Temperature Compensation mechanisms have been experimentally addressed across circadian model systems, but much less is known about the related process of Nutritional Compensation, where circadian period length is maintained across physiologically relevant nutrient levels. Known compensation effectors directly regulate core clock factors to buffer the oscillator's period length from variables in the environment. ![]() IP, immunoprecipitation.Ĭompensation is a defining principle of a true circadian clock, where its approximately 24-hour period length is relatively unchanged across environmental conditions. Asterisks: black, dHSP68 red, dHSP68R45A. Co-IPs were probed with anti-FLAG antibody, and WBs were performed using the indicated antibodies. (H) Co-IPs from S2 cells transfected with FLAG-dpPER (500 ng), dsRNA against dHsp68 5 0 and 3 0 UTRs (15 μg each), and either wild-type or mutant R45A V5-tagged dHSP68 (500 ng each). Co-IPs were probed with anti-MYC antibody, and WBs were performed using the indicated antibodies. (G) Co-IPs from S2 cells transfected with dsRNA against dTrx 5 0 and 3 0 UTRs and plasmids expressing dTRX-CT-V5 (500 ng) and either MYC-dpCLK (500 ng) or MYC-dpCLKΔ19r (500 ng) in the presence or absence of dsRNA against dHsp68 5 0 and 3 0 UTRs (15 μg each). (F) Co-IPs from S2 cells transfected with dsRNA against dHsp68 5 0 and 3 0 UTRs (15 μg each) and plasmids expressing either dpCLK-V5 (500 ng) or dpCLKΔ19r-V5 (500 ng) with either wild-type or mutant R45A FLAG-tagged dHSP68 (400 ng each). (E) Co-IPs from S2 cells transfected with dsRNA against the 5 0 and 3 0 UTRs of both dTrx and dHsp68 (15 μg each) plasmids expressing MYC-dpCLK, FLAG-dpPER, or dTRX-CT-V5 (500 ng each) and either wild-type or mutant R45A FLAG-tagged dHSP68 (400 ng each). (D) Co-IPs from S2 cells transfected with MYCdpCLK and FLAG-dpPER expressing plasmids (500 ng each) without or with dsRNA targeting dHsp68 5 0 and 3 0 UTRs (15 μg each) and with either wild-type or mutant R45A FLAG-tagged dHSP68 (500 ng each). For each panel, one-way ANOVA, Tukey post hoc: *P < 0.05, **P < 0.01, and ns is nonsignificant (in black for repression) + P < 0.05 and ++ P < 0.01 (in blue for activation). Each value is mean ± SEM of three replicates. ![]() Firefly luciferase activity was computed relative to renilla luciferase activity. (C) The monarch per E box luciferase reporter (dpPerEp-Luc 10 ng) was expressed in the presence of dpBMAL1 and dpCLK expression plasmids (5 ng each), increasing doses of dpPER (amounts are given in nanograms) without or with dsRNA targeting endogenous dHsp68 5 0 and 3 0 UTRs (7.5 μg each), and plasmids expressing either wild-type or R45A mutant dHSP68 (50 ng each). Among peptides pulled down in the presence (+) of TRX with spectral counts greater than four in each category (unique to +TRX or present in both conditions but enriched in +TRX), the ones methylated on arginine are listed. (B) Venn diagram of proteins identified using mass spectrometry that pulled down with MYC-dpCLK coexpressed with FLAG-dpPER in S2 cells in the presence or absence of endogenous dTRX. Co-IPs were probed with an anti-MYC antibody, and western blots (WBs) were performed using the indicated antibodies, including an antimethylated arginine antibody. (A) Co-IPs from S2 cells transfected with MYC-tagged dpCLK and FLAG-dpPER expressing plasmids (500 ng each) without or with dsRNA targeting dTrx 5 0 and 3 0 untranslated regions (UTRs) (15 μg each) and plasmids expressing either the wild-type or N3665A mutant dTRX-CT (500 ng each). ![]() TRX-dependent arginine methylation of HSP68 is necessary for PER repression and for CLK-PER interaction.
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