Skip to main content
. Author manuscript; available in PMC: 2020 Jul 13.
Published in final edited form as: Nat Cell Biol. 2020 Jan 13;22(2):187–199. doi: 10.1038/s41556-019-0450-y

Figure 7. LC3-dependent EV loading and secretion (LDELS) requires neutral sphingomyelinase 2 (nSMase2) and FAN.

Figure 7.

a, Whole cell (WCL) and EV lysates from cells treated in the absence or presence of 5μM GW4869 for 24h and immunoblotted for the indicated marker proteins (n=2 biologically independent experiments). b, Nanoparticle counting for a representative experiment in Panel a (line=mean; n=1, 3 technical replicates). c, Whole cell (WCL) and EV lysates harvested from equal numbers of HEK293T cells stably expressing non-targeting (NT), ATG7 or nSMase2 (nSM2) shRNAs were immunoblotted for indicated proteins (n=3 biologically independent experiments). d, Quantification of indicated protein levels in EVs from equal numbers of stable knockdown cells in Panel c relative to non-targeting (NT) shRNA (mean ±s.e.m.; n=3 biologically independent experiments). Statistical significance calculated by one-way analysis of variance (ANOVA) coupled with Tukey’s post hoc. e, HEK293T cells co-transfected with FLAG-tagged FAN and myc-tagged LC3A, LC3B, LC3C, GABARAP (GR), GABARAPL1 (GRL1), GABARAPL2 (GRL2) or myc-BirA* were lysed, immunoprecipitated (IP) with anti-myc antibody, and immunoblotted (WB) with indicated antibodies (n=2 biologically independent experiments). f, Whole cell (WCL) and extracellular vesicle (EV) lysates harvested from equal numbers of HEK293T cells stably expressing non-targeting (NT), ATG7 or FAN shRNAs were immunoblotted for indicated proteins (n=3 biologically independent experiments). g, Quantification of indicated protein levels in EVs from equal numbers of stable knockdown cells in Panel c relative to non-targeting (NT) shRNA (mean ± s.e.m.; n=3 biologically independent experiments). Statistical significance calculated by one-way ANOVA coupled with Tukey’s post hoc test. h, Domain map and primary LC3-interaction region (LIR) in FAN. i,, Cells co-transfected with FLAG-tagged FAN and myc-tagged LC3A, LC3B, LC3C, GABARAP (GR), GABARAPL1 (GRL1), GABARAPL2 (GRL2) or myc-BirA* were lysed, immunoprecipitated (IP) with anti-myc antibody, and immunoblotted (WB) with indicated antibodies (n=2 biologically independent experiments). j, Whole cell lysate (WCL) and EV fractions from cells stably co-expressing non-targeting (NT) or FAN shRNA along with FLAG-tagged wild-type FAN (WT) or mutant FAN (F602A) were immunoblotted for indicated markers (n=2 biologically independent experiments). k, Quantification of indicated proteins in EVs from equal numbers of FAN knockdown HEK293T cells expressing FLAG-tagged wild-type FAN (WT) versus mutant FAN (F602A) (mean ± s.e.m.; n=3 biologically independent experiments). Statistical significance calculated by paired two-tailed t-test. Data and unprocessed blots available in Source Data Fig. 7.