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Fig. 3 | Journal of Experimental & Clinical Cancer Research

Fig. 3

From: Surfaceome analyses uncover CD98hc as an antibody drug-conjugate target in triple negative breast cancer

Fig. 3

Internalization of an antibody against CD98hc. A Subcellular localization of CD98hc in MDA-MB231 and HCC3153 cells was analyzed by immunofluorescence. Scale bar = 25 μm. B Cell surface immunoprecipitation of CD98hc. The different TNBC cell lines treated or not with 10 nM of anti-CD98hc for 2 hours at 4°C were lysed and cell extracts precipitated with protein A-sepharose. CD98hc in those immunoprecipitates was analyzed by Western. C Protease protection experiments of CD98hc. Cells were treated with proteinase K, lysed and subjected to immunoprecipitation and Western analysis with the anti-CD98hc antibody. D FACS analyses of CD98hc cell surface expression. MDA-MB231 cells were incubated with 10 nM of anti-CD98hc for 20 minutes at 37°C. Cells were detached, incubated with an anti-mouse antibody conjugated to FITC, and fluorescence intensity was measured with a BD FACSAria TM III cytometer. The yellow histogram corresponds to signals from cells incubated with the secondary antibody alone, whereas the pink histogram represents the fluorescence due to the expression of CD98hc. E MDA-MB231 cells were seeded on coverslips and treated with 10 nM of anti-CD98hc for the times indicated. Scale bar = 25 μm. The images at the bottom of this section correspond to magnifications of a cell present in the images obtained at 24 hours. The white arrows indicate colocalization of CD98hc and LAMP1. Scale bar = 7.5 μm

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