Anti rabbit ig hrp

To detect the PAXX protein by western blot, we used rabbit polyclonal anti‐PAXX/C9orf142 IgG (NovusBio, Littleton, CO, USA, NBP1‐94172, dilution 1 : 500), which recognizes the C‐terminal half of the PAXX protein (amino acids 109‐204); anti‐PAXX/C9orf142 IgG (Abcam, Cambridge, UK, ab126353, 1 : 200) and swine polyclonal anti‐rabbit Ig‐HRP (Dako antibodies, #P0399, 1 : 3000). Anti‐GAPDH rabbit polyclonal (Sigma, St. Louis, MO, USA, #G9545, developed to recognize 314–333 amino acids of mouse GAPDH, 1 : 2000) and mouse monoclonal anti‐β‐actin (Abcam, ab8226, 1 : 3000) with rabbit polyclonal anti‐mouse Ig‐HRP (Dako antibodies, #P0260, 1 : 3000) were used to control protein loading.

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.