Filter retardation assay, preparation of protein extracts

For transiently transfected NSC34 cells, 1.5 µg of the PBS or NP40-soluble protein extracts (quantified used the BCA assay as described above) were loaded onto 0.2 µm cellulose acetate membrane (Whatman, GE Healthcare) and filtered through a Bio-Dot SF Microfiltration Apparatus (Bio-Rad). An equal volume of the corresponding NP40-soluble extract was loaded in the case of NP40-insoluble extracts. Slot-blots were probed as described for WB to detect GFP-TDPs or FLAG-TDPs retained insoluble species. For stably transfected GFP-TDPs-NSC34 cell lines, cells were harvested 72 h after induction with 1 μg/ml of doxycycline, and samples were prepared as described above for transiently transfected cells. 9 µg of the NP40-detergent soluble protein extracts were loaded onto cellulose acetate membranes and filtered. An equal volume of the corresponding NP40-soluble extract was loaded in the case of NP40-insoluble extracts. Slot-blots were probed with GFP antibody as described above for WB. Densitometric optical analysis of slot-blots was performed and represented as mean ± SEM (n = 3).