Figure S2.
Specific cysteine residues are required for activity and plasma membrane localization. (A) Activity of ABHD17A is not affected by individual mutation of N-terminal cysteine residues to serine. Acylation of GFP-NRas was detected by click-labeling with 17-ODYA (upper panel). Levels of immunoprecipitated NRas (middle panel) or total ABHD17A (lower panel) were detected by western blot using anti-GFP or FLAG antibody, respectively. (B) Quantification of GFP-NRas acylation in A. NRas acylation was normalized to vector. One-way ANOVA with Tukey’s multiple comparison test; n = 3, ns = P > 0.05, **** = P ≤ 0.0001. Statistical analysis is depicted with black font (comparison to vector) and blue font (comparison to WT). Error bars indicate STDEV. (C) Acylation of ABHD17A was not affected by mutating N-terminal cysteine residues individually. Acylation of ABHD17A-FLAG was detected by click-labeling with 17-ODYA (upper panel). Levels of immunoprecipitated ABHD17A were detected by western blot using anti FLAG antibody (lower panel). (D) Quantification ABHD17A-FLAG acylation in C. One-way ANOVA with Tukey’s multiple comparison test; n = 3, ns = P > 0.05. Statistical analysis shows comparison to WT ABHD17A. Error bars indicate STDEV. (E) Representative double cysteine mutant constructs show decreased plasma membrane association in BRET analysis. One-way ANOVA with Tukey’s multiple comparison test; n = 5, ns = P > 0.05, * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001, **** = P ≤ 0.0001. Error bars indicate STDEV. (F) Schematic of the predicted N-terminal helix of ABHD17A cysteine mutants showing acylation patterns of active and inactive constructs. Acyl groups are depicted with blue wavy lines. Source data are available for this figure: SourceData FS2. Refer to the image caption for details.

Specific cysteine residues are required for activity and plasma membrane localization. (A) Activity of ABHD17A is not affected by individual mutation of N-terminal cysteine residues to serine. Acylation of GFP-NRas was detected by click-labeling with 17-ODYA (upper panel). Levels of immunoprecipitated NRas (middle panel) or total ABHD17A (lower panel) were detected by western blot using anti-GFP or FLAG antibody, respectively. (B) Quantification of GFP-NRas acylation in A. NRas acylation was normalized to vector. One-way ANOVA with Tukey’s multiple comparison test; n = 3, ns = P > 0.05, **** = P ≤ 0.0001. Statistical analysis is depicted with black font (comparison to vector) and blue font (comparison to WT). Error bars indicate STDEV. (C) Acylation of ABHD17A was not affected by mutating N-terminal cysteine residues individually. Acylation of ABHD17A-FLAG was detected by click-labeling with 17-ODYA (upper panel). Levels of immunoprecipitated ABHD17A were detected by western blot using anti FLAG antibody (lower panel). (D) Quantification ABHD17A-FLAG acylation in C. One-way ANOVA with Tukey’s multiple comparison test; n = 3, ns = P > 0.05. Statistical analysis shows comparison to WT ABHD17A. Error bars indicate STDEV. (E) Representative double cysteine mutant constructs show decreased plasma membrane association in BRET analysis. One-way ANOVA with Tukey’s multiple comparison test; n = 5, ns = P > 0.05, * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001, **** = P ≤ 0.0001. Error bars indicate STDEV. (F) Schematic of the predicted N-terminal helix of ABHD17A cysteine mutants showing acylation patterns of active and inactive constructs. Acyl groups are depicted with blue wavy lines. Source data are available for this figure: SourceData FS2.

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