Anti laminin b1

Human embryonic kidney 293 T (HEK293T) cells were cultured as previously described and transfected using polyethylenimine. For western blotting analysis, cells were washed with ice-cold PBS and scraped in 100 ml lysis buffer (150 mm NaCl, 2% sodium dodecyl sulfate, 10 mm Hepes pH 7.4, 2 mm EDTA) plus protease inhibitor cocktail (Roche Diagnostics). Total lysates were sonicated and centrifuged at 13,000 rpm for 10 min at 4 C. Cells lysates were denatured at 95 C in sample buffer and processed for 4–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and electro-transferred onto nitrocellulose membranes (Millipore). Immunoblotting was done in 5% non-fat dry milk dissolved in Tris-buffered saline using the following antibodies: anti-FUS (1:2000, sc-25-540, Santa Cruz); anti-Tubulin (1:10000, Sigma); anti-Calnexin (1:5000, Enzo); anti-Laminin B1 (1:1000, Abcam). Immunoreactivity was detected using IRDye-conjugated Goat Anti-Rabbit or Anti- Mouse IgG (Li-Cor), and visualized using Odissey Imaging System (Li-Cor). Experiments were ran in triplicate using three independent lysate preparations from cultured cells.