Quantitative Western blotting for the AIM number associating with a pentameric IgM

HEK293T cells transfected with pCAGGS-AIM-HA were cocultured with HEK293T cells transfected with both pCAGGS-FLAG-mouse Fc and pCAGGS-mouse J chain–HA in DMEM, supplemented with 10% FBS for 1 day. From the culture supernatant, the Fc-pentamer/J-HA/AIM-HA complex was purified using the ANTI-FLAG M2 Affinity Gel (Sigma-Aldrich), followed by a rabbit anti-AIM polyclonal antibody (rab2, made in house) conjugated to a HiTrap NHS-activated HP column (GE Healthcare Life Sciences, PA). The eluate was analyzed for J chain and AIM by Western blotting in a reducing condition using an anti-HA antibody. The intensity of the signal was measured using the NIH ImageJ software.

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