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. 2021 Sep 10;10:e71335. doi: 10.7554/eLife.71335

Figure 1. Degron-tagged transmembrane insulin/insulin-like growth factor-1 (IGF-1) receptor DAF-2 is susceptible to auxin-mediated degradation.

(A) Schematic illustration of auxin-inducible degradation (AID)-mediated DAF-2 receptor depletion in daf-2(bch40) Caenorhabditis elegans. (B) Immunoblot of eft-3p::TIR1::mRuby::unc-54 3’UTR, daf-2(bch40), wild type (N2), and DAF-2::degron (eft-3p::TIR1::mRuby::unc-54 3’UTR; daf-2(bch40)). (C) Immunoblot of ‘DAF-2::degron’ animals that were grown on OP50 NGM and at L4 stage shifted to either empty vector control RNAi (L4440) or daf-2(RNAi) plates containing either DMSO or 1 mM auxin. After 48 hr on the second day of adulthood, animals were harvested for western blotting. (D) Densitometric quantification of (C) from n = 3 independent experiments. Error bars represent s.d. Two-sided t-test was used for statistical analysis. *: p < 0.05, **: p < 0.01, ***: p < 0.001. (E) Immunoblot of DAF-2::degron animals showed no decrease of DAF-2 levels at high temperatures. Animals were raised at 15°C and put as L4 for 24 hr at the indicated temperatures. (F) A representative immunoblot of ‘DAF-2::degron’ animals after 1% glucose and 36–48 hr starvation on the second day of adulthood. L4 DAF-2::degron animals were either placed on OP50 NGM plates with or without 1 mM auxin, or containing 1% glucose, or on empty (no bacteria) NGM plates. Animals were harvested 36–48 hr later. (G) Densitometric quantification of (F) from n = 3 independent experiments. Error bars represent s.d. Two-sided t-test was used for statistical analysis. *: p < 0.05, **: p < 0.01, ***: p < 0.001. (H) Immunoblot analysis of starved DAF-2::degron animals. Animals were raised on OP50 NGM at 20°C and shifted from L4 to L4440 containing FUdR. After 2 days, they were washed off, and either frozen as control or put on empty plates and harvested after 24 or 48 hr, respectively. (I) Immunoblot analysis of 1-day-old adult DAF-2::degron animals treated with 1 mM auxin for the indicated time periods. (J) Quantification of (I) from n = 3 independent experiments. Error bars represent s.d. One-sided t-test was used for statistical analysis. *: p < 0.05, **: p < 0.01, ***: p < 0.001. For (B–J), see Source data 1 and Source data 2 for raw data, full blots, and statistics.

Figure 1.

Figure 1—figure supplement 1. Degron-tagged DAF-2 is functional and susceptible to auxin-mediated degradation.

Figure 1—figure supplement 1.

(A) Schematic illustration of the daf-2 locus of daf-2(bch40 [daf-2::degron::3xFLAG::STOP::SL2::SV40::degron::wrmScarlet::egl-13NLS]) animals. Expression of the operon was not observed. Either it is not functional, or the levels are too low to be detected by confocal microscopy. (B) PCR of wild type (N2) and daf-2(bch40). Primer pair RV96 and RV97 span the 3’ of the insert, RV94 and RV99 span the 5’ of the insert, and RV94 and RV95 span the end of the gene of the wild type. The PCR product of RV96 and RV97 and RV94 and RV99 have been sequenced to verify the construct’s correct insertion. (C) Comparison of progeny number of wild type (N2) and DAF-2::degron from n = 3 independent experiments. Error bar represents s.d. (D) Comparison of the developmental speed of wild type (N2) and DAF-2::degron from n = 3 independent experiments. A timed egg lay for 2 hr was used for synchronization. After 4 days at 20°C, the developmental stages were scored. Error bar represents s.d. (E) Immunoblot analysis of DAF-2::degron and DAF-2::degron; germline TIR1 (eft-3p::TIR1::mRuby::unc-54 3’UTR; daf-2(bch40); sun-1p::TIR1::mRuby::sun-1 3'UTR) on DMSO and 1 mM auxin. The numbers below indicate values normalized to the respective control on DMSO. For (C–E), see Source data 1 and Source data 2 for raw data and statistics.