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. 2020 Sep 16;295(47):16058–16071. doi: 10.1074/jbc.RA120.014615

Figure 2.

Figure 2.

The transcription factor ELK1 is required by RAS-RAF-MEK1/2-ERK1/2 signaling to repress PARK2 expression. A, consensus ELK1 DNA-binding motif calculated using WebLogo3. Position of ELK1 DNA BS in the −635/+98 PARK2 promoter. Both WT (CCGGAAA) and mutagenized (mut, CCCCCAA) ELK1 BS are shown. B, Western blot analysis of pELK1 and pERK1/2 in A375 and SK-Mel-5 melanoma cells treated with SCH-772984 for the time indicated. HSP90 was used as loading control. C, ChIP assay showing that endogenous ELK1 binds to PARK2 promoter in HEK-293T cells. Two different specific anti-ELK1 antibodies were used. The y axis represents the relative promoter enrichment, normalized on the input material. IgG was used as negative control and set to 1. D, quantification of dual reporter luciferase assay in A375 and SK-Mel-197 cells transfected with the empty vector pCAG or ELK1 on PARK2 promoter carrying WT or mut ELK1 BS. Relative luciferase activities were firefly/Renilla ratios, with the level induced by control equated to 1. E, correlation between the expression of PARK2 and ELK1 using the TCGA melanoma cohort (n = 481). Pearson's correlation test. F, expression of PARK2, ELK1, and BRAF mRNA by qPCR in SK-Mel-28 melanoma cells transduced with LV-shELK1-1 and transiently transfected with BRAF-V600E. The y axis represents expression ratio of gene/(GAPDH and β-ACTIN average), with the level of control equated to 1. G, Western blot analysis of PARK2, ELK1, and BRAF in SK-Mel-28 cells transduced with LV-shELK1-1 and transiently transfected with BRAF-V600E. HSP90 was used as loading control. H, quantification of dual reporter luciferase assay in SK-Mel-28 cells showing that ELK1 silencing increases the transactivation of PARK2 promoter and partially prevents the inhibition by BRAF-V600E. Relative luciferase activities were firefly/Renilla ratios, with the level induced by control equated to 1. I, quantification of dual reporter luciferase assay in SK-Mel-28 cells showing that ELK1 decreases the transactivation of PARK2 promoter, whereas SCH-772984 treatment prevents the reduction of transactivation by ELK1. Relative luciferase activities were firefly/Renilla ratios, with the level induced by control equated to 1. Data shown are mean ± S.D. (C, D, H, I) or ± S.E. (F) of at least three biological replicates, each performed in triplicate. *, p < 0.05; **, p < 0.01.