Tp401

Human cell cultures were collected in non-denaturing lysis buffer (50 mm Hepes pH 7.4, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100) supplemented with EDTA-free protease inhibitor cocktail (Roche). Then, human cell samples were lysed by passing 10 times through a 27 G needle attached to a 1 ml syringe. Regarding C. elegans samples, the worms were collected with M9 buffer and worm pellets were frozen with liquid N₂. Frozen worm pellets were thawed on ice and worm extracts were generated by glass bead disruption. Then, either worm or cellular debris was removed with 8000g spin for 5 min and protein concentration was determined with BCA protein assay. Approximately 100 μg of protein extract was supplemented with SDS at a final concentration of 0.5% and loaded onto a cellulose acetate membrane assembled in a slot-blot apparatus (Bio-Rad). The membrane was then washed with 0.2% SDS. In human cells and C. elegans extracts, the retained polyQ-expanded proteins were assessed by immunoblotting for anti-polyQ-expansion diseases marker (Millipore, MAB1574, 1:5000) and anti-GFP antibodies (AMSBIO, #TP401, 1:5000), respectively. In addition, extracts were also analyzed by SDS–PAGE to determine the total levels of the corresponding proteins using the aforementioned antibodies.

Anti GFP and anti tubulin immunohistochemical stainings were performed as described previously (Punnamoottil et al., 2008; Wilson et al., 1990). An incross of the BAC‐FTO‐1‐GFP line was set up and the embryos were collected and screened for GFP expression at 3 dpf. The embryos were then fixed in 4%PFA and dehydrated through a series of methanol washes. If not stored at −20°C, the embryos were immediately rehydrated by reverse washes. Following permeabilization in 2 mg/ml collagenase for 20 min and refixation, the embryos were incubated overnight at 4°C in blocking solution containing Rabbit polyclonal anti GFP antibody (1:1000; AMSBio TP401) and mouse monoclonal anti acetylated tubulin antibody (1:500; Sigma T7451). Primary antibodies were detected through incubation with secondary antibodies (Life Technologies: Alexa Fluor® 488 goat anti‐rabbit, A‐11034; Alexa Fluor® 594 Chicken Anti‐Mouse, A‐21201) overnight at 4°C.