Fig. 4.

The LGMD2H disease mutant of TRIM32 is not degraded by autophagy. (A) HeLa cells were transfected with mCherry–EYFP–TRIM32^WT, mCherry–EYFP–TRIM32^P130S or mCherry–EYFP–TRIM32^D487N as indicated, kept in normal medium (full medium, FM) or starved with HBSS for 2 h (starvation medium, SM) at 1 day post transfection and imaged by a Zeiss780 confocal microscope. The graphs to the right display manual quantification of RedOnly dots and yellow dots in n=30 cells, and are representative of three independent experiments. Scale bars: 10 µm. (B) GFP fluorescence intensities (arbitrary units) of EGFP–TRIM32^WT, EGFP–TRIM32^P130S and EGFP–TRIM32^D487N as measured by flow cytometry 24 h after promoter shut off in full medium (FM) or HBSS for 6 h (SM), supplemented with BafA1 2 µm (Baf) or MG132 20 µM (MG) as indicated. Bars show average mean±s.d. GFP intensity from three independent experiments (n=10,000 cells). P-values are as indicated; n.s., not significant (Student's t-test). (C) Representative images of the Flp-In EGFP-TRIM32 cells analyzed by the proximation ligation assay DuoLink using antibodies against GFP and p62. The graph represents the mean±s.d. relative number of colocalization dots with from three independent experiments. Each experiment includes z-stack images of n≈100 cells per condition, and was quantified using the Volocity software. P-values are as indicated; n.s., not significant (Student's t-test). Scale bars: 10 µm.