Fig. 5. Redistribution of eNOS-GFP in response to BK stimulation is abrogated by overexpression of the dominant negative dynamin-2 mutant, K44A. (A) To confirm protein separation by buoyant density gradient centrifugation, equal amounts of protein from each fraction were analyzed by SDS-PAGE and western blotting using caveolin pAb, and ß-COP pAb. Enrichment of caveolin, a marker of low buoyant density cellular membranes, was detected in the early fractions (#2-4), while ß-COP, a marker coat protein of Golgi and trans-Golgi, was enriched in the higher buoyant density fractions (#5-7). (B) eNOS-GFP ECV 304 cells were cotransfected with pcDNA V5 empty vector (E) or alternatively a V5 epitope-tagged dyn-2 K44A construct (K). 24 hours later, cells were stimulated with BK (10 µM for 10 minutes; +) or sham treatment (-). Four 100 mm dishes of cells from each experimental group were then prepared for sucrose gradient subcellular fractionation. Equal volumes of each fraction were analyzed for eNOS and V5-K44A expression by SDS-PAGE and western blot analysis using eNOS mAb and V5 mAb, respectively. As seen in the rectangular box in Fig. 5B, in cells transfected with empty vector (E), stimulation with BK (+), was associated with an enrichment of eNOS protein levels in the early fractions of the gradient as compared to cells in the absence of BK stimulation [see eNOS signal in lanes labeled E in fractions #1-4; (-) vs (+)]. In cells transfected with V5-K44A, the relative enrichment of eNOS in early fractions in cells after BK stimulation was no longer apparent [see eNOS signal in lanes labeled (K) in fractions #1-4; (-) vs (+)]. No major changes were observed in the distribution of eNOS in the heavy fractions in response to the various experimental conditions and Coommassie staining of SDS-PAGE gels demonstrated similar protein loading amongst the experimental groups within each fraction (not shown). Overexpression of K44A was confirmed by the detection of a 90 kDa band in total cell lysates from dishes transfected with the vector encoding V5-K44A, and overexpression did not influence total eNOS protein levels from cell lysates (see eNOS and V5 western blot bands under lanes labeled lysate). All experiments presented here were performed three times independently, with similar results.

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