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Fig. 1 Mind bomb ubiquitylates Jagged 1 and is required for Jagged 1-induced Notch activation. (A) Jag1a and Jag1b were ubiquitylated by Mib. COS7 cells were transfected with HA-tagged Jag1a (Jag1a-HA), HA-tagged Jag1b (Jag1b-HA), HA-tagged DeltaD (DelD-HA), Myc-tagged wild-type Mib (WT), C1001S Mib (CS) and Flag-ubiquitin (Ub), as indicated. Cell extracts were then subjected to immunoprecipitation with an anti-HA antibody. The immunoprecipitated complexes were immunoblotted with anti-Flag (top) and anti-HA (top and middle) antibodies. Whole cell extracts (WCEs) were immunoblotted with an anti-Myc antibody (bottom). Molecular weight marker sizes in kDa are indicated on the left. (B) Mib was required for Jag1-induced Notch activation in Jag1-expressing cells. Jag1-3T3 cells were transfected with each Mib siRNA and then co-cultured with Notch1-3T3 cells. Notch activity was measured in Notch1-3T3 cells transfected with TP1, a Notch reporter plasmid. (C) Mib was not required for Jag1-induced Notch activation in Notch1-expressing cells. Notch1-3T3 cells were transfected with each Mib siRNA and the TP1 reporter plasmid, and then co-cultured with Jag1-3T3 cells. Error bars in B,C represent the mean ± s.d. of three independent experiments. (D) The expression of Jag1 on the cell surface was not altered by Mib knockdown. GFP-positive Jag1-3T3 cells were transfected with Mib siRNAs and, after 48 hours, the expression of Jag1 was examined by flow cytometry with an anti-Jag1 antibody. Hamster IgG was used as a control. (E) The Jag1 endocytosis rate was not dramatically altered by Mib knockdown. The surface proteins of Jag1-3T3 cells were labelled with Sulfo-NHS-SS-Biotin and incubated for 20 minutes at 37°C. The remaining cell-surface biotin was then stripped, and WCEs were prepared. Internalized biotinylated Jag1 and total Jag1 was measured by an ELISA. The values shown are mean ± s.d. (%).