Fig. 6.

Detection of MG132-induced HSP30 localization by LSCM. Cells were grown on glass coverslips and were maintained at 22°C or treated with 5, 10, or 30 μM MG132 at 22°C for 24 h. Additionally, cells were treated with 30 μM MG132 for 24 h followed by a 48 h recovery in fresh media (bottom row). Actin and nuclei were stained directly with phalloidin conjugated to TRITC (red) and DAPI (blue), respectively. HSP30 was indirectly detected with an anti-HSP30 antibody and a secondary antibody conjugated to Alexa-488 (green). The fourth row contains an example of some cells treated with 30 μM MG132 for 24 h which showed distinct patterns of HSP30 staining. An area of HSP30 staining delineated by a red rectangle was enlarged (fourth row, third panel). The white arrows indicate large circular cytoplasmic foci of HSP30 while the yellow arrow points to an example of distinct areas within the cytoplasm where HSP30 was not detected. The 5-, 10-, or 20-μm white scale bars are indicated at the bottom right section of each panel. These data are representative of four different experiments