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. 2016 Aug 30;11(8):e0162050. doi: 10.1371/journal.pone.0162050

Fig 5. Comparison of WT, Site 1 and Site 2 mutated AP180 and CALM in a transferrin uptake assay.

Fig 5

COS-7 cells were transfected with GFP-tagged WT, Site 1, Site 2 and Site 1&2 mutants for (A) AP180 and (B) CALM. Alexa Fluor 594 labelled Tfn (red) was added and the amount of uptake was measured as an assay of endocytosis function and clathrin sequestration. Note that in (A) and (B), respectively, GFP-tagged WT AP180 and CALM exhibit a dominant negative effect due to sequestration of clathrin. Data is expressed as a fraction of Tfn uptake in untransfected control cells ± SEM (n = 4; ***, P < 0.001 compared to WT; ns = not significant). The approximately equal expression level of AP180 and CALM fusions with GFP is shown in panels (C) and (D), respectively, which was produced from COS-7 cell lysis, SDS-PAGE and Western blotting with anti-GFP or anti-actin (control for protein content).