Figure 4

RABEP2 does not associate with Rab5, early endosomes or macropinosomes. (A) HEK293 cells were transfected with Flag-tagged-Wild-type (WT), Ser204Glu (204E) or Ser200Ala/Ser204Ala (DBL) RABEP2 expression vectors. Cell lysates were prepared 24 h later and 0.5 mg subjected to immunoprecipitation with anti-FLAG. The entire immunoprecipitate (IP), and 7.5% of the remaining supernatants (SN), underwent SDS-PAGE and transfer to nitrocellulose. The membrane was cut into three by molecular mass and the upper section probed with anti-total RABEP2, the middle section with anti-GSK3 and the lower with anti-rab5 antibodies (original images of membrane sections are provided in Supplementary Information). (B) H9C2 cells were co-stained for endogenous Rab5 (green) and endogenous RABEP2 (red) along with DAPI staining of nuclei. Channels are shown individually and after merging images. The average Pearson’s coefficient for 26 different images was 0.219 ± 0.143, suggesting little, if any, co-localisation. (C) GFP-tagged WT RABEP2 (green) was overexpressed in HEK293 cells and cells loaded with Transferrin-Alexa Fluor594 (red) to label early endosomes. Channels are shown individually and after merging images. (D) To image F-actin in the cytoskeleton H9C2 cells were incubated for 5 mins in PBS containing 0.33 uM (final) AlexaFluor488 Phalloidin (Green), and counterstained for RABEP2 (red). Coverslips were visualised using a Leica SP5 laser scanning confocal microscope. Two representative images are provided. (E) H9C2 cardiomyoblasts were serum starved for 3 hours before incubation with DMSO vehicle, PMA or CT99021 for 1 hr as indicated. FITC-dextran (green) was added to the cells to label macropinosomes, endogenous RABEP2 was visualised by immunofluorescence (red), and nuclei were stained using DAPI. Two representative images for each condition are shown. The data indicates that GSK3 inhibition (CT) does not cause internalisation of FITC-dextran into macropinosomes, while the PKC inhibitor (PMA) is a known inducer of macropinocytosis (arrowheads indicate macropinosomes). These very distinct intracellular structures do not co-stain with RABEP2 implying that RABEP2 is not localised onto macropinosomes.