Figure 5. The RPEL-like tail of GJA1-20k... | Rockefeller University Press

Figure 5.

$The RPEL-like tail of GJA1-20k is sufficient to inhibit actin polymerization. (A) Representative immunofluorescence images of actin (phalloidin) and nuclei (Hoechst) in HeLa cells transfected with 0.5 µg of GJA1-11k-V5; scale bar = 10 µm. (B) Representative western blotting of actin in nuclear and cytoplasmic subcellular fractions of HeLa cells transfected with 0.5 µg of either GJA1-11k-V5 or a GFP-V5–negative control. Lamin-B1 was used as a loading control for the nuclear fraction and tubulin was used as a loading control for the cytoplasmic fraction. (C) Quantification of western blots of actin density in the nuclear and cytoplasmic fraction of HeLa cells transfected with either GJA1-11k-V5 or GFP-V5, normalized to either Lamin-B1 (nucleus) or tubulin- (cytoplasm) loading controls. Data are presented as mean ± SEM (n = 5 separate independent fractionation experiments) *P < 0.05 by two-tailed Mann–Whitney U test. (D) Representative TIRFM images of cell-free Alexa Fluor 488–labeled actin in the presence of increasing concentrations of the αCT11 peptide (RPRPDDLEI) or a reverse control (IELDDPRPR); scale bar = 5 µm. Reduction in actin polymerization is quantified by total fluorescence (A.U.) (E) Data are presented as mean ± SEM (n = 3–5 independent experiments per concentration). ns = P > 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by multiple t tests comparing the αCT11 peptide to the reverse control for each concentration with Holm–Sidak correction for multiple comparisons. No significant difference was found between the actin only group and the reverse control groups (all concentrations), ns = P > 0.05 by one-way ANOVA with multiple comparisons. (F) Representative TIRFM images of Alexa Fluor 488–labeled actin (green) in the presence of 250 nM of Alexa Fluor 555–labeled αCT11 (red) or reverse control (red) to show colocalization (merge); scale bar = 5 µm, images taken 2–3 min after the initiation of polymerization. Images were quantified by using Pearson’s correlation to measure colocalization. (G) Data are presented as box and whisker plots with boxes showing median, 25th, and 75th percentile, with whiskers spanning the 10th to 90th percentile (n = 35 frames from three independent experiments) *P < 0.05, ****P < 0.0001 by two-tailed Mann–Whitney U test. Source data are available for this figure: SourceData F5.$

The RPEL-like tail of GJA1-20k is sufficient to inhibit actin polymerization. (A) Representative immunofluorescence images of actin (phalloidin) and nuclei (Hoechst) in HeLa cells transfected with 0.5 µg of GJA1-11k-V5; scale bar = 10 µm. (B) Representative western blotting of actin in nuclear and cytoplasmic subcellular fractions of HeLa cells transfected with 0.5 µg of either GJA1-11k-V5 or a GFP-V5–negative control. Lamin-B1 was used as a loading control for the nuclear fraction and tubulin was used as a loading control for the cytoplasmic fraction. (C) Quantification of western blots of actin density in the nuclear and cytoplasmic fraction of HeLa cells transfected with either GJA1-11k-V5 or GFP-V5, normalized to either Lamin-B1 (nucleus) or tubulin- (cytoplasm) loading controls. Data are presented as mean ± SEM (n = 5 separate independent fractionation experiments) *P < 0.05 by two-tailed Mann–Whitney U test. (D) Representative TIRFM images of cell-free Alexa Fluor 488–labeled actin in the presence of increasing concentrations of the αCT11 peptide (RPRPDDLEI) or a reverse control (IELDDPRPR); scale bar = 5 µm. Reduction in actin polymerization is quantified by total fluorescence (A.U.) (E) Data are presented as mean ± SEM (n = 3–5 independent experiments per concentration). ns = P > 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by multiple t tests comparing the αCT11 peptide to the reverse control for each concentration with Holm–Sidak correction for multiple comparisons. No significant difference was found between the actin only group and the reverse control groups (all concentrations), ns = P > 0.05 by one-way ANOVA with multiple comparisons. (F) Representative TIRFM images of Alexa Fluor 488–labeled actin (green) in the presence of 250 nM of Alexa Fluor 555–labeled αCT11 (red) or reverse control (red) to show colocalization (merge); scale bar = 5 µm, images taken 2–3 min after the initiation of polymerization. Images were quantified by using Pearson’s correlation to measure colocalization. (G) Data are presented as box and whisker plots with boxes showing median, 25th, and 75th percentile, with whiskers spanning the 10th to 90th percentile (n = 35 frames from three independent experiments) *P < 0.05, ****P < 0.0001 by two-tailed Mann–Whitney U test. Source data are available for this figure: SourceData F5.

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