Journal: Scientific Reports

Article Title: Inactivation of TRPM7 kinase in mice results in enlarged spleens, reduced T-cell proliferation and diminished store-operated calcium entry

doi: 10.1038/s41598-018-21004-w

Figure Lengend Snippet: TRPM7/TRPM6 protein expression and TRPM7 kinase activity in splenic T cells. ( A ) Western blot analysis of immunoprecipitated TRPM7 from whole cell lysates of WT and KD splenic T cells. T cells were stimulated with PMA/ionomycin or anti-CD3/CD28 antibody coated beads for 48 hrs. Mouse embryonic fibroblasts were used as a positive control. Equal amounts of protein before immunoprecipitation were ensured by probing for actin. ( B ) Incorporation of 32 P into exogenous myelin basic protein (MBP) by TRPM7 immunoprecipitated from WT and KD resting T cells. Equal quantities of MBP were verified by coomassie blue staining. ( C ) Control experiment showing that anti-TRPM6 antibody was able to recognize TRPM6, by immunoprecipitation using anti-TRPM6 antibody in GFP-TRPM6 transfected HEK cells ( D ). Western blot analysis of TRPM6 immunoprecipitated from WT and KD mouse T cells and kidneys. Full gel images are provided in Supplementary Fig. .

Article Snippet: Extracts of T cells, mouse embryonic fibroblasts (MEF) or HEK293 cells (untransfected or heterologously expressing GFP tagged human TRPM6 in pEGFP-C1 vector) were obtained as described above for the TRPM7 kinase assay and incubated with anti-TRPM7 antibody (1:500) or anti-TRPM6 antibody (1:500, ACC-046; Alomone labs, Israel) overnight at 4 °C, followed by incubation with protein A sepharose beads for 1 hr.

Techniques: Expressing, Activity Assay, Western Blot, Immunoprecipitation, Positive Control, Staining, Transfection