Figure 5

Study of the GM2 hydrolytic process in BRV5CA1 and GRP94-silenced clones. (A) HexA, HexB and total activities in the cellular models. HexA activity is calculated by the difference of total activity and HexB activity by thermal denaturation. The percentages of HexA activity respect to the total activity are specified. (B) HexA activity measured using the specific substrate for HexA. The percentages of HexA of silenced clones are written down; (C) Western Blot for HexA and GM2-AP and relative quantification; Hela cells are used as positive control of HexA, JAR cells are used as a positive control of GM2-AP; the bands of interest have been cropped from the original gel (available as Fig. S4); (D) Immunofluorescence assay for GM2 and GM3 and relative quantification. Nuclei were stained in blue and GM2 and GM3 in red, ***p < 0.005; (E) Immunofluorescence assay of GM2 and co-localization with Lysotracker, in control and shGRP94-2 cells. Scale bars = 20 µm; (F) Immunofluorescence assay of LC3B and GM2, in control and shGRP94-2 cells. Scale bars = 20 µm; (G) q-RT-PCR of GM2A gene in the cell clones, ***p < 0.005.