Nbp1 48337

Cells were lysed using RIPA lysis buffer (Beyotime, Shanghai, China) in the presence of Cocktail protease inhibitor (Abmole, USA) and Cocktail phophatase inhibitor (Abmole, USA). Lysate protein concentrations were determined by BCA Protein Assay Kit (Beyotime, Shanghai, China). Equivalent protein from different samples was separated by 10% SDS-PAGE protein electrophoresis and the separated protein samples were transferred onto the Immobilon PVDF membranes. Then, the membranes were incubated with 5% skim milk to block the non-specific sites. Rabbit polyclonal anti-human FPR3 antibody (orb608040, Biorbyt, UK), rabbit polyclonal anti-human CCR2 antibody (NBP1-48337, Novus Biologicals, USA) and HRP-conjugated secondary antibody (1:2000, Proteintech, IL, USA) were used. GAPDH (1:5000, Proteintech, IL, USA) was simultaneously used as an internal control. Signals were detected and visualized with Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore, Darmstadt, Germany).

For immunofluorescence staining, monocytes were seeded into culture wells containing glass coverslips. After 1 h attached monocytes were washed with warm PBS and fixed with 3.7% paraformaldehyde for 15 min at room temperature. After a washing step with PBS, signal enhancer ImageIT solution (Invitrogen, Carlsbad, CA, USA) was added for 30 min at room temperature, followed by cell permeabilization with Tween 20 for 15 min at room temperature. Dilutions of primary antibodies were prepared in 1% BSA (anti-Tie-2, 1:100, Abcam, ab24859, Cambridge, UK; anti-CCR2, 1:50, Novus biological, NBP1-48337, Littleton, CO, USA). Samples were incubated with the respective antibody dilution for 1 h at room temperature. After 3 washing steps in PBS, incubation with respective secondary antibodies coupled with Alexa Fluor 488 (1:1.000 in 1% BSA; Thermo Fisher, A32723, Waltham, MA, USA) or rhodamine (1:125 in 1% BSA; Thermo Fisher, #31685) were performed in the dark for 90 min at room temperature. The glass cover slips containing stained monocytes were placed upside down on a drop of mounting solution (DAPI-containing Fluoromount; Invitrogen) located on microscope glass slides. Analyses were performed using a Leica DM2000 microscope (Leica, Wetzlar, Germany) with fluorescence filters for DAPI, FITC and rhodamine.