Tim23 sc 514463

Immunoblotting was performed as previously described [36 (link)]. Cells were lysed with ice-cold RIPA lysis buffer in the presence of a protease inhibitor cocktail. Protein quantification, electrophoresis, and transfer were performed, followed by membrane blocking with 5% nonfat milk. The human-specific antibodies used included GAPDH (ab128915) from Abcam; Tom20 (sc-11415), Parkin (sc-133167), and Tim23 (sc-514463) from Santa Cruz; p62 (23214), PINK1 (6946), and LC3 (3868) from Cell Signaling Technology (Boston, MA, USA); and MFN1 (66776-1-Ig) and MFN2 (67487-1-Ig) from Proteintech (Chicago, IL, USA). Then they were incubated with secondary antibodies (KPL) for 1 h at room temperature. Protein bands were visualized using ECL blotting detection reagents (Life-iLab, Shanghai, China).

Chloroquine diphosphate (CQ, Sigma, C6628). Tris [(1-benzyl-1H-1,2,3-triazol-4-yl) methyl] amine (TBTA), Tris (2-carboxyethyl) phosphine (TCEP), CuSO₄ were purchased from Sigma. Rhodamine-azide was obtained from Jinglan Co. (Guangzhou, China) and biotin-azide was purchased from Thermo Fisher Scientific. The ART-probe (ART-P) was synthesized as previous description [20] (link). MitoSOX™ Red Mitochondrial Superoxide Indicator (M36008), JC-1 Mitochondrial Membrane Potential Dye (65-0851-38), MitoTracker™ Red (M7513), Pacific Blue™ Annexin V Apoptosis Detection Kit (A35122), ThiolTracker™ Violet (glutathione, T10095) were purchased from Thermo Fisher Scientific.
The following antibodies were used: autophagy-related gene 7 (ATG7) (2631), cytochrome c oxidase Ⅳ (COX Ⅳ) (4850), dynamin-related protein 1 (DRP1) (8570), phospho-DRP1 (Ser616) (3455), heat shock protein 60 (HSP60) (12165), lysosomal-associated membrane protein 1 (LAMP1) (9091), mitofusin 1 (MFN-1) (14739), MFN-2 (11925), Dynamin-like 120 kDa protein (OPA-1) (67589), PINK1 (6946), Parkin (4211), SQSTM1/P62 (5114), Ubiquitin (3936), were purchased from Cell Signaling Technology. α-tubulin (sc-23948), translocase of outer mitochondrial membrane 20 (TOMM20) (sc-11415), translocase of the inner membrane 23 (TIM23) (sc-514463) were purchased from Santa Cruz Biotechnology. LC3 (L7543) was purchased from Sigma.