RPM-1 Uses Both Ubiquitin Ligase and Phosphatase-Based Mechanisms to Regulate DLK-1 during Neuronal Development
Figure 5
CoIP from transgenic whole worm lysates showing that (A) PPM-2::GFP and PPM-2::GFP D59N bind to FLAG::DLK-1 K162R (upper panel) (B) PPM-2::GFP R185A shows increased binding to FLAG::DLK-1 K162R compared to wild-type PPM-2::GFP (upper panel). (C) Quantitation of PPM-2::GFP coIP with FLAG::DLK-1 K162R. Note that data was acquired from 2 independently derived transgenic lines for each genotype, and histograms represent the ratio of the amount of PPM-2::GFP or PPM-2::GFP R185A in coIP to the amount of FLAG::DLK-1 K162R that was immunoprecipitated. (D) Immunoblots of whole worm lysates generated solely from transgenic worms. Catalytically inactive PPM-2::GFP D59N was consistently expressed at elevated levels compared to wild-type PPM-2::GFP (upper panel). (E) Immunoblots of whole worm lysates generated solely from transgenic worms. The level of wild-type PPM-2::GFP was elevated when coexpressed with FLAG::DLK-1 K162R, compared to when it was coexpressed with mCherry (upper panel). (F) Quantitation of PPM-2::GFP levels from lysates of the indicated transgenic genotypes. Shown are the average levels of PPM-2 acquired from 4 independently derived transgenic lines for each genotype normalized to MPK-1 (loading control). Error bars represent the standard error of the mean, and significance was determined using an unpaired t-test. **p<0.01, ***p<0.001, ns = not significant.
