Mouse monoclonal antibodies against SMN were acquired from BD Transduction Laboratories and used at a dilution of 1:100 for immunofluorescence. Protocols for immunofluorescence, image acquisition and western blotting were conducted as described previously . Cell lysate generation and immunoprecipitations were performed as previously described , except RIPA buffer was used. Reduction of endogenous coilin message was accomplished using a siRNA that targets the 39 untranslated region of coilin, obtained from Integrated DNA Technology . The 39 UTR of coilin has been deleted in the ABT-263 citations GFP-coilin WT and phosphomutant constructs, allowing for the specific knockdown of the endogenous message. The non-targeting siRNA#2 was obtained from Thermo Scientific . Lipofectamine 2000 was used to introduce the coilin and control siRNAs into cells according to the manufacturer��s directions. Proliferation assays were performed using the cell titer blue reagent from Promega according to the manufacturer. For proliferation assays employing transient transfections, cells were transfected with the various GFP-tagged coilin constructs as described above. 24 h after Y-27632 transfection, 5000 cells per well of a 96-well dish were seeded. The fluorescence was read 24 h, 48 h and 72 h after seeding with a FLx800 Spectrophotometer using a 490/540 filter set. The readings obtained from 72 h were divided by the readings obtained from the 24 h time point, and all values were normalized to WT. For proliferation assays using the stable cell lines in the presence of endogenous coilin, 5000 cells per well of a 96-well dish were seeded in the presence of 1 ug/ml doxycycline to induce expression of the various GFP-coilin proteins or left untreated. Plates were then read 24 h, 48 h, and 72 h after seeding. The values from the 72 h reading were divided by the 24 h reading, and each line was normalized to the untreated condition for that cell line. For proliferation assays using the stable cell lines and siRNA transfection, cells were seeded in the presence of 1 mg/ml doxycycline to induce expression or left untreated. 18 h later, doxycycline treated and untreated cells were transfected with control or coilin siRNA. 24 h post-transfection, 5000 cells per well of a 96-well dish were seeded.