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. 2013 Dec 20;8(12):e83096. doi: 10.1371/journal.pone.0083096

Figure 8. Degradation of C99 in acidic compartments is independent of its ubiquitination.

Figure 8

(A) H4 cells stably expressing GFP-tagged C99-F/P-D/A (C99) or C99-5K/R-F/P-D/A (C99-5K/R) were left untreated or treated for 16 h either with 100 µM CQ, 1 µM MG132, or with a combination of 100 µM CQ and 1 µM MG132. After cells were biotinylated on the cell surface with Sulfo-NHS-LC-Biotin and soluble extracts pulled down with NeutrAvidin-agarose, total and biotinylated proteins were analyzed by immunoblot with anti-GFP antibody. Immunoblot with anti-β-actin or anti-transferrin receptor (TfR) antibodies was used as loading control for total or biotinylated proteins, respectively. The positions of molecular mass markers are indicated on the left. (B) Confocal fluorescence microscopy of H4 cells stably expressing GFP-tagged C99-F/P-D/A (C99) or C99-5K/R-F/P-D/A (C99-5K/R) treated with a combination of 1 µM MG132 and 100 µM CQ for 16 h. Bar, 10 µm. (C) Densitometric quantification of the ratio AICDγ/C99 of H4 cells stably expressing GFP-tagged C99-F/P-D/A (C99) or C99-5K/R-F/P-D/A (C99-5K/R) left untreated or treated for 16 h with 1 µM MG132. (D) Densitometric quantification of the levels of C99 in H4 cells stably expressing GFP-tagged C99-F/P-D/A (C99) or C99-5K/R-F/P-D/A (C99-5K/R) left untreated or treated for 16 h with 1 µM MG132, or with a combination of 100 µM CQ and 1 µM MG132. (E) Densitometric quantification of the levels of C99 at the plasma membrane (PM) in H4 cells stably expressing GFP-tagged C99-F/P-D/A (C99) or C99-5K/R-F/P-D/A (C99-5K/R) left untreated or treated for 16 h with 1 µM MG132, or with a combination of 100 µM CQ and 1 µM MG132. Bar represents the mean ± SD (n = 3). *P<0.05; **P<0.01.