Fig. 3.

Co-localization studies of CA IX and its mutants with endoplasmic reticulum and Golgi apparatus markers in SH-SY5Y neuroblastoma cells. A) CA IX (green) and calreticulin (red) were visualized by confocal fluorescence microscopy using specific antibodies. The merged images show clear CA IX co-localization with calreticulin only in CA IX MUT1. B) In this panel CA IX is shown in green and golgin in red. The two mutants do not show significant co-localization with golgin while a modest presence is detected for the WT protein. C) Unfolded protein response in the endoplasmic reticulum is shown by GRP78 expression levels in lysates of SH-SY5Y cells transfected with mock, CA IX WT, MUT1 and MUT4 vectors. The Western blot analysis shows a significant increase of GRP78 levels in SH-SY5Y cells overexpressing CA IX MUT1. CA IX protein in the same lysates is used as normalizer. Statistic was performed by unpaired t-test (***p < 0.005). Bars in 3A and 3B = 10 μm (enlargements = 5 μm). Uncropped images of the Western blot analysis are available as supplementary material (Succoio_supplementary_materials).