Figure 5. Effects of TDD on AP2-clathrin... | Rockefeller University Press

Figure 5.

$Effects of TDD on AP2-clathrin interactions.(A–D) TIRF imaging of ARPE cells expressing α-eGFP-AP2. Data presented were obtained from a single experiment that is representative of three independent repeats. n = 10 videos for each condition. Number of analyzed tracks: 105,498 for –TDD and 102,207 for +TDD. (A and B) Single frame from TIRFM video (7.5 min/video, see Videos 3 and 4) and corresponding kymographs without (A) and with (B) TDD expression. Arrowheads point to examples of dynamic structures subjected to cmeAnalysis. (C) Mean fluorescence intensity traces of lifetime cohorts of analyzed AP2-labeled structures. Dashed lines indicate the elevation of background fluorescence intensity of AP2 as quantified in D. (E) Representative Western blot of α-AP2 from subcellular fractionation of ARPE cells with or without TDD expression. (F–K) Dual-color TIRFM imaging of ARPE cells expressing both mRuby-CLCa and α-eGFP-AP2. (F–H) Dual-color cmeAnalysis was performed using AP2 as primary channel. Data presented were obtained from a single experiment that is representative of two independent repeats. n = 10 videos for each condition. Number of analyzed tracks: 106,310 for control and 77,932 for +TDD. The percentage of AP2 structures with (CLC+) and without (CLC−) detectable clathrin is shown for –TDD and +TDD cells. (G and H) Comparison of lifetime distributions of all detected AP2 structures and those with (CLC+) or without (CLC−) detected clathrin in –TDD (G) and +TDD (H) cells. (I–K) Same as F–H, except CLC was assigned as the primary channel. Number of analyzed tracks: 172,166 for –TDD and 133,604 for +TDD. Error bars are SD. ***, P ≤ 0.001.$

Effects of TDD on AP2-clathrin interactions. (A–D) TIRF imaging of ARPE cells expressing α-eGFP-AP2. Data presented were obtained from a single experiment that is representative of three independent repeats. n = 10 videos for each condition. Number of analyzed tracks: 105,498 for –TDD and 102,207 for +TDD. (A and B) Single frame from TIRFM video (7.5 min/video, see Videos 3 and 4) and corresponding kymographs without (A) and with (B) TDD expression. Arrowheads point to examples of dynamic structures subjected to cmeAnalysis. (C) Mean fluorescence intensity traces of lifetime cohorts of analyzed AP2-labeled structures. Dashed lines indicate the elevation of background fluorescence intensity of AP2 as quantified in D. (E) Representative Western blot of α-AP2 from subcellular fractionation of ARPE cells with or without TDD expression. (F–K) Dual-color TIRFM imaging of ARPE cells expressing both mRuby-CLCa and α-eGFP-AP2. (F–H) Dual-color cmeAnalysis was performed using AP2 as primary channel. Data presented were obtained from a single experiment that is representative of two independent repeats. n = 10 videos for each condition. Number of analyzed tracks: 106,310 for control and 77,932 for +TDD. The percentage of AP2 structures with (CLC+) and without (CLC−) detectable clathrin is shown for –TDD and +TDD cells. (G and H) Comparison of lifetime distributions of all detected AP2 structures and those with (CLC+) or without (CLC−) detected clathrin in –TDD (G) and +TDD (H) cells. (I–K) Same as F–H, except CLC was assigned as the primary channel. Number of analyzed tracks: 172,166 for –TDD and 133,604 for +TDD. Error bars are SD. ***, P ≤ 0.001.