Figure 2: Human FCMR mediates endocytosis of hIgM.
From: Glycan-independent binding and internalization of human IgM to FCMR, its cognate cellular receptor
(a) To investigate hIgM internalization by FCMR, GFP+ hFCMR-transfected cells were incubated with media alone (grey trace) or media supplemented with 15 μg/ml purified hIgM for 1 hr at 4 °C. After washing, cells were left at 4 °C (black trace) or incubated at 37 °C for the indicated times. Internalization was halted with the addition of ice-cold media, and cells were washed extensively to remove unbound IgM. Cells were then labeled with F(ab’)2-anti-human IgM-RPE and analyzed by flow cytometry. (b) IgM cell surface levels (geometric MFI) were normalized to time 0. The mean ± SD from five experiments are shown. (c) Following incubation at 37 °C, hFCMR-transfected cells were cytospun, fixed and permeabilized, and incubated with F(ab’)2-anti-human IgM-RPE for 1 hr. Cells were washed, and labeled with DAPI prior to visualization. Red labeling depicts IgM levels (anti-hIgM-RPE); blue colour, nuclei (DAPI); and green colour shows hFCMR-transfected cells (GFP). Permeabilization of hFCMR-transfected cells resulted in partial quenching of the GFP signal. (d) hFCMR-transfected cells were either left at 4 °C (grey) or incubated for 60 min at 37 °C in the presence or PAO (30 μM, red) or carrier control DMSO (blue). Data shown is one of three representative repeat experiments. (e) IgM cell surface levels (GMFI) indicate enhanced binding and internalization of heat-aggregated IgM (IgM-IC) to hFCRM-transfected cell lines, when compared to hIgM (n = 3).
