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. Author manuscript; available in PMC: 2015 Sep 1.
Published in final edited form as: Nat Cell Biol. 2015 Feb 16;17(3):300–310. doi: 10.1038/ncb3112

Figure 6. ATG12-ATG3 conjugation promotes multiple Alix functions.

Figure 6

(a) Whole cell lysates (WCL) and exosomal fractions (Exo) from indicated cell types were resolved by SDS-PAGE and stained by Coomassie (left) or immunoblotted for exosomal markers anti-Alix, anti-TSG101, anti-GAPDH, and anti-HSC70 (right) to measure relative exosome release. Bottom: Quantification of exosomal protein release (mean ± SEM; n = 7 independent experiments for Alix and GAPDH, n = 6 for HSC70, n = 5 for TSG101). Statistical significance calculated using ANOVA, followed by Tukey’s HSD test (***P < 0.001, **P < 0.01, *P < 0.05). (b) Whole cell lysates (WCL) and exosomal fractions (Exo) from wild-type MEFs transfected with siRNA against Alix (ALIX) or a non-targeting control (CTL) were resolved by SDS-PAGE and immunoblotted for exosomal markers anti-Alix and anti-HSC70 to measure relative exosome release. Bottom: Quantification of exosomal HSC70 protein levels (mean ± SEM; n = 3 independent experiments). Statistical significance calculated using unpaired two-tailed t test (***P < 0.001). (c) Indicated cell types were transfected with YFP-tagged murine leukemia virus Gag (MLV Gag-YFP). Virus-like particles (VLPs) and whole cell lysates (WCL) were resolved by SDS-PAGE and immunoblotted for anti-Gag to measure relative viral budding. Bottom: Quantification of Gag release (mean ± SEM; n = 4 independent experiments). Statistical significance calculated using ANOVA, followed by Tukey’s HSD test (**P < 0.01, *P < 0.05). Uncropped images of blots are shown in Supplementary Figure 6.