TRPM7 protein, human

Mouse embryonic fibroblasts (MEFs) were obtained from WT or TRPM7^R/R mouse embryos. Peritoneal macrophages were isolated from adult mice34 (link). Cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 120 mM NaCl, 0.5 mM DTT, 1.5 mM MgCl₂, 0.2 mM EDTA, 1% Triton X-100 supplemented with protease inhibitors) on ice for 20 min and insoluble materials were removed by centrifugation at 15,000 rpm for 10 min. Cell extracts were incubated with rabbit polyclonal anti-TRPM7 antibody (Millipore) overnight at 4°C, followed by incubation with protein A sepharose beads for 1 hr. Subsequently, the beads were washed three times in the lysis buffer and once in kinase buffer (50 mM HEPES, pH 7.0, 4 mM MnCl₂, 5 mM DTT). Immunoprecipitates were suspended in kinase buffer with 50 μg/ml myelin basic protein (MBP). The kinase reaction was initiated by adding 0.1 mM ATP in combination with 5 μCi [γ-³²P] ATP and proceeded for 30 min at 30°C. Proteins bound to the beads were eluted in Laemmli sample buffer, subjected to SDS-PAGE, and analyzed by autoradiography.

All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) and adhered to principles outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Experiments in A. Mazur laboratory were performed according to the guidelines of the INRA Ethics Committee, in accordance with decree no. 87–848.
The knockout mice were obtained using a gene-targeting vector technique for TRPM7 with further mice colony generation. The gene-targeting vector was prepared using an in vivo recombination strategy. To disrupt the kinase domain of the TRPM7, the targeting vector was constructed by using a short arm 1.3 kb DNA fragment of an intron located between exons 36 and 37. The short arm was a PCR fragment from AKASA2 to AKASA1. AKASA2 is located 222 bp downstream of exon 36, with a sequence of 5′-GTAACTTTCCTGCCCGAGTCTC-3′. AKASA1 is located 116 bp upstream of exon 37 with a sequence of 5′-CTTATCTCTCAAGCCAATTTAGGAG-3′. The short arm was inserted into the 5′ of the Neo gene cassette using MluI site. The long arm was a 9.7 kb genomic fragment containing exons 28–31. In this strategy, exons 32–36 encoding the large portion of the kinase domain were replaced by the Neo gene cassette (Fig. 1). As a result, the TRPM7 protein is truncated immediately upstream of the alpha-kinase domain. The reverse transcription–PCR analysis of ES knockout cells (see Supplementary Fig. S2) showed that TRPM7Δkinase protein contains TRPM7 amino acids 1–1,537 and an additional stretch of 48 amino acids with the following sequence: DLQRIDPLRRTRQEGDRRRCAANRERRYRKARGSGQPIRRQALQQYHG.
The targeting vector was linearized by NotI and then transfected by electroporation into 129 SV ES cells. After selection with G418, surviving colonies were expanded, and PCR analysis was performed to identify clones that had undergone homologous recombination. The correctly targeted ES cell lines were microinjected into C57BL/6J blastocysts. The chimeric mice were generated and resulted in germline transmission of the trpm7 gene with the disrupted kinase domain. The knockout of one allele in heterozygous TRPM7^+/Δkinase mice was confirmed by Southern blot analysis (Fig. 1b). Preparation of ES cells for construction of TRPM7^Δkinase mice and implantation were performed at inGenious Targeting Laboratory, Stony Brook, NY.