Fig 1: Expression analysis of Siglec-3, Siglec-5 and Siglec-10 using IFA and Western blotting. (a) PK-15 cells were transfected with Siglec-3-, Siglec-5- and Siglec-10-encoding vector. Twenty-four hours post-transfection, the cells were fixed with 4 % PF and permeabilized with Triton X-100 for cytoplasmic staining, or not permeabilized for surface staining. IFA was performed using V5-specific antibody (Siglec; green) and Hoechst 33 342 (nuclei; blue). Scale bar: 25 µm (b) Western blot identification. HEK-293T cells were transfected with Siglec-3, Siglec-5 and Siglec-10. Twenty-four hours post-transfection, the cells were collected and lysed with lysis buffer. Afterwards, cellular lysates were treated or mock-treated with sialidase, EndoH or PNGase F and analysed by SDS-PAGE and Western blot. Primary antibody V5-specific mAb (GenScript; A00641) and secondary peroxidase-labelled goat anti-mouse IgG antibodies (Dako) were used for immunoblotting. For the detection of tubulin, an HRP-conjugated anti-alpha tubulin monoclonal antibody (Abcam; ab40742) was used.