Fig. 7. Knocking down gskt significantly increases TIM protein level.

a Potential substrates of GSKT filtered by PPI. Substrates that are involved (or potentially involved) in circadian regulation are labeled in green. b Western blots of proteins from whole-head extracts of gskt-RNAi (timG4/+; Udcr2/Ugskt-RNAi) and control (timG4/+; Udcr2/+) collected at the indicated circadian time. ACTB was used as a loading control. Repeated seven times independently with similar results. c Quantification of PER and TIM protein levels of blots in b (n = 7). PER and TIM protein levels were normalized to that of ACTB. For each time series, the value of the control at CT0 was set to 1. Two-tailed Student’s t test, CT0, *p value =  0.0154, CT4, *p value = 0.0395. d Plots of relative mRNA abundance vs. circadian time for clock genes determined by qRT-PCR in whole-head extracts of gskt-RNAi and control flies collected on DD1 (n = 3). For each time series, the value of the control at CT0 was set to 1. Two-tailed Student’s t test, **p value = 0.0025. e Brains from gskt-RNAi and control flies collected at indicated circadian time and were immunostained with TIM (green) and PDF (red) antisera. The scale bar represents 10 µm. f Quantification of TIM protein levels in the s-LNvs of images in e (From left to right: n = 50, 48, 31, and 39; one-way ANOVA, CT0 p value = 6.84042 × 10⁻¹², CT16 p value = 0.09995). g Left panel: western blots of proteins from S2 cells co-transfected with pActin-tim and pActin-gskt or empty vector. Repeated five times independently with similar results. Right panel: quantification of TIM protein levels from the blots on the left (n = 5, two-tailed Student’s t test, **p value = 0.0089). ACTB was used as a loading control. Owing to large differences in molecular weight, ACTB was not ran on the same blot with PER/TIM although they were from the same sample. Error bars represent SEM. G4, GAL4; U, UAS. Source data are provided as a Source Data file.