Fig. 6.

SMYD2 Cys13 glutathionylation induces dissociation of SMYD2 from N2A and Hsp90. a, b SMYD2 glutathionylation disrupts its interaction with Hsp90. Purified SMYD2-SH and SMYD2-SSG were incubated with GST-Hsp90 bound to glutathione beads, and eluted sample was analyzed (a). Hsp90 was co-immunoprecipitated with SMYD2 WT or C13S from HEK293 cells in response to AMA with glucose deprivation (b). c, d SMYD2 glutathionylation disrupts its interaction with N2A. Purified SMYD2-SH and SMYD2-SSG were incubated with GST-N2A bound to glutathione beads, and eluted sample was analyzed (c). FLAG-N2A was co-immunoprecipitated with SMYD2 WT or C13S in HEK293 cells in response to AMA with glucose deprivation (d). e SMYD2 subjected to glutathionylation decreases its binding with N2A. SMYD2 WT or C13S was pre-incubated with H₂O₂ in the absence or presence of glutathione for 15 min, then mixed with GST-N2A bound to glutathione beads for 1 h. Eluted samples were analyzed. f, g Colocalization of titin and SMYD2 decreases upon incubation of AMA in rat neonatal cardiomyocytes expressing SMYD2 WT versus C13S. Immunostainings of cardiomyocytes with antibodies to titin (α-titin-NT, red), HA, or SMYD2 (green) are shown with enlarged areas for details (the red boxes) (f). Pearson’s correlation coefficients were calculated to determine colocalization of titin and SMYD2 (g). About 30 cells were analyzed in individual conditions. Images represent the major colocalization pattern in individual experiments. Scale bars, 10 µm. Data represent the mean ± SD, n = 3 independent experiments