Western blot analysis of IgM was performed by loading 1 µg of purified Human IgM, Human IgA, and Human IgG and 10 µL of PageRuler Prestained Protein Ladder (Product # 26616) per well onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane using the G2 Fast Blotter (Product # 62288), and blocked with 5% BSA in TBST for at least 1 hour at room temperature. IgM heavy chain was detected using a mouse anti-human IgM monoclonal antibody (Product # MA5-14729) diluted to a concentration of 2 µg/mL in blocking buffer, overnight at 4C on a rocking platform, followed by an HRP-conjugated goat anti-mouse light chain secondary antibody diluted 1:40,000 for at least 30 minutes at room temperature (panel A). To verify the presence of all isotypes, a second blot was probed with a goat anti-human IgM + IgG +IgA polyclonal antibody (Product # 31128) at a concentration of 0.5 µg/mL in blocking buffer overnight at 4C on a rocking platform, followed by an HRP-conjugated mouse anti-goat IgG secondary antibody (Product # 31400) diluted 1:40,000 in blocking buffer for at least 30 minutes at room temperature (panel B). Chemiluminescent detection was performed using SuperSignal West Pico.

Flow cytometry analysis of cytoplasmic IgM expression was performed on Ramos (surface and cytoplasmic IgG/lambda-expressing B cells), Raji (cytoplasmic IgG/kappa-expressing B cells), and Jurkat (T cells) cell lines. Fixed and permeabilized cells were stained with a mouse anti-human IgM monoclonal antibody (Product # MA5-14729, red histogram) or mouse IgG1 isotype control (black histogram), each at a concentration of 1 µg/mL. After incubation of the primary antibody on ice for 1 hour, the cells were stained with a FITC-conjugated goat anti-mouse IgG Fc secondary antibody (Product # 31630) at a dilution of 1:500 for 30 minutes on ice. The representative 10,000 cells were obtained for each sample.

Flow cytometry analysis of surface IgM expression was performed on Ramos (surface and cytoplasmic IgG/lambda-expressing B cells), Raji (cytoplasmic IgG/kappa-expressing B cells), and Jurkat (T cells) cell lines. Cells were stained with a mouse anti-human IgM monoclonal antibody (Product # MA5-14729, red histogram) or mouse IgG1 isotype control (black histogram), each at a concentration of 1 µg/mL. After incubation of the primary antibody on ice for 1 hour, the cells were stained with a FITC-conjugated goat anti-mouse IgG Fc secondary antibody (Product # 31630) at a dilution of 1:500 for 30 minutes on ice. The representative 10,000 cells were obtained for each sample.