Fig. 3. Glutamate triggered LCRs by increasing the oxidation of Ca²⁺-handling proteins.

a, b To assess the oxidation status of RyR2, the free thiol content of immunoprecipitated RyR2 was measured using the anti-DNP antibody. Western blot showing increased oxidation of RyR2 under 5 mM glutamate treatment. a Representative western blot bands. b Pooled data from a. n = 6 per group. c, d To assess the oxidation status of SERCA2a, the free thiol content of immunoprecipitated SERCA2a was measured using the DNP antibody. Western blot showing increased oxidation of SERCA2a under glutamate treatment. c Representative western blot bands. d Pooled data from c. n = 6 per group. e, f To assess the oxidation status of CaMKII, the free thiol content of immunoprecipitated CaMKII was measured using the DNP antibody. Western blot showing increased oxidation of CaMKII under glutamate treatment. e Representative western blot bands. f Pooled data from e. n = 6 per group. g Representative confocal line-scan images of LCRs in permeabilized SANPCs. h–k Pooled data from g demonstrated that oxidation inhibitors (5 mM DTT or 10 μM dantrolene) reversed glutamate-induced increases in LCR number (h), LCR size (i), LCR duration (j) and LCR amplitude (k) in permeabilized SANPCs compared to the vehicle group (n = 4–8 per group, cells were isolated from at least 4 rats). DAN, dantrolene. *P < 0.05, calculated by unpaired Student’s t-test (b, d, f) or one-way ANOVA with Dunnett post-hoc test (h–k).