Dcp1a

The CCHCR1-HEK293 cell lines, vector control, and wild type HEK293 cells were grown on cover slips coated with rat tail collagen I (Gibco, Invitrogen) and fixed with 4% paraformaldehyde solution. After fixation cells were permeabilized with 0.1% Triton-×100 in PBS. The samples were incubated 1 h at room temperature with the antibodies against P-body markers EDC4 (rabbit polyclonal, Cell Signaling) and DCP1A (mouse monoclonal, Abnova), and centrosome marker γ-tubulin (mouse monoclonal, Sigma). Alexa Fluor 555 and 488 conjugated IgGs (Invitrogen, Molecular Probes) were used as secondary antibodies and the nuclei stained with DAPI (4′,6-diamidino-2-phenylindole, Sigma-Aldrich). The pictures were taken with Zeiss LSM 5 Duo confocal microscope. Differences in localization of CCHCR1 isoforms with centrosomal P-bodies were determined by counting colocalized staining of CCHCR1 and EDC4 and DCP1A in each CCHCR1-overexpressing cell line blindly (Additional file 8: Supplementary Information about qPCR and co-localization of CCHCR1 with P-body markers).

Cells (SKOV3) were cultured on glass coverslips for 24 h. Cytoplasmic mRNP granule formation was triggered by treatment with 5 μg/ml sodium arsenite (Sigma-Aldrich) for one hour. Cells were washed before being incubated with PHEM fixative (4% PFA, 60 mM PIPEs, 25 mM HEPES, 10 mM EGTA, 4 mM MgCl₂) for 10 min. Fixative was removed and cells were washed and blocked in PBSTB buffer (1% BSA, 0.1% TritonX-100) for 1 h. Cells were stained with antibodies to DCP1A (Abnova), PABP (Abcam) and LARP1 (SDIX- Novus Biologicals). Primary antibody solution was applied and incubated overnight at 4°C. After washing, Alexa Fluor-conjugated secondary antibodies (Life Technologies) were applied and incubated at room temperature for 1 h. Cells were washed and mounted with ProLong Gold mounting medium with DAPI (Life Technologies). Immunofluorescence staining was analysed using Leica 500 confocal microscope and images processed with Leica LAS AF lite software.