Figure 1.
Inducible β-Catenin Knockout Alleles Produce N-Terminally Truncated Isoforms in mESCs
(A) Schematic representation of murine β-catenin (Ctnnb1) locus and the two loxP alleles used for β-catenin studies in mESCs. Black boxes represent exons, yellow boxes coding exons, dashed red lines indicate loxP sites, and white boxes represent exons excised upon CRE-mediated recombination of loxP sites.
(B and C) Western blot (B) and relative quantification (C) of NLC1 and SR18 cell lines upon 72-h 4′-hydroxytamoxifen treatment (+4OHT) and respective untreated controls (CTRs). SR18 untreated cell line is heterozygous for full-length β-catenin deletion. Western blot band intensities (C) are normalized on NLC1 full-length CTTNB1.
(D) β-Catenin immunofluorescence staining on fixed SR18 or NLC1 parental cell lines or upon 72-h +4OHT treatment. A primary antibody raised against the C-terminal portion of β-catenin was used. DAPI was used to counterstain nuclei. Scale bar represents 50 μm.
(E) Multichannel fluorescence intensity measurement of immunofluorescence images in (D). Image quantification has been performed across the dashed yellow line depicted in (D), merge panel.
(F) Schematic representation of short-hairpin targeted regions (red triangles, β1, β2, and β3) and qRT-PCR amplicons (blue lines, #1 and #2) along the Ctnnb1Tm4Wbm allele.
(G) Western blot of β-catenin of mESCs harboring the Birchmeier β-catenin allele after (Ctnnb1Tm4Wbmdel/del; left) or before (Ctnnb1Tm4Wbm fl/fl; right) CRE-mediated recombination of the loxP sites. Cells were transduced with a control short hairpin (shCtr) or three different short hairpins against β-catenin mRNA (β1, β2, or β3).
(H) qRT-PCR on total mRNA extracts of Ctnnb1Tm4Wbm fl/fl or Ctnnb1Tm4Wb del/del cells transduced with the short-hairpin constructs used in Figure 1D. Two different amplicons were amplified to monitor deleted region (Ctnnb1 #1) or 3′ UTR (Ctnnb1 #2). GAPDH was used as housekeeping control. Error bars represents standard deviation of technical triplicates.