The RPEL-like motif of GJA1-20k facilitates Cx43 trafficking. (A) Representative frame of a live-cell video of a HeLa cell transfected with LifeAct (0.5 µg) to visualize the cytoskeleton and EB1-YFP (0.5 µg) tracks to visualize dynamic microtubule movement. These cells were also expressing GJA1-WT (1 µg) with no fluorescent tag in order to express full-length Cx43 and its downstream isoforms, or GJA1-43k to express full-length Cx43 in the absence of GJA1-20k; scale bars = 10 µm. White arrows indicate location of intracellular actin puncta. (B) Quantification of EB1 local track thickness (n = 13–15 videos, 1 cell per video). *P = 0.0222 by two-tailed Mann–Whitney U test. (C) Quantification of actin puncta density for cells in A (n = 15 and 13 cells for GJA1-WT and GJA1-43k). Data are presented as mean ± SD. ***P < 0.001 by two-tailed Mann–Whitney U test. (D–G) Representative images of Cx43-NT, N-cadherin, and DAPI in HeLa cells transfected with GJA1-WT or GJA1-ΔαCT11 with or without GJA1-20k (GST used as control). Cx43 is located on cell border–labeled N-cadherin (arrowheads) but αCT11 mutation resulted in less trafficking (asterisks). Scale bars, 10 and 5 µm (insets). Cx43 localization is quantified as Cx43 intensity at cell border (E), total Cx43 (F), and Cx43 ratio of border to total (G). n = 47 (GJA1-WT + GST), 45 (GJA1-WT + GJA1-20k), 49 (GJA1-ΔαCT11 + GST), and 47 (GJA1-αCT11 + GJA1-20k) cells from three independent experimental repeats. ns = P > 0.05, *P < 0.05, **P < 0.01, ****P < 0.0001 by two-way ANOVA with Tukey’s multiple comparisons test.