Epr9048b

Total protein lysates were extracted from tissue culture cells using Laemmli buffer, boiled and the protein concentration determined (BCA protein assay, Pierce). Thirty micrograms of protein was resolved on SDS-PAGE gels, transferred to PVDF membranes and detected with a rabbit monoclonal antibody for NOVA1 (Abcam, EPR13847, ab183024, 1:1000 dilution in 5% NFDM), PTBP1 (Abcam, EPR9048B, ab133734, 1:10,000 dilution in 5% NFDM), or PTBP2 (Abcam, EPR9891, ab154853, 1:1000 dilution in 5% NFDM). Protein loading was determined with antibodies against with histone H3 (Anti-Histone H3 antibody produced in rabbit, H0164; Sigma). Blots were imaged with Bio-Rad Chemidoc XRS + Molecular Imager and quantified with Bio-Rad Image Lab software. Analysis shown in Fig. 2b was one cell lysate with the blot repeated twice. Blots shown in Fig. 5b were repeated twice from two separate pulldowns. Figure 5d–f were performed twice in the laboratory with three biological replicates. Figure 6a and b blots were from 1 biological replicated repeated twice in the laboratory. Figure 6c–h are from biological triplicates repeated twice in the laboratory.