Mouse Anti-FCMR Recombinant Antibody (clone HM7) (CAT#: FAMAB-0547WJ)

Figure 1 SDS-PAGE analysis of biotinylated cell surface proteins.

FcμR⁺/GFP⁺ or control BW5147 cells were surface biotinylated, quenched, and incubated with the indicated FcμR-specific MAbs (HM14, HM10, HM2, HM7, and HM3), isotype-matched control MAbs (IgG1κ, IgG3κ, and IgG2bκ) or mouse myeloma IgMκ (TEPC183), before washing and solubilization in 1% NP-40 lysis buffer containing protease inhibitors. The MAb- or IgM-bound cell surface proteins were captured by addition of beads coupled with rat anti-mouse κ MAb (187.1 clone) and resolved on SDS-10% PAGE under reducing and non-reducing conditions (not shown), followed by transfer onto membranes, blotting with horseradish peroxidase-coupled SA, and visualization with ECL. Essentially the same results were obtained with the HM6 MAb (not shown). These experiments were performed at least three times. Mr is shown in kDa.

Kubagawa, Y., Honjo, K., Kang, D. W., & Kubagawa, H. (2014). Monoclonal antibodies specific for human IgM Fc receptor inhibit ligand-binding activity. Monoclonal antibodies in immunodiagnosis and immunotherapy, 33(6), 393-400.

Figure 2 Epitope mapping analysis.

BW5147 cells stably expressing a chimeric FcμR protein made by swapping of the Ig-like domain of human (H) or mouse (M) origin, MHH- or HMM-type FcμR, were incubated with the indicated receptor-specific MAbs (thick black lines) or isotype-matched control MAbs (thin gray lines), before developing with PE-labeled goat anti-mouse IgG antibodies. Stained cells were analyzed by flow cytometry. The acronym MHH indicates the Ig-like domain of mouse origin and the remaining stalk/transmembrane and cytoplasmic regions of human origin, and the HMM indicates the reverse. The flow cytometric analyses were performed three times.

Kubagawa, Y., Honjo, K., Kang, D. W., & Kubagawa, H. (2014). Monoclonal antibodies specific for human IgM Fc receptor inhibit ligand-binding activity. Monoclonal antibodies in immunodiagnosis and immunotherapy, 33(6), 393-400.

Figure 3 Binding characteristics of FcμR-specific MAbs.

Effects of pre-incubation of anti-FcμR MAbs. A mixture of FcμR⁺/GFP⁺ and control BW5147 cells was first incubated with: the indicated anti-FcμR MAb (10 μg/mL) (+) or PBS (-) at 4°C for 20 min, washed, and then with biotin-IgM (2 μg/mL). Stained cells were analyzed by flow cytometry. Horizontal lines indicating the peak fluorescence in PBS-treated cells are included for comparison.

Kubagawa, Y., Honjo, K., Kang, D. W., & Kubagawa, H. (2014). Monoclonal antibodies specific for human IgM Fc receptor inhibit ligand-binding activity. Monoclonal antibodies in immunodiagnosis and immunotherapy, 33(6), 393-400.

Figure 4 Binding characteristics of FcμR-specific MAbs.

Effects of pre-incubation of IgM (B). A mixture of FcμR⁺/GFP⁺ and control BW5147 cells was first incubated with: IgM (10 μg/mL) (+) or PBS (-), washed, and then with the indicated, biotin-anti-FcμR MAbs (2 μg/mL), before developing with PE-SA. Stained cells were analyzed by flow cytometry. Horizontal lines indicating the peak fluorescence in PBS-treated cells are included for comparison.

Kubagawa, Y., Honjo, K., Kang, D. W., & Kubagawa, H. (2014). Monoclonal antibodies specific for human IgM Fc receptor inhibit ligand-binding activity. Monoclonal antibodies in immunodiagnosis and immunotherapy, 33(6), 393-400.

Figure 5 Competition of IgM-ligand and anti-FcμR MAbs in binding to FcμR.

Similar mixture of cells was simultaneously incubated with biotin-IgM (2 μg/mL) and various concentrations of anti-FcμR MAbs, washed, and then with SA-PE. The % IgM binding was determined by: 100×[mean fluorescence intensity (MFI) of IgM binding by FcμR⁺/GFP⁺ cells with anti-FcμR MAb÷MFI of IgM binding by control cells with anti-FcμR MAb]÷[MFI of IgM binding by FcμR⁺/GFP⁺ cells in PBS÷MFI of IgM binding by control cells in PBS]. These experiments were performed at least twice.

Kubagawa, Y., Honjo, K., Kang, D. W., & Kubagawa, H. (2014). Monoclonal antibodies specific for human IgM Fc receptor inhibit ligand-binding activity. Monoclonal antibodies in immunodiagnosis and immunotherapy, 33(6), 393-400.

Figure 6 Representative immunofluorescent profiles.

Control GFP⁺ or FcμR⁺/GFP⁺ Jurkat cells were cultured at 37°C for 15 h in the absence (-) or presence (+) of agonistic IgM anti-Fas MAb (CH11 clone) at 10 ng/mL, along with (+) or without (-) HM3 or HM7 MAb at 10 μg/mL. Cells were stained with 7-AAD and APC-labeled annexin V to detect early (7-AAD-/annexin V+) and late (7-AAD+/annexin V+) apoptotic cells. Profiles of background apoptosis [IgM α-Fas(-)/MAb(-)], another control [IgM α-Fas(-)/HM3(+)] and other concentrations of HM3 or HM7 MAb with IgM anti-Fas MAb were done but are not shown.

Kubagawa, Y., Honjo, K., Kang, D. W., & Kubagawa, H. (2014). Monoclonal antibodies specific for human IgM Fc receptor inhibit ligand-binding activity. Monoclonal antibodies in immunodiagnosis and immunotherapy, 33(6), 393-400.

Figure 7 Induction of apoptosis in FcμR⁺/GFP⁺ Jurkat cells by addition HM7 MAb.

The % apoptosis was determined by: 100×[% annexin V+ cells with IgM α-Fas(+)/MAb(+) - % annexin V+ cells with IgM α-Fas(-)/MAb(-) in FcμR⁺/GFP⁺ cells]÷[% annexin V+ cells with IgM α-Fas(+)/MAb(-) - % annexin V+ cells with IgM α-Fas(-)/MAb(-) in GFP+ cells]. These experiments were performed twice.

Kubagawa, Y., Honjo, K., Kang, D. W., & Kubagawa, H. (2014). Monoclonal antibodies specific for human IgM Fc receptor inhibit ligand-binding activity. Monoclonal antibodies in immunodiagnosis and immunotherapy, 33(6), 393-400.