Normal goat igg

Pyrrolidine dithiocarbamate (PDTC), minocycline and fluorocitrate were obtained from Sigma (St. Louis, MO, USA). The normal goat IgG, anti-CCL5 neutralizing antibody and recombinant rat CCL5 were purchased from R&D Systems (Minneapolis, MN, USA). Anti-rat CCL5, rabbit anti-rat NF-κB p65 and mouse anti-rat β-actin were obtained from Santa Cruz (Santa Cruz, CA, USA). Fluorescein isothiocyanate (FITC)-conjugated IgG and tetraethyl rhodamine isothiocyanate (Jackson Immunolab, West Grove, PA, USA), glial fibrillary acidic protein (GFAP, Millipore, Bedford, MA, USA), ionized calcium–binding adapter molecule 1 (Iba-1, Abcam), and neuronal specific nuclear protein (NeuN, neuronal marker, NOVUS) were purchased. The dosages of intrathecal drugs and peptides were chosen according to former studies [17 (link), 29 (link)] and our preliminary tests.

Reagents and culture mediums for cell cultures were purchased from MilliporeSigma and Thermo Fisher Scientific. Goat anti-mouse IL-6R polyclonal neutralizing Ab (catalog AF1830) and normal goat IgG (catalog AB-108-C) were purchased from R&D Systems, Bio-Techne. Anti-mouse TLR4/MD2 neutralizing Ab (clone MTS510, catalog 117608) was from BioLegend. STAT3 inhibitor (Stattic) was purchased from Santa Cruz Biotechnology and rmIL-6 was from R&D Systems, Bio-Techne. Abs for flow cytometry were PE anti-mouse p-STAT3 (Tyr705, clone 13A3-1, catalog 651004), PE/Cy7, Pacific blue anti-mouse F4/80 (clone BM8, catalog 123114 and 123124), and PE/Cy7 anti-mouse IL-6R (clone D7715A7, catalog 115814) from BioLegend. Abs for Western blotting included anti-mouse p-STAT3 (Tyr705, catalog 9131) and total STAT3 (catalog 9139) from Cell Signaling Technologies. β-actin Ab (clone AC-15, catalog A5441) from MilliporeSigma. Infrared dye–labeled secondary Abs were from Li-Cor Biosciences. For immunocytochemistry staining and FRET analysis, rabbit anti-mouse CIRP Ab (catalog 10209-2-AP) from ProteinTech was used. Goat anti-mouse CD11b Ab (catalog MBS420973) was from MyBiosource. Fluorescence-labeled secondary Ab Cy3-conjugated donkey anti–rabbit IgG (catalog 711-166-152) and Cy5-conjugated donkey anti–goat IgG (catalog 705-175-147) were from Jackson ImmunoResearch Laboratories.

Anti-Osterix (# ab22552), anti-CD31 (# ab28364), anti-VEGF (# ab46154) and anti-TGF-β (# ab66043) antibodies were obtained from Abcam (Cambridge, UK). Anti-alkaline phosphatase (ALP, # PAB12279), β-actin (# 4967) and anti-F4/80 (# MCA497R) antibodies were purchased from Abnova (Taipei, Taiwan), Cell Signaling Technology (Danvers, MA, USA) and AbD Serotec (Raleigh, NC, USA), respectively. Anti-bone morphogenetic protein (BMP)-2 (# sc-6895) and anti-hypoxia-inducible factor (HIF)-1α (# sc-10790) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). A neutralizing goat anti-CCL3/macrophage inflammatory protein-1α antibody (# AB-450-NA) and normal goat IgG were obtained from R&D Systems (Minneapolis, MN, USA).

Chromatin was prepared as indicated for ChIP-seq and immunoprecitated with either normal goat IgG (R and D Systems) or anti-HNF1A (C-19, Santa Cruz Biotechnology) overnight using the SimpleChIP Plus Enzymatic Chromatin IP kit and protocol. Quantitative PCR was performed using immunoprecipitated DNA and 2% chromatin input DNA as described earlier for qRT-PCR using modified thermocycling conditions: 95°C hold for 10 min, 45 cycles of 95°C for 15 s and 60°C for 60 s. Percent Input for immunoprecipitated DNA was calculated using the formula 2% x 2^{(Ct 2% Input Sample - Ct IP Sample)}. Primers for POU5F1/OCT4 regulatory regions were as follows: half-site #1 (HS1) (5’-GTGAAATCTTTAGTGTTGTGAG-3’ and 5’-CCAAGAAATGTAGCAGGACGAGCCCC-3’), half-site #2 (HS2) (5’-AACCTTTTACATGAGCAGGTTTG-3’ and 5’-AATGGTGGAAAGAATTACATGG-3’), half-site #3 (HS3) (5’-GGGCACTCAGTTTATTGTTAGG-3’ and 5’-TTTCCTGTCACAGGGGTTTAGTG-3’), and distal enhancer (DE) (5’-GAGAGGCCGTCTTCTTGGCAGAC-3’ and 5’-GTTCACTTCTCGGCCTTTAACTGCCC-3’). MYOD (primers 5’-AGACTGCCAGCACTTTGCTATC-3’ and 5’-ATAGAAGTCGTCCGTTGTGGC-3’) was used as a non-HNF1A target gene control.

PTC-209, pomalidomide and carfilzomib were obtained from SelleckChem, dissolved in DMSO and stored at −80 °C. Dexamethasone was obtained from Sigma-Aldrich, dissolved in PBS and stored at −20 °C. Recombinant human IGF-1, IL-6, receptor activator of nuclear factor-kappa B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) were obtained from Peprotech, dissolved in PBS/BSA 0.1 % and stored at −20 °C. Goat anti-human DKK1 neutralizing antibody and normal goat IgG were purchased from R&D Systems, dissolved in PBS and stored at −20 °C.

Cells grown on coverslips were fixed briefly in paraformaldehyde and nonspecific binding was blocked with 5% BSA in Tris-buffered saline containing 0.05% Tween. Coverslips were incubated with a rabbit polyclonal anti-RAGE primary antibody (Abcam, Cambridge, MA) diluted in blocking buffer for 2 hours at room temperature and then with Alexa 488-labeled goat anti-rabbit Fab secondary antibody (Life Technologies, Grand Island, NY) for 1 hour in the dark at room temperature. Control slides were incubated with normal goat IgG (R&D Systems, Minneapolis, MN) at a protein concentration equivalent to the primary antibody before addition of secondary antibody. Cells were examined by confocal microscopy.