Figure S3.

CHN-1/CHIP-Dependent Ubiquitylation of the INSR, Related to Figure 3

(A) CHN-1 protein level does not significantly change during aging. CHN-1 protein level was evaluated by western blotting using CHN-1 specific antibodies in whole worm lysates of adult worms (representative immunoblots are shown of n = 3 experiments).

(B and C) Ubiquitylation of INSR was carried out as indicated using CHN-1, CHN-1(ΔU-box), CHN-1(U-box), (TPR)CHN-1, CHIP, UFD-2, and WWP-1 as ubiquitin ligases.

(D) Mutations in the TPR domain of CHN1 ((TPR)CHN-1) abrogate INSR ubiquitylation. The ubiquitin ligase WWP-1 is unable to ubiquitylate INSR. Ubiquitylation reaction was carried out using FLAG-tagged ubiquitin (FLAGUb) in the presence of E. coli BL21 lysates. Activity of used E3 enzymes was evaluated by western blotting using anti-FLAG specific antibodies showing ubiquitylated bacterial proteins.

(E) In vitro ubiquitylated DAF-2, INSR were subjected to mass spectrometry to identify the lysine residues used for ubiquitin attachment. Conserved residues are indicated in red. Previously published residues are marked as follows: #O’Rahilly et al., 1991; +Kim et al., 2011; ^∗Udeshi et al., 2013.