Anti 14 3 3 β

Proteins extracts (20 mg) from each of the two conditions compared (control and infected samples) were loaded on 12% SDS-PAGE and transferred onto a PVDF membrane (Millipore, Bedford, MA, USA). Membrane were blocked for 1 h with 5% dried milk in TBS buffer with 0.05% Tween-20 (TBS-T) and incubated overnight at 4°C with rabbit polyclonal antibody anti-14-3-3 β (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) or anti-FKBP4 (Abcam, Cambridge, UK) at 1:1000 v/v dilution in 5% skimmed milk in TBS-T. Secondary peroxidase-linked anti-rabbit antibody (1:10000 dilution) was used to generate immunocomplexes, that were visualized with Immobilon Western chemiluminescent HRP substrate (Millipore). Triplicate blots were carried out for both proteins. Optical densities of the immunoreactive bands were measured using ImageJ 1.43u software. Statistical differences between groups were evaluated using a two-tailed t-test. To confirm equal sample loading, all membranes were reblotted with anti-GAPDH monoclonal antibody (GenScript, Picastaway, NJ, USA).

The following antibodies were used for immunoblotting, immunocytochemistry and immunoprecipitation: anti-SRPK1 (611072, BD Biosciences), anti-Hsp90 (610418, BD Biosciences), anti-Tip60 (DR1041, Calbiochem), anti-CLK1 (ab74044, Abcam), anti-myc (#2276, Cell Signalling Tech.), anti-Flag (#14793, Cell Signaling Tech.), anti-PARP (#9542, Cell Signaling Tech.), anti-phospho-H2AX (Ser139) (#9718, Cell Signaling Tech.), anti-β-actin (sc-47778, Santa Cruz), anti-GAPDH (#5174, Cell Signaling Tech.), anti-14-3-3β (sc-59419, Santa Cruz) and anti-Lamin A (sc-6214, Santa Cruz). The antibodies were diluted 1:100 for immunofluorescence and immunoblotting. For Monoclonal antibody MAb104, hybridoma cells (ATCC® CRL-2067™) were raised in 10% FBS-containing RPMI, and the conditioned medium was collected every two days, which could be directly used for immunoblotting.