The expression of Mib is efficiently knocked down by two independent siRNAs, but Jagged 1 expression is not affected by the Mib knockdown. (A) Jag1-3T3 cells were transfected with two Mib siRNAs (#1 and #2) at 2 nM or 10 nM, and whole-cell extracts were immunoblotted with anti-Mib (Mib, upper panel), anti-Flag (Jag1, middle panel), and anti-α tubulin (tubulin, bottom panel) antibodies. (B) Notch-3T3 cells were transfected with two Mib siRNAs (#1 and #2) at 10 nM, and whole-cell extracts were immunoblotted with anti-Mib (Mib, upper panel) and anti-α tubulin (tubulin, lower panel) antibodies. (C) The surface proteins of Jag1-3T3 cells were labelled with Sulfo-NHS-LC-Biotin for 15 minutes, and whole-cell lysates were prepared. Flag-tagged Jag1 was immunoprecipitated with a Flag antibody from the whole-cell extract (WCE). Immunoprecipitates were immunoblotted with NeutrAvidin and the anti-Flag antibody. WCE was immunoblotted with anti-Flag, anti-Mib, and anti-α tubulin antibodies. α tubulin was used as a loading control. IP, immunoprecipitation; IB, immunoblot.
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