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. 2008 May 14;79(3):546–561. doi: 10.1095/biolreprod.108.068106

FIG. 3.

FIG. 3.

1110005A23RIK represses expression of the Fshb gene. A) An 1110005A23RIK expression vector (RIK) was transfected into LβT2 cells, and RT-PCR carried out for 1110005A23Rik, Fshb, and Lhb mRNAs, and the levels of 1110005A23RIK overexpression were evaluated using antisera to the HA tag in Western analysis. The effect of 1110005A23RIK overexpression was tested in LβT2 cells on transiently transfected mouse Fshb (intron and promoter (IP): includes first intron), Lhb, and Cga promoter-luciferase reporter genes with pRL-SV40 as internal control (B), or endogenous mRNA levels by real-time PCR, using Actb as internal control (C). For both types of experiment, levels are expressed relative to those in control cells after normalization with levels of the internal controls. Mean ± SEM; n = 3–4. Student t-test compared the promoter activity of each gene with or without 1110005A23RIK overexpression; *P < 0.05; ***P < 0.001, NS: P > 0.05. D) An siRNA construct targeting 1110005A23Rik (siRik) was transfected into αT3–1 cells together with each of the subunit promoter-luciferase constructs. Luciferase activity was measured after 48 h and is presented as in B. The effect of the knockdown was verified by RT-PCR and quantified relative to the controls after normalization with the levels of Actb. E) The 1110005A23RIK contains three domains, two of which were removed individually and in combination, and truncated fusion proteins created fused to LGALS4 DBD (pM vector), as shown before testing their effects on a SV40-LGALS4 reporter gene; mean ± SEM; n = 4–6. ANOVA compared means; those that are not significantly different (P > 0.05) share the same letter. F) The effect of the wild-type or C-terminus-truncated 1110005A23RIK construct on the Fshb promoter activity was tested similarly in LβT2 cells; mean ± SEM; n = 4–6. Statistical analysis is as in (E). G) The 1110005A23RIK was overexpressed in LβT2 cells before ChIP using antisera to the HA tag to detect association with the Fshb proximal promoter. Also shown are the input samples before precipitation, and the negative Actb control, which was not precipitated by the antisera. H) The effect of GnRH (10 nM, 24 h) on the 1110005A23Rik expression level in LβT2 cells was tested using RT-PCR. I) The ChIP analysis was repeated as in G after GnRH treatment (10 nM, 4 h), and the association of Fshb and Lhb proximal promoters was assessed.