Mortalin sc 133137

AF568-Annexin V, MitoTracker Deep Red, Protein A/G magnetic beads and AF680 conjugated goat anti-rabbit IgG (A21109) were purchased from Invitrogen (Burlington, ON, Canada). FluidMAG-DXS beads were from Chemicell (Berlin, Germany). Mortalin (sc-133137) and β-actin (sc-1616-R) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). PARP antibody (9542) was from Cell Signaling (Denvers, MA, USA). FITC-conjugated goat anti-rabbit IgG (L43001) was from Caltag (Burlingame, CA, USA). DLST antibody (HPA003010), MKT-077 and paraformaldehyde (PFA) were from Sigma-Aldrich (St. Louis, MO, USA). Vir S antibody (ab22743) was purchased from Abcam (ON, Canada). Ultra-small goat anti-rabbit IgG (25101) was from Electron Microscopy Sciences (Hatfield, PA, USA). Staurosporine was purchased from Calbiochem (San Diego, CA, USA). Recombinant Cpn60.2 protein and Cpn60.2 antibody (kindly provided by Dr Richard W. Stokes, University of British Columbia, Canada) were described earlier (Hickey et al., 2010 (link)) and Dos R antibody was kindly provided by Dr Yossef Av-Gay, University of British Columbia, Canada.

Cells were harvested in lysis buffer containing 62.5 mM Tris-HCl (pH 6.8)/2% SDS, and protease and phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich, St. Louis, MO, USA, P8340, P5726, P0044). Protein concentrations were measured using the bicinchoninic acid reagent (Pierce, Waltham, MA, USA, 23228, 1859078). Proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane filter (Bio-Rad, Hercules, CA, USA, 1620177). After transfer, membranes were blocked at 25 °C for 1 h in buffer containing 0.1 M Tris (pH 7.4), 0.9% NaCl, 0.05% Tween 20, and 5% nonfat dry milk.
Membranes were then incubated with the appropriate antibodies overnight at 4 °C, at the dilutions indicated as follows: poly(ADP-ribose) polymerase (PARP, #9542), 1:1000; cleaved lamin A (#2035), 1:2000; cytochrome c oxidase subunit IV (COX IV, #4850), 1:2000 (Cell Signaling, Danvers, MA, USA); β-actin A2228) 1:5000 (Sigma, St. Louis MO, USA, A1978); E2F-1 (sc-193), 1:1000; RET (sc-167), 1:1000; mortalin (sc-133137), 1:5000; p27^KIP1 (sc-1641) 1:1000 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). SuperSignal West Pico and Femto chemiluminescence kits (Pierce, Waltham, MA, USA, 34579 and 34094) were used for visualization of the signal. For densitometry, immunoblots were analyzed using Image Lab software (Bio-Rad, Hercules, CA, USA).