Figure 6. SCRAPS motif and the stutter... | Rockefeller University Press

Figure 6.

$SCRAPS motif and the stutter cysteine are required to interconvert YAP1 interaction between K14 and K15. (A) Representative co-immunoprecipitations of HeLa cells transfected with untagged K5 and EGFP-tagged WT K14, K15, and mutant K15. Pull-down of endogenous 14-3-3σ is depicted. (B) Differential levels of WT K15, relative to WT K14 or mutant K15, were observed in the soluble fraction. Transfection of keratin(s) did not affect endogenous 14-3-3σ levels. WB intensity was normalized to a loading control (histone H3). Dots represent three independent replicates, displaying mean and standard error. Comparisons were made using one-way ANOVA, ****P < 0.0001, ns = not significant. (B′) Endogenous 14-3-3σ pulls down more efficiently with WT K14 and mutagenized K15, as compared to WT K15. Equation to calculate 14-3-3σ pull-down efficiency is described in Materials and methods. Dots represent three independent replicates. Comparisons were made using one-way ANOVA, **P = 0.0055, *P = 0.0237, ns = not significant. (C) Representative micrographs of YAP1-14-3-3σ PLA performed on transfected HeLa cells. Cells were transfected with untagged K5 and EGFP-tagged WT and mutant K14 or K15. Merge panels show PLA punctae (red), EGFP-tagged keratin autofluorescence (green), and DAPI counterstain (blue). Scale bar = 10 µm. (D) Scatter plots representing YAP1-14-3-3σ PLA punctae counted per cell in three pooled independent replicates, displaying mean and standard deviation. Dots represent a sc. Comparisons were made using Mann–Whitney tests. ****P < 0.0001, **P = 0.0033, ns = not significant. (E) Transcription of a YAP1 target gene, CYR61, as measured via RT-qPCR in HeLa cells transfected with untagged K5 and WT and mutant K14 or K15. Dots represent six independent replicates. Comparisons were made using Mann–Whitney tests, **P = 0.0033, ns = not significant. Source data are available for this figure: SourceData F6.$

SCRAPS motif and the stutter cysteine are required to interconvert YAP1 interaction between K14 and K15. (A) Representative co-immunoprecipitations of HeLa cells transfected with untagged K5 and EGFP-tagged WT K14, K15, and mutant K15. Pull-down of endogenous 14-3-3σ is depicted. (B) Differential levels of WT K15, relative to WT K14 or mutant K15, were observed in the soluble fraction. Transfection of keratin(s) did not affect endogenous 14-3-3σ levels. WB intensity was normalized to a loading control (histone H3). Dots represent three independent replicates, displaying mean and standard error. Comparisons were made using one-way ANOVA, ****P < 0.0001, ns = not significant. (B′) Endogenous 14-3-3σ pulls down more efficiently with WT K14 and mutagenized K15, as compared to WT K15. Equation to calculate 14-3-3σ pull-down efficiency is described in Materials and methods. Dots represent three independent replicates. Comparisons were made using one-way ANOVA, **P = 0.0055, *P = 0.0237, ns = not significant. (C) Representative micrographs of YAP1-14-3-3σ PLA performed on transfected HeLa cells. Cells were transfected with untagged K5 and EGFP-tagged WT and mutant K14 or K15. Merge panels show PLA punctae (red), EGFP-tagged keratin autofluorescence (green), and DAPI counterstain (blue). Scale bar = 10 µm. (D) Scatter plots representing YAP1-14-3-3σ PLA punctae counted per cell in three pooled independent replicates, displaying mean and standard deviation. Dots represent a sc. Comparisons were made using Mann–Whitney tests. ****P < 0.0001, **P = 0.0033, ns = not significant. (E) Transcription of a YAP1 target gene, CYR61, as measured via RT-qPCR in HeLa cells transfected with untagged K5 and WT and mutant K14 or K15. Dots represent six independent replicates. Comparisons were made using Mann–Whitney tests, **P = 0.0033, ns = not significant. Source data are available for this figure: SourceData F6.

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