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Fig. 6. Ectopic expression of Varp induces giant vacuoles in HeLa cells. (A) Lysates from HeLa cells stably expressing Myc-tagged Varp, VarpN, or Rabex5 were separated by SDS-PAGE and immunoblotted (IB) with an anti-Myc antibody. (B) Phase-contrast micrographs of living HeLa cells stably expressing Varp (a), VarpN (b), Rabex5 (c), or transiently expressing EGFP-Rab5Q79L (d). Bars, 50 µm. (C) Late endosomal and/or lysosomal markers are localized on the giant vacuoles in HeLa cells expressing the Varp protein. HeLa cells expressing Varp were labeled with (a-c) anti-Varp (green) and anti-EEA1 antibodies (red), or (g-i) anti-LAMP3 antibody (red). (d-f) HeLa cells expressing Varp were transfected with an EGFP-Rab7 construct and labeled with an anti-Varp antibody (red). (j-l) HeLa cells expressing EGFP-tagged Varp were labeled with a rabbit anti-p115 antibody (red). Bars, 10 µm. (D) HeLa cells expressing Varp were transfected with an EGFP-Rab21 construct and labeled with an anti-Varp antibody (red). Colocalization of Varp with EGFP-Rab7, LAMP3 or EGFP-Rab21 on the giant endosomes is also shown (arrows). ^*, giant endosomes induced by Varp. Bars, 10 µm.

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