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. 2022 Apr 6;42(14):3011–3024. doi: 10.1523/JNEUROSCI.1508-21.2022

Figure 4.

Figure 4.

Mutant p62(C331A) increases α-syn and p62/LC3 secretion. A, Immunoblots for p62, LC3, and Flotillin1 in supernatants or EVs obtained after ultracentrifugation from CM collected from V5-α-syn-transfected WT p62 or mutant p62(C331A) knock-in cells. LC3-I was the predominant form in supernatants, while LC3-II was the major form in EVs. B, C, V5-α-syn levels in EVs (B) and supernatants (C) measured by ELISA. Data are mean ± SEM; n = 4. *p < 0.05, Student's t test. D, Vesicle flow cytometry of CM (concentrated with 100 kDa cutoff filter) from mNeonGreen-tagged human α-syn (mNG-α-syn)-transfected WT p62 or mutant p62(C331A) knock-in cells. mNG-α-syn+ events were determined using a gate set on a bivariate plot of diameter versus mNG fluorescence for untransfected WT cell-derived EVs. E, Quantification of mNG-α-syn-positive EVs by flow cytometry. Inset, Immunoblot of CM used for flow cytometry. Data are mean ± SEM; n = 3. *p < 0.05, Student's t test. F, G, α-syn levels by ELISA in EVs (F) and supernatants (G) ultracentrifuge fractions of conditioned media from isogenic control or A53T hiPSC-DA neuronal cultures in the presence or absence of 1 mm l-NAME. Data are mean ± SEM; n = 3. *p < 0.05, **p < 0.01, Sidak's correction. See Extended Data Figures 4-1, 4-2, and 4-3.