Immunoblotting was performed as previously described for splenic T cells [11 (link)]. TRPM7 protein isolated from splenic macrophages was immunoprecipitated, and macrophage lysates were incubated with rabbit polyclonal anti-TRPM7 antibody (1:500) (AB15562; Millipore, Billerica, MA) overnight at 4°C, followed by incubation with protein A sepharose beads for 1 hour. The beads were then washed three times in lysis buffer. Protein bound to sepharose A beads was eluted using Laemmli sample buffer. SDS-PAGE analysis was performed on the eluted protein. PVDF membrane was used to transfer the protein from the SDS gel (Millipore) and blocked with Blocking One (Nacalai Tesque, Kyoto, Japan). The blocked membrane was probed with anti-TRPM7 antibody overnight at 4°C, washed, and incubated for 1 hour with the secondary HRP-conjugated anti-rabbit IgG (1:2000) antibody (Dako, Carpenteria, CA). The membrane was washed afterwards and incubated with Amersham ECL Prime (GE Healthcare, Piscataway, NJ). TRPM7 protein was visualized using ImageQuant400 (GE Healthcare).