Fig. 4: Phosphorylation of S126 influences the phase separation behavior of the LSD1 IDR. | Communications Biology

Fig. 4: Phosphorylation of S126 influences the phase separation behavior of the LSD1 IDR.

From: The kinase NEK6 positively regulates LSD1 activity and accumulation in local chromatin sub-compartments

Fig. 4

a Turbidity measurements at OD600 comparing the optical density increase of IDR-mV S126E with IDR-mV WT. (n = 3, mean ± SEM). b Quantification of partial droplet fluorescence recovery after photobleaching (FRAP) over time for IDR-mV WT and S126E. (n = 2 biological replicates with NWT = 72 and NS126E = 75 individual bleach events, error bars = median ± SD). c Box plots depicting of fluorophore recovery half-time during FRAP experiments of IDR-mV WT vs S126E. (n = 2 biological replicates with NWT = 72 and NS126E = 75 individual bleach events, box plot: 5-95 percentile; mean as center line, ****:p < 0.0001; unpaired t-test). d Box plots depicting fluorophore mobile fraction during FRAP experiments of IDR-mV WT compared to S126E. (n = 2 biological replicates with NWT = 72 and NS126E = 75 individual bleach events, box plot: 5-95 percentile; mean as center line, ***:p < 0.001; unpaired t-test). e Box plot depicting the size of phase-separated IDR-mV WT vs S126E droplets. Pictures were analyzed with Cell Profiler. (n = 2 with NS126E = 308, NWT = 363, NmV = 15 mV, box plot: 5-95 percentile; mean as center line, *:p < 0.1, ***:p < 0.001; ordinary one-way ANOVA followed by Dunnett’s multiple comparison). f Box plot showing the mean droplet intensity of phase-separated IDR-mV WT vs S126E condensates. Pictures were analyzed with Cell Profiler. (n = 2 with NS126E = 308, NWT = 363, NmV = 15, box plot: 5-95 percentile; mean as center line, ***:p < 0.001, ****:p < 0.0001, ordinary one-way ANOVA followed by Dunnett’s multiple comparison). g Bar graphs depicting the reversibility of droplet formation. The dilute phase concentration (csup) of the indicated LSD1 IDR variants was determined after addition of increasing KCl concentrations to determine alterations in droplet aging. (n = 3, mean ± SEM; **:p < 0.01, ordinary one-way ANOVA of data at 125 mM KCl followed by Dunnett’s multiple comparison). h Quantification of dilute phase concentration (csup) of IDR-mV WT compared to S126A/E. The saturation concentration (csat) was determined by increasing concentrations of the respective LSD1 variants. (n = 3, mean ± SEM).

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