Monoclonal mouse anti hsp60

Confirmation of protein identities was performed by Western blotting. Protein extracts were prepared in lysis buffer and each cell lysate sample (1 μg/μL, 10 μL) was electrophoresed through a precast gel (NuPAGE®Novex® 4–12% Bis-Tris Gel, 1.5 mm, 10 wells, Invitrogen™, Carlsbad, CA). Proteins were transferred from the gel to a polyvinyldifluoride (PVDF) membrane (Millipore, Bedford, CA) by means of the semidry technique using the Criterion Blotter (Bio-Rad) at 100 V for 60 min and blocked with 5% milk in PBS (adjusted to pH 7.4) containing 0.05% Tween-20. The membranes were then separately incubated overnight with primary rabbit antibodies (1 μg/μL). The commercially available primary antibodies used in this study included the following: monoclonal mouse anti-HSP60 (Stressgen, USA) and polyclonal rabbit anti-RanBP2 (Abcam, USA). After washing, the membrane was incubated with HRP-conjugated goat anti-mouse IgG antibodies (purchased from Jackson ImmunoResearch, USA) for 1 hour (1 : 10000). Proteins were detected with an enhanced chemiluminescent (ECL) system, and quantitative analysis of Western blotting was carried out using the ImageQuant-TL-7.0 software, version 2010 (Amersham Biosciences).

The SH-SY5Y cells were grown on coverslips in 12-well culture plates. After 24 hours' incubation, the cells were fixed (60% methanol and 40% acetone) at −20°C for 30 min and then permeabilized (0.5% Triton X-100) at room temperature for 5 min. After rinsing with PBS, the cells were blocked (6% bovine serum albumin) and then incubated with primary and secondary antibodies. The nuclei and cytoskeleton of the cells were stained with DAPI (Sigma-Aldrich, USA), vimentin (Vimentin DyLight 488 Antibody, Epitomics, USA), monoclonal mouse anti-HSP60 (Stressgen, USA), and polyclonal rabbit anti-RanBP2 (Abcam, USA), respectively. After rinsing with PBS, the cells were mounted with ProLong® Gold Antifade Reagent (Invitrogen). The images were acquired by a microscope equipped with fluorescence light source (FLoid Cell Fluorescence Imaging Station, Invitrogen).