Redox-Sensitive Fluorescent Probes Reveal CypD Oxidation

Sodium hydrogen sulfite and sodium sulfite (NaHSO₃/Na₂SO₃, freshly mixed at 1:3 M ratio, pH 7.4) were used as SO₂ donors and purchased from Sigma (Zhang et al., 2021 (link)). Isoprenaline hydrochloride was purchased from Sigma (I5627). A chemically selective fluorescent probe SS-1 was provided by Professor Kun Li and Xiaoqi Yu. DAz-2 was used as a protein sulphenylation probe (13382, Cayman, Michigan, USA) to capture and enrich the sulphenylated protein. The primary antibodies in the present study included CypD (abcam, USA), AAT1 (Sigma, USA), AAT2 (Sigma, USA), β-actin (Zsbio, China), caspase9 (CST, USA), caspase3 (Beyotime, China), cleaved caspase3 (Beyotime, China), cytc (santa, USA), His (Zsbio, China), and β-tubulin (Zsbio, China). Human CypD wild type (WT), and C104S, C82S, C157S, and C203S mutant plasmids were constructed by Sangon Biotech. The information of three kinds of ATP plasmids used in this study are as follows: EcAT3.10 (Conley et al., 2017 (link)) was deposited at Addgene by Mathew Tantama (Addgene plasmid #107215); pm-iATPSnFR1.1 (Lobas et al., 2019 (link)) (Addgene plasmid #102549) and cyto-iATPSnFR1.0 (Lobas et al., 2019 (link)) (Addgene plasmid #102550) were deposited at Addgene by Baljit Khakh.