Fig 1: Complex formation of the Rab proteins with C9orf72, analyzed by size-exclusion chromatography.Complex formation of Rab5A (A), Rab7A (B) and Rab11A (C) with full-length C9orf72 was analyzed on a Superdex 200 10/30 column (GE Healthcare, Buckinghamshire, UK) by monitoring the UV absorption at 280 nm (left). Eluted fractions were subjected to denaturing polyacrylamide gel electrophoresis (SDS–PAGE) to verify Rab and C9orf72 co-elution (right). An amount of 21 µM C9orf72 and 48 µM Rab were applied per gel filtration run. (D) Western blot analysis of the eluted complexes: C9orf72-Rab5A (left panel), C9orf72-Rab11A (middle panel) and C9orf72-Rab7A (right panel). The band around the 55 kDa mark is C9orf72 and the band at around 25 kDa mark all the three panels is the Rab protein from the respective complex. Detection was carried out using an affinity purified rabbit polyclonal anti-C9orf72 (primary) antibody (1:1,000) from Santa Cruz Biotechnology (sc-138763) in combination with: anti-Rab5A (primary) antibody (1:400) for C9orf72-Rab5A complex, anti-Rab7A (primary) antibody (1:200) for C9orf72-Rab7A complex and anti-Rab11A (primary) antibody (1:200) for C9orf72-Rab11A complex. The Rab antibodies were from a sampler kit bought from Cell Signaling Technology (9385).