Journal: Cell Death Discovery

Article Title: ALS-associated mutant FUS inhibits macroautophagy which is restored by overexpression of Rab1

doi: 10.1038/cddiscovery.2015.30

Figure Lengend Snippet: Formation of autolysosomes is inhibited in cells expressing mFUS. ( a ) Neuro2a cells were co-transfected with HA-FUS (WT or mutant) and mCherry-GFP-LC3 for 18 h before being examined using confocal microscopy. White arrows indicate mCherry-LC3 (indicating lysosomes). Scale bar=10 μ m. ( b ) Quantification of the percentage of vesicles with mCherry-LC3 from at least 10 cells of each sample, n =3. ( c ) SHSY5Y cells were co-transfected with HA-FUS (WT or mutant) and pcDNA-LAMP2C vectors for 18 h. Cell lysates were collected and subjected to immunoblotting using anti-LAMP2 antibodies. Blots were stripped and re-probed with β -actin as a loading control. ( d ) Quantification of the relative intensities of LAMP2 in ( c ), normalised to untransfected cells, n =2. Mean±S.E.M. One-way ANOVA with Tukey post hoc test. * P <0.05, ** P <0.0001 versus Untr, ## P <0.0001, ### P <0.00001 versus WT.

Article Snippet: The antibodies used in this study included rabbit polyclonal antibody to HA (Sigma, Sydney, NSW, Australia; H6908) for immunocytochemistry (1 : 100), mouse monoclonal antibody to HA (Sigma; H3663) for immunocytochemistry (1 : 100) and immunoblotting (1 : 1000); rabbit polyclonal antibody to LC3 (Novus Biologicals, Littleton, CO, USA; NAB100-2220) for immunohistochemistry (1 : 100) and immunoblotting (1 : 1000), rabbit polyclonal antibody to Rab1 (Santa Cruz, Dallas, TX, USA; sc-28566) for immunoblotting (1 : 400), rabbit polyclonal antibody to NBR1 (Santa Cruz; sc-130380) for immunocytochemistry (1 : 50), rabbit monoclonal antibody to ATG9 (Abcam, Melbourne, VIC, Australia; ab108338) for immunocytochemistry (1 : 100), mouse monoclonal antibody to Myc (Santa Cruz; sc-40) for immunocytochemistry (1 : 50), mouse monoclonal antibody to HuR (Life Technologies, Mulgrave, VIC, Australia; 39-0600) for immunocytochemistry (1 : 100), mouse monoclonal antibody to TIAR (BD Transduction Laboratories, North Ryde, NSW, Australia; 610352) for immunocytochemistry (1 : 1000), mouse monoclonal antibody to LAMP2 (H4B4) (Abcam; ab25631) for immunoblotting (1 : 1000), mouse monoclonal antibody to β -actin (Sigma; A5441) for immunoblotting (1 : 4000), rabbit polyclonal antibody to GFP (Professor Mike Ryan’s laboratory) for immunoblotting (1 : 1000), Bafilomycin A1 was obtained from Sigma (B1793), sodium arsenite was obtained from Sigma (71287).

Techniques: Expressing, Transfection, Mutagenesis, Confocal Microscopy, Western Blot, Control