Mouse monoclonal anti ubiquitin

For imaging, 4 × 10⁴ HeLa cells were seeded in chamber slides (Lab-Tek chambered coverglass; Thermo Fisher Scientific) and transfected 24 h later with 0.3 µg of any indicated DNA with X-tremeGENE 9 (Roche). After treatment, cells were fixed with 4% (vol/vol) paraformaldehyde (Electron Microscopy Services) in PBS for 20 min at room temperature and washed three times in PBS. Cells were permeabilized in 0.5% Triton X-100 in PBS and stained with the primary antibodies as follows: mouse monoclonal anti-Parkin (catalog no. sc-32282; Santa Cruz Biotechnology, Inc.), rabbit polyclonal anti-Tom20 (catalog no. sc-11415; Santa Cruz Biotechnology, Inc.), mouse monoclonal anti-ubiquitin (catalog no. MAB1510; EMD Millipore), and mouse monoclonal anti-HA (catalog no. MMS-101R; Covance) in 5% (wt/vol) BSA (Thermo Fisher Scientific) for 18 h at 4°C, followed by anti–mouse or anti–rabbit Alexa Fluor 488– or Alexa Fluor 594–conjugated secondary antibodies (Life Technologies). For Parkin translocation, counts of Ser/Thr mutant samples were manually counted for translocation phenotype (50 cells/mutant in each of two independent replicates). All images were acquired using LSM software (Carl Zeiss) with fixed cells in PBS at room temperature on an inverted confocal microscope (LSM510 Meta; Carl Zeiss) using a 63×/1.4 NA oil immersion Plan Apochromat objective.