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Fig. 5. Ectopically expressed Varp colocalizes with endosomal markers. (A) HeLa cells stably expressing Varp-Myc were used in these assays. (a-c) Cells were fixed and double labeled with a rabbit polyclonal anti-Varp antibody (green) and a mouse anti-EEA1 antibody (red). (d-f) Cells were transfected with an EGFP-Rab5a construct and labeled with anti-Myc antibody (red). (g-h) Cells were labeled with 25 µg/ml Alexa 594-conjugated human transferrin in DMEM at 37°C for 10 minutes and washed with ice-cold PBS, and then fixed and immunostained with an anti-Varp antibody (green). (B) Control HeLa cells or HeLa cells expressing the Varp protein were transfected with an EGFP-Rab5a construct and labeled with an anti-Varp antibody (red) and an anti-EEA1 antibody (blue). Arrows indicate the colocalization of Varp, EEA1 and Rab5. (C) Control HeLa cells or HeLa cells expressing the Varp protein were transfected with an EGFP-Rab21 construct and labeled with an anti-Varp antibody (red) and an anti-EEA1 antibody (blue). Arrows indicate the colocalization of Varp, EEA1 and Rab21. (D) Control HeLa cells or HeLa cells expressing the Varp protein were transfected with an EGFP-Rab7 construct and labeled with an anti-Varp antibody (red) and an anti-EEA1 antibody (blue). Arrows indicate colocalization of Varp, EEA1 and Rab7, and arrowheads indicate colocalization of Varp and Rab7, but not EEA1. Bars, 10 µm.
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