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. 2010 Nov 8;120(12):4493–4506. doi: 10.1172/JCI42674

Figure 1. Enigma regulates p53 and p21 protein levels through MDM2.

Figure 1

(A) Enigma stabilizes MDM2. HLK3 cells were transfected with F-Enigma vector (0, 2, 5 μg [designated –, +, ++]), incubated for 12 hours in the presence or absence of 10 μM MG132, and immunoblotted as indicated. (B) p53 levels are reflected in p53 activity. We transfected cells with the indicated reporter constructs (0.5 μg each) and with or without F-Enigma vector, and incubated them with or without MG132 for 12 hours. A luciferase assay was performed. Rel. luc. unit, relative luciferase units. (C) The effects of Enigma on mRNA levels of MDM2, p53, and p21. We transfected HLK3 cells with or without F-Enigma vector, prepared cell lysates or extracted total RNAs after 48 hours, and performed Northern blotting (NB) or IB as indicated. We stained 28S RNA with ethidium bromide as a control to indicate equivalent loading. (D) Enigma regulates p53 levels Mdm2-dependently. We transduced MEFs with Ad-p53 at an MOI of 100. After 16 hours, we transduced them with Ad–F-Enigma or Ad-LacZ at an MOI of 50, or Ad-siEnigma or Ad-siControl at an MOI of 100, and incubated them for 32 hours before IB as indicated. (E) Enigma regulates p53 and p21 levels MDM2-dependently. We transfected cells with F-Enigma (5 μg), F-EniΔLIM3 (5 μg), siEnigma (10 μg), or siControl (10 μg) vectors, and incubated them for 48 hours before IB as indicated.