PMC

Mutant tubby aggregates co-localize with Hsp70 (an aggresome marker), but not with markers for lysosomes (LysoTracker), the ER (ER-Tracker), mitochondria (MitoTracker), or the Golgi apparatus (GM130). COS-7 cells expressing GFP-mutant tubby were fixed and stained. Scale bars: 20 μm.

Overexpression of mutant tubby leads to aggresome formation. (A) COS-7 cells were transfected with GFP-mutant tubby. At 12, 24, 48, and 72 h post-transfection, the cells were fixed and imaged for GFP. (B) At 12 h post-transfection, the cells were incubated with or without 20 ng/ml nocodazole for 24 h and then imaged for GFP. Scale bars: 20 μm.

The C-terminal region of tubby is important for its nuclear translocation. Neuro-2A cells were transfected with vectors encoding GFP-tubby or GFP-mutant tubby, and grown for 24 h. After being starved for overnight, the cells were treated with 10 μM UTP, 100 nM BK, or 1 μM insulin for 2 h, and the cellular localizations of wild-type and mutant tubby were observed under confocal microscopy. Scale bars: 20 μm.

The C-terminal region of tubby is essential for its subcellular localization. (A) Frozen sections of mouse hippocampus were subjected to immunohistochemical analysis using an anti-tubby antibody. (B) A schematic representation of the utilized GFP-fused wild-type tubby, mutant tubby, and truncated tubby proteins. Numbers indicate positions with respect to the amino acid sequence. (C) Representative fluorescence micrographs of COS-7 cells are expressing GFP-fused proteins. COS-7 cells were transfected with constructs encoding GFP-tagged tubby proteins. Twenty-four hours later, the cells were starved overnight, fixed, and visualized by confocal microscopy. Nuclei were visualized with propidium iodide (PI). Scale bars: 20 μm. (D) At 48 h post-transfection, the cells were lysed and separated into Tx-100 soluble (supernatant) and insoluble (pellet) fractions. The fractions and total lysates were analyzed by Western blotting using anti-GFP and anti-actin antibodies.