon YM-30 spin filters were from Millipore. MS-grade Trypsin, Asp-N, and Arg-C were from Promega. Tris, SDS, methanol, chloroform, 2,2′-dithiodipyridine (DTDP), dime-thylformamide (DMF), hydroxylamine, sodium chloride, ammonium bicarbonate, and ammonium formate were from Sigma-Aldrich. Arginine (Arg0), lysine (Lys0), ¹³C₆¹⁵N₄-arginine (Arg10), ¹³C₆¹⁵N₂-lysine (Lys8), dialyzed fetal bovine serum, RPMI 1640 medium for SILAC, 660 nm Protein Assay Kit, TCEP, NEM, high-capacity streptavidin agarose beads, SuperSignal chemiluminescent substrate, trap columns, EASY-Spray analytical columns, and Horseradish peroxidase (HRP)-conjugated streptavidin were from Thermo Fisher Scientific. Primary antibodies were from Cell Signaling Technology, Novus Biologicals, Santa Cruz Biotechnology, Sigma-Aldrich, and R&D Systems. HRP-conjugated species-specific secondary antibodies were from Cell Signaling Technology and Jackson ImmunoResearch Laboratory.

Immunoblot analysis was performed as described previously (Campana et al., 2006a (link); Inoue et al., 2010 (link)). Briefly, extracts of sciatic nerve tissue distal to the crush injury site were prepared in RIPA buffer. The protein content of each extract was determined by bicinchoninic acid (BCA) assay. An equivalent amount of protein (20–40 μg per lane) was subjected to 10% SDS-PAGE and electro-transferred to nitrocellulose membranes. Blots were blocked with 5% nonfat dry milk and subsequently incubated with primary polyclonal antibodies to GAP43 (1:1000; Sigma), STMN2 (1:1000; Novus Biologicals), total ERK1/2 (t-ERK) (1:1000; Cell Signaling), GAPDH (1:2000; Sigma), NF200 (1:800; Sigma), p75^NTR (1:5000; Millipore) or βIII neuronal tubulin (Tuji; 1:10,000; BioLegend) overnight at 4° C. Antibody-binding was detected by HRP-conjugated species-specific secondary antibodies (1:2000; Cell Signaling) and enhanced chemiluminescence (GE Healthcare). Blots were scanned (Canoscan) and densitometry was performed using Image J software.

Five μg of protein samples were resolved by SDS-PAGE with reducing condition and transferred to nitrocellulose membrane. After blocking with 5% skim milk in TBS supplemented 1% Tween-20 (TBST), membranes were incubated with primary antibodies in 5% BSA/TBST for overnight at 4°C followed by incubation with appropriate HRP-conjugated species-specific secondary antibodies (Cell Signaling Technology). Bands were detected using SuperSignal West Dura or Femto chemiluminescent substrate (Thermo Scientific) and visualized by the ImageQuant LAS-4000 (GE Healthcare). Antibodies against MMP2 (ab37150) and MMP14 (ab53712) were obtained from abcam. Anti-SH3BP2 monoclonal antibody (clone 1E9) was purchased from Abnova. Antibody against NFATc1 (7A6) was obtained from Santa Cruz Biotechnology. Antibodies against phospho-IKKα/β (#2697), IKKβ (#2678), phospho-JNK (#4668), JNK (#9258), phospho-ERK (#4370), ERK (#4695), phospho-p38 (#4511), and p38 (#9212) were obtained from Cell Signaling Technology.

Sciatic nerves were harvested 2 days after PNL to identify early molecular and cellular changes. Approximately 0.5 cm of sciatic nerve was collected distal from the ligation site. Ipsilateral and contralateral nerves were collected. Nerves were lysed in RIPA buffer and equal amounts of protein (20 μg) from nerves lysates, as determined by BCA Protein Assay (Bio-Rad, Hercules, CA, USA), were subjected to 10% SDS-PAGE and electro-transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat dried milk and then incubated with anti-TLR4(CD284)/MD2 (BioLegend, San Diego, CA, USA; cat#117601, 1:1000), anti-CD11b (Abcam, Cambridge, MA, USA, cat#Ab1333357) and anti-β-actin (Cell Signaling Technology, Danvers, MA, USA; cat#1:1000). Primary antibodies were detected with HRP-conjugated species‐specific secondary antibodies (Cell Signaling Technology, Danvers, MA, USA; cat#7076S or 4S; 1:5000). Immunoblots were developed using SuperSignal West Pico PLUS chemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA, USA), and the Protec Ecomax X-ray film processor. Densitometry analysis was performed using Image J software (U. S. National Institutes of Health, Bethesda, MD, USA).