Anti drp1 antibody

All lysates were prepared with protein sample buffer (62.5 mM Tris–HCl, pH 6.8, 25% glycerol, 2% SDS, 5% β-mercaptoethanol, 0.01% Bromophenol blue) (BioRad, Hercules, CA). Then the samples separated by SDS-PAGE were transferred to PVDF membrane (BioRad). After blocking with 4% skim milk in TBST (25 mM Tris, 3 mM 140 mM NaCl, 0.05% Tween-20), the membranes were incubated overnight with specific primary antibodies at 4 °C. Anti-Drp1 antibody was from BD (San Jose, CA); anti-hnRNP A1 was from Santa Cruz Biotechnologies (Santa Cruz, CA); anti-cleaved caspase-3 was from Cell Signaling Technology (Danvers, MA); anti-Actin antibody was from Millipore (Temecula, CA). For protein detection, the membranes were incubated with HRP-conjugated secondary antibodies (Pierce, Rockford, IL).

For Western blotting, all lysates were prepared with protein sample buffer (62.5 mM Tris-HCl, pH 6.8, 25% glycerol, 2% SDS, 5% β-mercaptoethanol, 0.01% Bromophenol blue) (BioRad, Hercules, CA). Then the samples were separated by SDS-PAGE and transferred to PVDF membrane (BioRad). After blocking with 4% skim milk in TBST (25 mM Tris, 3 mM 140 mM NaCl, 0.05% Tween-20), the membranes were incubated over-night with specific primary antibodies at 4°C. Anti-Drp1 antibody was from BD (San Jose, CA); anti-Cpn10 antibody was from BD (San Jose, CA); anti-Actin antibody was from Millipore (Temecula, CA). For protein detection, the membranes were incubated with HRP-conjugated secondary antibodies (Pierce, Rockford, IL).

Acr-treated or control cells on coverslips of 6-well chambers were incubated with MitoTracker Red CM-H2XRos (Invitrogen, M7513) or with MitoTracker Green FM (Invitrogen, M7514), or Lysotracker Red DND-99 (Invitrogen, L7528), as indicated by the manufacturer’s instructions. After staining, cells were washed with PBS followed by immunofluorescent staining procedures, as previously described [59 (link)]. The following antibodies were used at the noted dilutions: anti-LC3 antibody (1:100, Cell Signaling, # 2775), anti-PINK1 antibody (1:100, BD, # BC100-494), anti-Drp1 antibody (1:100, BD, # 611112) at 4 °C for overnight. Monodansylcadaverine (MDC) has been proposed as a tracer for autophagic vacuoles [63 (link)]. A549 and MRC-5 cells were cultured on coverslips overnight, treated with Acr for 8 h, rinsed with PBS, then stained with MDC (Sigma, 50 μM) and MitoTracker Red CM-H2XRos (Invitrogen, M7513), at 37°C for 30 min. The cells were fixed for 15 min with ice-cold 4% paraformaldehyde at 4°C, washed with PBS, and examined under Olympus FV1000 confocal laser microscopy. Morphology of mitochondrial was stained with MitoTracker Red CM-H2XRos (Invitrogen, M7513) described the above and quantified byMicroP3D software [64 ].