Figure 8. Parkin translocation to mitochondria is neither affected by laforin silencing nor by overexpression of laforin and malin.

SH-SY5Y cells were transfected for 24 h with pGFP-siRNA-control and HA-Parkin plasmids (first and second rows of images), with pGFP-siRNA-laforin (to silence laforin expression) and HA-Parkin plasmids (third and fourth rows) or with pEGFP-Parkin and Myc-laforin and HA-malin plasmids (fifth and six rows). Then, they were treated (+CCCP) or not with 10 μM CCCP in full medium for 4 h. Proteins were localized either by direct fluorescence (GFP-expressing plasmids) or by immunofluorescence using anti-HA, anti-Myc, anti-TOM20 or anti-CYTC as indicated. Cell nuclei were stained with DAPI and the fourth column shows the corresponding merge images. A representative image is presented in each case (A). Bar: 10 μm. To test the laforin silencing, SH-SY5Y cells were transfected with pGFP-siRNA-control and pGFP-siRNA-laforin plasmids. After 24 h of transfection, cellular extracts were prepared and the levels of endogenous laforin were analysed by Western blot using anti-laforin antibody and also anti-tubulin antibody as loading control (B). We found around 60% reduction in the levels of endogenous laforin. To check the overexpression levels, cellular extracts were prepared and the levels of laforin and malin were analysed by Western blot using anti-laforin and anti-HA antibodies, respectively (C). Tubulin was used as loading control. No differences were found in cells treated or not with CCCP.