ere separated by centrifugation at 15,000 rpm for 15 min. M2 flag beads was equilibrated on a rotary shaker using 50 mM TrisCl pH 7.4 with 150 mM NaCl. The clarified lysate was loaded on the beads and bound on rotary shaker for 2h at 4C. The beads were washed with the above buffer 3 times and then incubated with sample buffer at 70C. The samples were loaded onto SDSPAGE gels immuno-blotted and detected using anti-Flag and anti-HA antibodies. For endogenous protein IP, 50 g anti DAZAP1 antibody was immobilized onto protein A/G agarose beads overnight at 4C on a rotary shaker. HEK293T cells were grown in DMEM media at 6080% confluence and lysed using lysis buffer. Binding and washing conditions are same as above. Elution was performed using two 70 l 1M glycine wash and neutralized with 1M Tris prior to immunoblotting. Uncropped images of Western Blots are shown in Supplementary Fig. 12. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Nat Commun. Author manuscript; available in PMC 2014 August 27. Choudhury et al. Page 16 Phosphorylation assay Author Manuscript Author Manuscript Author Manuscript Author Manuscript Phosphorylation studies were performed on HEK 293T cells. The cells were transfected with reporter and protein expression constructs and further serum starved overnight prior to addition of 1mM PMA for 40 min. The RNA was extracted using Trizol method and the splicing assay was performed as mentioned above. Addition of MEK inhibitor was followed 2 h post transfection and cells were collected after 1618hpost treatment for protein and splicing analysis. The phospho-mutants were prepared using site directed mutagenesis kit. The microscopy analysis was done on Olympus confocal microscope by counter staining the nucleus with DAPI. DAZAP1 or double mutants were made as a fusion construct with pDsRed2. Cells were cultured on poly Llysine coated slide and transfected using lipofectamine 2000 and fixed using 4% formaldehyde. Immuno fluorescence was performed using anti-flag M2 primary antibody and subsequently detected by a secondary anti-mouse goat IgG antibody against flag tagged hnRNPA1. Phospho DAZAP1 was resolved on PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19844160 PhostagTM Gels 54, immuno-blotted and detected using anti Flag mouse antibody. Total Erk, phospho Erk1/2 and DAZAP1 antibody were from Santacruz Biotechnology Inc. hnRNPA1 antibody was purchased from Cell Signaling. HA-tagged proteins were detected using anti mouse mAb from Cell Signaling. Uncropped images of Western Blots are shown in Supplementary Fig. 12. High throughput sequencing and analysis Cell lines stably transfected with shRNA 3 or shRNA4 vector against DAZAP1 were created. Total RNA from shRNA knock down lines or a mock transfected lines were purified using TrizolTM method and subsequently cleaned using RNAeasy Kit the bound RNA digested with RNAse free DNAse in column as per manufacturer’s instruction. Total RNA not exceeding 3 g was further used to purify 
poly A RNA using Illumina buy Vatalanib TruSeq Total RNA Sample Prep kits with Ribo-Zero Human for removal of cytoplasmic rRNA. The mRNA purified was further analyzed using Bioanalyzer prior to generation of cDNA with bar coded ends. The TruSeq SR Cluster Kit v3-cBot-HS was next used to cluster generation reagents for the cBot cluster amplification system in a pre-mixed, 96-well plate format that requires minimal reagent preparation. The cluster generation and sequencing were carried out by standard procedures in HiSeq 2000 Illumina platform, and
