Fig. 4.

Mg²⁺ inhibits GSDMD-NT membrane localization and oligomerization in HEK293T cells. a−f HEK293T cells transfected with indicated cDNA were cultured for 16 h, in the absence or presence of MgCl₂, MgSO₄, or MgGluc₂ as indicated. a Percentage of LDH release. b Immunoblots for Flag in the whole cell lysate, cytosolic fraction, and membrane fraction. Cytosolic and membrane-bound proteins were separated by using a Plasma membrane protein isolation kit. α-tubulin and TFR1 are used as loading controls respectively. c Representative confocal immunofluorescence images of cells. Bar = 5 μm. d Oligomer formation assessed by Flag immunoblot of nonreducing gel (left) and reducing gel (right). e, f Cells transfected with CFP-GSDMD-NT and YFP-GSDMD-NT were cultured for 16 h in the presence of 10 mM MgSO₄. The fluorescence resonance energy transfer (FRET) signal was then detected before and after a washout of MgSO₄. Shown are representative images (e) and quantification of FRET efficiency (f). Bar = 10 μm. For panels (a) and (f), data are presented as mean ± SD; *P < 0.05, **P < 0.01, compared to the cells transfected with Flag-GSDMD-NT in the absence of Mg²⁺ (a) or the cells before the washout (f). Each panel is a representative experiment of at least three replicates. See also Figs. S5 and S6. LDH lactate dehydrogenase