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. 2015 Sep 4;290(42):25595–25608. doi: 10.1074/jbc.M115.661413

FIGURE 4.

FIGURE 4.

HLXB9 suppresses the expression of Cblb mRNA by suppressing Cblb promoter activity. A and B, among targets identified by anti-HB9-PO4 ChIP-Seq, the expression of only Arid1b and Cblb was affected by HLXB9. Quantitative RT-PCR is shown of the indicated genes using RNA prepared from MIN6-4N cells transfected with control siRNA (siC) or HLXB9 siRNA (siHB9), empty vector, or mh-HB9-WT. A, Western blot confirming knockdown or overexpression of HLXB9 with anti-HLXB9 or anti-myc-tag, respectively, and β-actin as the loading control. B, HLXB9 knockdown or overexpression regulated the relative mRNA level of only two genes (Arid1b and Cblb). Both were reduced upon HLXB9 overexpression, but only Cblb was increased upon HLXB9 knockdown. Error bar = mean and S.D. from three experiments. * = p < 0.05. C and D, Nono did not regulate the expression of HLXB9 target genes. Shown is quantitative RT-PCR of the indicated genes using RNA prepared from MIN6-4N cells transfected with control shRNA (shC) or Nono shRNA (shNono), FLAG-vector, or FLAG-Nono. A, Western blot confirming knockdown or overexpression of Nono with anti-Nono or anti-FLAG-tag, respectively, and β-actin as the loading control. B, Nono knockdown or overexpression did not regulate the relative mRNA level of any gene. Error bar = mean and S.D. from three experiments. E and F, Cblb promoter activity is suppressed by HLXB9. The promoter region of Cblb (−1078 to +219) located near the sequence identified by anti-HB9-PO4 ChIP-Seq was cloned in the promoter-less luciferase reporter vector PG02 (GeneCopoeia) and analyzed for promoter activity in MIN6-4N cells. RLU for each of the transfections are shown. Compared with the empty vector PG02, the PG02-Cblb plasmid showed significantly high RLU, and co-expression of increasing amounts of HLXB9 suppressed the Cblb promoter activity. Error bar = mean and S.D. from three experiments. * = p < 0.05. A representative Western blot shows the expression of HLXB9 (with anti-myc-tag) in the MIN6-4N cells analyzed for luciferase activity. p84 was used as the loading control. G and H, Cblb expression is down-regulated by the phosphorylated isoform of HLXB9. WCE and RNA were prepared from MIN6-4N cells transfected with control-shRNA (C−) or Men1-shRNA (M−) together with empty vector or mh-HB9-WT. G, Western blot shows the extent of menin knockdown (anti-menin blot) and transfected HLXB9 and increased phospho-HLXB9 in M− (anti-myc-tag blot, top band of the doublet band). H, quantitative RT-PCR shows significantly reduced Cblb mRNA upon menin knockdown in mh-HB9-WT-transfected cells. Error bar = mean and S.D. of a representative experiment performed in triplicate. * = p < 0.05.