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. 2012 Aug 28;7(8):e44150. doi: 10.1371/journal.pone.0044150

Figure 5. KCa2.3 enters Rab5 positive endosomes.

Figure 5

BLAP-tagged KCa2.3 was co-expressed with DsRed-tagged WT (A), S34N (B) or Q79L (C) Rab5 in HEK293 cells. KCa2.3 was labeled with streptavidin-Alexa488 and localization evaluated by confocal fluorescence after 0 or 3 hrs at 37°C. KCa2.3 is localized to the plasma membrane at 0 hrs (upper panels) in each case, as expected. After 3 hrs (lower panels), endocytosed KCa2.3 co-localizes with WT Rab5 (A) as is clear in the merge (yellow color), whereas S34N appears to slow endocytosis (B). As is apparent, Q79L Rab5a induces the formation of intracellular vacuoles (C) and endocytosed KCa2.3 accumulates on these vacuoles; confirming co-localization of KCa2.3 and Rab5. D. Co-IP of myc-tagged KCa2.3 with DsRed-tagged Rab5. KCa2.3 was immunoprecipitated using an anti-myc Ab (lanes 1, 2, 4, 5, 6) or an anti-V5 Ab as IgG control (lane 3) and subsequently IB using an anti-Rab5 Ab. Q79L Rab5 was detected by IB (lane 6), confirming an association between KCa2.3 and Rab5. Note that an association was not detected between KCa2.3 and either WT or Q79L Rab5 suggesting these associations are very transient in nature. Bottom Panel confirms equivalent Rab5 expression (20 µg total protein loaded per lane). Data are representative of 3 experiments.