Fig. 5
NEU1 alters α5β1 and FN distribution and expression on cell membrane. a Membrane and cytoplasmic proteins of YTS-1/Ctrl and YTS-1/NEU1 cells were extracted with a kit. NEU1 expression was analyzed by Western blotting and presented as IOD as in Fig. 2c. Tubulin was the cytoplasmic protein control and caveolin was the membrane control. b DIM and DSM proteins were extracted as described in Supplementary information/Methods, and expression of FN, NEU1, α5, and β1 was analyzed by Western blotting. Caveolin (caveolae marker of DIM) was used as reference. Representative results from two independent experiments are shown. c Co-IP assay: cells were lysed by modified RIPA buffer, incubated with anti-FN antibody (which binds to protein A/G Plus agarose) overnight at 4 °C with gentle rotation. FN, β1, and NEU1 in precipitate were assayed by Western blotting. Tubulin was used as loading control. d FN immunoprecipitated from YTS-1 cell lysate or purified from cells were analyzed by Western blotting and Lectin blotting. e FN (50 μg/mL) was coated on 12-well plates and treated with 1 U/mL sialidase for 1 h at 37 °C. KK47 cells (2 × 105) were cultured on plates coated with normal FN and desialylated FN for 18 h, lysed with RIPA buffer, and subjected to SDS-PAGE and Western blotting. f Binding affinity between FN and integrin α5β1 was detected by modified ELISA method (see Methods). FN were fixed on to ELISA plates and treated with sialidase. Then integrin α5β1 were coated onto plates to interreact with FN. HRP conjugated anti-integrin β1 antibody and TMB-ELISA Substrate Solution were used to detect binding ratio. g YTS-1/Ctrl and YTS-1/NEU1 cells were seeded onto glass cover slips (diameter 12 mm) in 24-well tissue culture plates, fixed with 2% fresh paraformaldehyde/ PBS, blocked with 1% BSA/ PBS, and stained with anti-FN, anti-caveolin, anti-α5, anti-NEU1, or anti-LAMP2, as described in Supplementary information /Methods. For each antibody staining, clear DAPI staining of nuclei and merged photos are shown