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. 2006 Aug;26(15):5603–5614. doi: 10.1128/MCB.01845-05

FIG. 2.

FIG. 2.

Expression of a luciferase reporter linked to the 3′-UTR DICE-2R (fLuc-2R) determinant fails to be repressed by αCPs endogenous to HeLa cells and is unaffected by overexpression of exogenous αCP1. (A) Diagram of the fLuc-2R and fLuc-2Rm mRNAs. Diagram details are as for Fig. 1. (B) The DICE-2R determinant fails to repress expression of an fLuc reporter in transfected HeLa cells. Equimolar amounts of plasmids encoding the fLuc-2R or fLuc-2Rm mRNAs were transfected into HeLa cells along with an internal control rLuc reporter. The ratio of fLuc and rLuc activities for 12 independent studies is represented on the histogram. The means and standard deviations are shown. (C) Overexpression of myc-tagged αCP1 in HeLa cells cotransfected with fLuc-2R or fLuc-2Rm expression vector. Top panel: Western blots of the cells cotransfected with the indicated vectors (empty vector [vect] and myc-epitope-tagged αCP1 vector [myc-αCP1]). The membranes were probed with monospecific antibody to αCP1 (anti-αCP1; lab designation FF1 (3). This antibody detects both endogenous αCP1 and myc-αCP1 (light lower and dark upper bands, respectively). Recombinant myc-tagged αCP1 (rαCP1) was included in the analysis (right lane) as a positive control. Lower panel: Western blots from a parallel gel were probed with anti-myc antibody to specifically detect the epitope-tagged exogenous αCP1 protein. Molecular weight markers are indicated to the right of the gel figure. (D) RNA-EMSA analysis demonstrating increased levels of α-complex formation in HeLa cells cotransfected with the myc-αCP1 expression vector. The identity of the sample in each lane is as indicated by the legend above the autoradiograph. The comparison is of extract isolated from HeLa cells transfected with the empty vector to HeLa cells transfected with the myc-αCP1 expression cassette. The positions of the α-complex and the supershifted α-complex are indicated to the right of the gel. (E) Expression of fLuc-2R fails to be repressed in HeLa cells and is unresponsive to elevated levels of exogenous αCP1. HeLa cells were cotransfected with fLuc-2R or fLuc-2Rm expression vectors along with either empty vector (vect) or the same vector containing the myc-αCP1 expression cassette (myc-αCP1). The graded input of expression vector used in the transfections (see Materials and Methods) is indicated by the wedge. The data represent five studies.