Fig_5.tif (2.17 MB)

Endogenous spastin-M1 is detected in purified LDs.

figure

posted on 2015-04-13, 06:19 authored by Chrisovalantis Papadopoulos, Genny Orso, Giuseppe Mancuso, Marija Herholz, Sentiljana Gumeni, Nimesha Tadepalle, Christian Jüngst, Anne Tzschichholz, Astrid Schauss, Stefan Höning, Aleksandra Trifunovic, Andrea Daga, Elena I. Rugarli

(A) Endogenous spastin in NSC34 cells was downregulated with siRNA oligonucleotides targeting either all spastin isoforms (Spast), or exon-4 containing isoforms (Ex4). Hereby identified isoforms are indicated. C, control siRNA. (B) NSC34 cells were treated with OA overnight. LDs were purified by sucrose gradient centrifugation and fractions from top (LD) to bottom (2–11) were analyzed by immunoblotting. The whole LD fraction and 1/5 of other fractions were loaded on the gel. PLIN2 was used as marker for LD, and BiP for the ER. LD, lipid droplet fraction; I, input; P, pellet. (C) The relative amount of spastin-M1 to spastin-M87 in the input (I) or in the LD fraction has been quantified using ImageJ. Results shown are the means ± SEM of three independent experiments. (D) HeLa cells co-expressing mCherry-spastin-M87 (red) and Flag-spastin-M1 (blue) were incubated with OA. LDs were stained with BODIPY 493/503 (green). Spastin-M1 recruits spastin-M87 to LDs. Enlargements of boxed areas are shown. Images are individual Z-stacks. Scale bar, 10 μm.