Turbo blotting system

IIA1.6 FcγR transfected cells (5 × 10⁶) were stimulated with non-blocking agonist mAb 8.2 (20 µg/mL), lysed, then membrane isolated according to manufacturer’s specifications (Qproteome Cell Compartment Kit, Qiagen). Briefly, the lysate was clarified by centrifugation at 10,000 g for 10 min (4°C) and receptors immunoprecipitated with human IgG (IVIg) (Intragam, CSL, Parkville, Melbourne, VIC, Australia) coated Sepharose beads for 1 h (4°C). The Sepharose beads were washed and bound proteins analyzed by SDS-PAGE. The proteins were transferred to PVDF membranes using a Turbo-blot. Turbo Blotting System (BioRad Laboratories) and FcγRII detected using rabbit anti-FcγRIIa antiserum followed by anti-rabbit Ig/HRP (DakoCytomation). Band signal intensities were enumerated using image J open source Java application (https://imagej.nih.gov/ij/) of precipitated receptor from unstimulated cells was taken as 100% and the intensities of receptor band signals from later time points adjusted accordingly for each replicate.

Control and treated cells were lysed in NP40 cell lysis buffer (cat. no. #FNN0021; Invitrogen) supplemented with the Halt protease and phosphatase inhibitor cocktail, centrifuged at 17 950 g for 10 min at 4 °C. Protein concentrations of cell lysates were determined by the BCA assay. Cell lysates (50 μg of protein) were resolved on 10% SDS/PAGE and transferred onto PVDF membranes (cat. no. #548IPVH00010; GE Healthcare Biosciences, Philadelphia, PA, USA) with the help of Turbo Blotting System (Bio‐Rad Laboratories). Membranes were first blocked with 5% bovine serum albumin (BSA) in TBS (Tris‐buffered saline) buffer for 1 h followed by an overnight incubation with primary antibodies (CCL2, 1 : 200 dilutions; HIF‐1A, 1 : 500 dilutions) in a rotating shaker at 4 °C. The membranes were then washed three times with TBST (TBS containing 0.1% Tween 20) buffer at 10‐min intervals and incubated with peroxidase‐conjugated specific secondary antibodies (1 : 2000 dilution) for 2 h at room temperature. Membranes were then washed three times with TBST at 10‐min intervals and subjected to Clarity Western ECL Substrate incubation for 5 min at room temperature. Protein bands were visualized in Chemidoc XRS+ System (Bio‐Rad Laboratories) using image lab Software.