Ab120812

Recombinant human CCL17 and CCL22 were purchased from R&D Systems (Minneapolis, MN). Recombinant human CCL2 was obtained from BioLegend (San Diego, CA). Antibodies used in western blotting, IHC, immunocytochemistry, and flow cytometry were as follows: rabbit anti-human CCR2 antibody (ab21667), rabbit anti-human CCR4 antibody (ab83250), goat anti-human CCR4 antibody (ab1669), rabbit anti-goat immunoglobulin (Ig)G H&L (fluorescein isothiocyanate; FITC) antibody (6737), goat anti-rabbit IgG H&L (FITC) antibody (6717), anti-mannose receptor antibody (ab64693), and anti-CCR7 antibody (ab103404) obtained from Abcam (Cambridge, MA); anti-Akt antibody (9272), anti-phospho-Akt (Thr308) antibody (9275), anti-phospho-Akt (Ser473) antibody (9271), and anti-rabbit IgG HRP-linked antibody (7074) obtained from Cell Signaling Technology (Danvers, MA); and anti-GAPDH antibody (NB300-221) obtained from Novus Biologicals (Littleton, CO). The antagonists used in the neutralizing assay were a selective CCR2 antagonist (ab120812) obtained from Abcam and a CCR4 antagonist (SC-221406) obtained from Santa Cruz Biotechnology (Dallas, TX). The Akt inhibitor AZD5363 (S8019) was obtained from Selleckchem (Houston, TX).

HBPCs were seeded at 4 × 10⁵ cells per well in collagen I-coated 12-well plates and grown in DMEM/F-12 medium containing 10% FBS. After a 6-h incubation with serum-free low-glucose DMEM, cells were treated with 100 ng/mL LPS and/or 100 ng/mL PDGF-BB for 1 h. Cells were washed with phosphate-buffered saline (PBS) to remove these stimulants, cultured in fresh low-glucose DMEM for 24 h, and CM was collected and stored at − 80℃ until used. Cytokine release from pericytes in CM was analyzed with the Proteome Profiler Human XL cytokine array kit (ARY022B, R&D Systems, USA). In the inhibition assay of pericyte-derived factors, BMDMs were pretreated with 1 μM SB225002, a C-X-C motif chemokine receptor (CXCR)1/2 antagonist (13336, Cayman), 5 μM RS102895, a C–C motif chemokine (CCR)2 antagonist (ab120812, Abcam), or 100 nM C-021, a CCR4 antagonist (21885, Cayman) for 1 h, followed by a treatment with each CM for 12 h. Cells were then stimulated with 10 ng/mL LPS for 24 h.