(A) Representative whole-cell recordings from TRPV2-expressing HEK293T cells showing the responses to 3 mM 2-aminoethyl diphenylborinate (2-APB) before and after the treatment by 0.3 mM 2-APB plus 5 mM Mg2+ (left). Average peak responses to 3 mM 2-APB before and after Mg2+ application (right). The dotted line indicates zero current level. The holding potential was –60 mV. p = 0.12 by one-sample t-test. (B) Tyrosine phosphorylation and serine/threonine phosphorylation of immunoprecipitated TRPV2-Flag transiently transfected in HEK293T cells in the absence and presence of 5 mM Mg2+ were determined by immunoblotting with anti-phosphotyrosine antibody (pTyr) and anti-Phospho-(Ser/Thr) Phe antibody (pSer/Thr). Inset, Protein amounts of tyrosine-phosphorylated or serine/threonine-phosphorylated immunoprecipitated TRPV2 proteins were quantified, and phospho-Tyr TRPV2/total TRPV2 and phospho-Ser/Thr TRPV2/total TRPV2 were calculated from at least three independent experiments. Error bars indicate SD. (C) Left, representative whole-cell currents at –60 mV in a TRPV2–expressing HEK293T cell treated with 0.3 mM 2-APB, 0.3 mM 2-APB plus 5 mM Mg2+ and 3 mM 2-APB. The pipette solution contained adenosine triphosphate ( ATP) nonhydrolyzable analog adenylyl imidodiphosphate (AMP-PNP). Right, summary of relative changes under different conditions. p = 9.29E-6 by unpaired Student’s t-test. (D) Whole-cell currents in response to 2-APB under inhibition of JAK1 by Ruxolitinib. (E) Summary plot of Mg2+ effects on TRPV2 currents under the various conditions. ***p < 0.001. (F) In vitro kinase assay with [32P]-γ-ATP, tyrosine kinase JAK1, and recombinant His-tagged rat TRPV2 N-terminus. Phosphorylation signals were detected by autoradiography. Loading amount of different TRPV2 proteins was accessed by coomassie blue staining. (G) Flow cytometry analysis for phagocytosis. Flow cytometry analysis was employed to determine the phagocytosed level of green fluorescent protein (GFP)-expressing Escherichia coli (GFP E. coli) by bone marrow-derived macrophages (BMDMs) treated with varying concentrations of Ruxolitinib or transfected with shTRPV2#1. Bar graph displaying the effects on phagocytosis under different conditions. *p < 0.05, **p < 0.01, ***p < 0.001. (H) Immunoblot analysis (with anti-JAK1 or anti-β-actin) of BMDM cells transfected for 72 hr with JAK-1-targeting shRNA (shJAK1#1, shJAK1#2, and shJAK1#3) or shControl to test knockdown efficiency of shRNA. (I) Immunoblot analysis of the tyrosine phosphorylation levels of TRPV2 in BMDM cells transfected with shJAK1#3 or shControl for 72 hr in the absence and presence of Mg2+, respectively. (J) Whole-cell recordings in BMDM cells transfected with shJAK1#3 showing the responses to 0.3 mM 2-APB, 0.3 mM 2-APB plus 5 mM Mg2+ and 3 mM 2-APB. (K) Comparison of relative increase under different conditions. p = 4.49E-6 by unpaired Student’s t-test. Error bars indicate standard error of the mean (SEM).