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. 2010 May 3;285(27):20532–20540. doi: 10.1074/jbc.M110.109298

FIGURE 4.

FIGURE 4.

CRD-BP is a positive regulator of MITF expression. A, 451Lu cells were transfected with either control shRNA or shRNA against CRD-BP. 48 h after transfection, the cells were collected and assayed for levels of MITF mRNA by quantitative RT-PCR, normalized to GAPDH expression, and presented as percentages of control (scrambled shRNA). B, 1241 Mel and Mel IM cells were co-transfected with the indicated shRNA-expressing plasmids, β-galactosidase-expressing plasmid, and pHTRPL4, where the luciferase gene is expressed under the MITF-dependent promoter. The values corresponding to luciferase activity normalized to β-galactosidase expression are presented. C, NHMs were electroporated using AMAXATM with either CRD-BP-overexpressing plasmid or empty vector. 72 h after transfection, the cells were collected, assayed for levels of MITF mRNA by quantitative RT-PCR normalized to GAPDH expression, and presented as percentages of control (pBABE). D, NHM cells were electroporated using AMAXATM with indicated plasmids. 48 h after electroporation, the cells were stained for senescence-associated β-galactosidase, and the percentages of β-galactosidase-positive cells were calculated. E, 451Lu cells were co-transfected with Tet-off plasmid, p-BIG-MITF, and either pcDNA control or CRD-BP-expressing plasmid. The turnover of MITF transcript was analyzed by real time RT-PCR after stopping transcription by doxycycline treatment for the indicated time points. Normalization was done with respect to GAPDH expression. All of the results are representative of three separate experiments and are expressed as the mean values ± S.D. (error bars). The average half-lives of mRNAs are presented in supplemental Table S3. F, FLAG immunoprecipitation of UV cross-linked ribonuclear protein complexes. Protein extracts from 293T cells, transfected with FLAG-CRDBP, were incubated with internally 32P-labeled RNA transcripts of three fragments of the MITF mRNA coding region, MITF full-length mRNA, and the short 3′-UTR. Fragment 1 contains nucleotides 1–421 of the coding region, fragment 2 consists of nucleotides 422–841, and fragment 3 consists of nucleotides 842–1260 of the MITF coding region. Ribonuclear protein complexes were precipitated with anti-FLAG antibodies, analyzed on PAGE, and autoradiographed.