Alexa fluor 555 and 488 conjugated iggs

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).

For immunofluorescence studies, we cultured cells extracted from full-thickness samples12 (link). The cells were grown on cover slips with Rat Tail Collagen I (Gibco, Invitrogen) coating, and fixed with methanol for 5 min at −20 °C⁵⁶. The cover slip samples were incubated one hour at room temperature with the MTCO2 (Abcam, ab3298) and CARD6 antibodies. Alexa Fluor 555 and 488 conjugated IgGs (Invitrogen, Molecular Probes) were used as secondary antibodies and the nuclei stained with DAPI (Sigma-Aldrich). The pictures were taken with Zeiss LSM 5 Duo confocal microscope.