Fig. 4

Lhx8 suppresses Lin28a expression. a Immunofluorescence with anti-LIN28A antibodies shows that LIN28A is preferentially expressed in oocytes within the ovary. LIN28A abundance is higher in Lhx8 conditionally deficient oocytes (Lhx8 ^flx/flx Gdf9Cre) compared to controls (Lhx8 ^flx/flx). b and c Lin28a transcripts and protein are significantly more highly expressed in Lhx8 ^flx/flx Gdf9Cre oocytes (G9cKO) unlike controls (Ctrl). a–f Chromatin immunoprecipitation (ChIP) assays with anti-LHX8 affinity purified antibodies on oocytes. d A putative LHX8 DNA binding site, TGATTG [22], which perfectly fits the LHX8 binding consensus sequence, was identified at position −536 to −531 relative to the Lin28a transcription initiation site. e Anti-LHX8 antibodies precipitate genomic DNA containing the TGATTG binding sequence from the Lin28a promoter region as shown by ChIP-quantitative PCR (qPCR). Immunoglobulin G (IgG) antibodies served as control. The percentage input method is used to analyze the qPCR data. “Input” is the PCR product from chromatin pellets before immunoprecipitation. A triplicate average Ct normalized to an adjusted input was used for the calculation of percentage input. Two sets of primers (F1 and R1) and (F2 and R2) were used to perform ChIP-qPCR. f PCR amplification of the oocyte input DNA, as well as DNA precipitated by normal guinea pig IgG and anti-LHX8 antibodies by the F1/R1 and F2/R2 primer sets. Scale bars: 50 μm (A)