Figure 1

ITCH is induced under various stress conditions and redistributes with perinuclear cytoplasmic misfolded aggregates.

(A) Cos-7 cells were plated into six-well culture plates and treated with different doses of chloroquine (CQ) for 8 h and bafilomycin (Baf) for 12 h, as shown in section A. Effect of heat stress (HS) on ITCH levels. Cells were exposed to 43°C for 30 minutes and then returned to normal incubation conditions for 3 h. The cell lysates were then collected and processed for immunoblotting using anti-ITCH and anti-actin antibodies. (B) Quantification of ITCH band intensities collected from three different experiments as shown in A using NIH image analysis software. *, P < 0.05 compared with the untreated control. (C) Cells were plated into two-well chamber slides and on the subsequent day, they were treated with 10 μM MG132 for 8 h, 50 nM Baf for 10 h and 40 μM CQ for 5 h. (D–G) The Cos-7 cells were transiently transfected with the indicated (GFP-wtCAT and GFP-Δ9CAT) plasmids. After 48 h of transfection, the cells were subjected to immunofluorescence staining using antibodies against ITCH (D), ubiquitin (E), p62 (F) and 20S proteasome (G). The arrows indicate the colocalization of ITCH with perinuclear cytoplasmic aggregates of GFP-Δ9CAT misfolded proteins. A rhodamine-conjugated secondary antibody was used to stain ITCH (D), Ub (E), p62 (F) and 20S (G). Full-length blots are presented in Supplementary Figure-S1. Scale bar, 20 μm.