Figure S5.
Roles of the PTEN-like domain and clathrin-binding domain of auxilins in the endocytic and secretory pathways. (A) AP2-TagRFP+/+ cells with or without GAK (AP2-TagRFP GAK-KO) treated with control siRNA or siRNA targeting Aux1 for 3 d (two sequential transfections), then subjected to transient expression of the indicated EGFP-tagged constructs for additional 1 d followed by measurements of Alexa Fluor 647–conjugated transferrin uptake by flow cytometry. The plots (left) and equivalent histograms (right) show comparisons of the internalized transferrin (37°C with acid wash) in the absence or presence of low and high levels of ectopic expression of the indicated constructs. (B) The GAK-KO AP2-TagRFP+/+ cells were treated with siRNA targeting endogenous Aux1 and then transfected for transient expression of EGFP-tagged full-length Aux1 (left) or Aux1 lacking the PTEN-like domain (right). The cells were imaged at 1-s intervals for 300 s by TIRF microscopy. The averaged fluorescence intensity traces (mean ± SE) for AP2-TagRFP (red) and EGFP-tagged constructs (green) were identified in nine and seven cells, respectively, and then grouped in cohorts according to lifetimes. The numbers of analyzed traces are shown above each cohort. (C) The GAK-KO AP2-TagRFP+/+ cells were treated with siRNA targeting endogenous Aux1 and then transfected for transient expression of EGFP-tagged constructs as indicated. The cells with EGFP expression at a similar level as the endogenous auxilins were imaged at 1-s intervals for 300 s by TIRF microscopy. Distribution of the maximum number of EGFP-tagged molecules recruited during the uncoating burst (from left to right: 363 traces from five cells, 348 traces from six cells, 587 traces from five cells, 221 traces from five cells). (D) Genomic PCR analysis showing biallelic integration of TagRFP into the AP1S1 genomic locus to generate the clonal gene-edited cell line AP1-TagRFP+/+. (E) AP1-TagRFP+/+ cells were treated with lentivirus containing control shRNA or shRNA targeting GAK for 4 d, then subjected to transient expression of the indicated EGFP-tagged constructs for an additional 1 d, and volumetrically imaged by spinning-disk confocal microscopy (34 sequential optical sections spaced at 0.3 µm). Maximum-intensity z projections acquired using the same acquisition parameters as with gene-edited EGFP-GAK+/+ cells, making it possible to identify cells ectopically expressing at the same level as endogenous GAK. Scale bars, 10 µm.

Roles of the PTEN-like domain and clathrin-binding domain of auxilins in the endocytic and secretory pathways. (A) AP2-TagRFP+/+ cells with or without GAK (AP2-TagRFP GAK-KO) treated with control siRNA or siRNA targeting Aux1 for 3 d (two sequential transfections), then subjected to transient expression of the indicated EGFP-tagged constructs for additional 1 d followed by measurements of Alexa Fluor 647–conjugated transferrin uptake by flow cytometry. The plots (left) and equivalent histograms (right) show comparisons of the internalized transferrin (37°C with acid wash) in the absence or presence of low and high levels of ectopic expression of the indicated constructs. (B) The GAK-KO AP2-TagRFP+/+ cells were treated with siRNA targeting endogenous Aux1 and then transfected for transient expression of EGFP-tagged full-length Aux1 (left) or Aux1 lacking the PTEN-like domain (right). The cells were imaged at 1-s intervals for 300 s by TIRF microscopy. The averaged fluorescence intensity traces (mean ± SE) for AP2-TagRFP (red) and EGFP-tagged constructs (green) were identified in nine and seven cells, respectively, and then grouped in cohorts according to lifetimes. The numbers of analyzed traces are shown above each cohort. (C) The GAK-KO AP2-TagRFP+/+ cells were treated with siRNA targeting endogenous Aux1 and then transfected for transient expression of EGFP-tagged constructs as indicated. The cells with EGFP expression at a similar level as the endogenous auxilins were imaged at 1-s intervals for 300 s by TIRF microscopy. Distribution of the maximum number of EGFP-tagged molecules recruited during the uncoating burst (from left to right: 363 traces from five cells, 348 traces from six cells, 587 traces from five cells, 221 traces from five cells). (D) Genomic PCR analysis showing biallelic integration of TagRFP into the AP1S1 genomic locus to generate the clonal gene-edited cell line AP1-TagRFP+/+. (E) AP1-TagRFP+/+ cells were treated with lentivirus containing control shRNA or shRNA targeting GAK for 4 d, then subjected to transient expression of the indicated EGFP-tagged constructs for an additional 1 d, and volumetrically imaged by spinning-disk confocal microscopy (34 sequential optical sections spaced at 0.3 µm). Maximum-intensity z projections acquired using the same acquisition parameters as with gene-edited EGFP-GAK+/+ cells, making it possible to identify cells ectopically expressing at the same level as endogenous GAK. Scale bars, 10 µm.

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