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. Author manuscript; available in PMC: 2015 Jan 10.
Published in final edited form as: Nature. 2008 Jun 25;454(7208):1137–1141. doi: 10.1038/nature07066

Figure 4.

Figure 4

Expression of E2f3b or E2f1 from the E2f3a locus suppresses phenotypes due to loss of E2f3a. a. Targeting strategies for replacement of E2f3a with E2f3b (E2f3a3bki; top schematic) or E2f3a with E2f1 (E2f3a1ki; bottom schematic). Relevant exons are labeled; (E2f1) E2f1 ORF. LoxP sites are indicated as solid triangles; PCR genotyping primers are indicated by thin arrows; primers for measuring the expression of E2f3a3bki and E2f3b alleles by real-time (RT) PCR are indicated by thick arrows. Note that RT primer 1 spans a region that is complementary to exon 1a and exon 1b, as indicated by the red/black colored arrow. b, c. Southern blot (top panels) and PCR (bottom panels) genotyping of genomic DNA derived from livers of one month-old E2f3a3bki (b) and E2f3a1ki (c) mice. d. Real-time PCR gene expression analysis of the E2f3b (left panel) and E2f3a3bki (right panel) alleles in E2f3a+/+ (grey square) or E2f3a+/3bki (red/blue square) MEFs. Primers used for each allele are indicated (for primer sequence information see Supplementary Fig. 6). Note that RT primers used to detect E2f3a3bki also detect endogenous E2f3b allele since the 3′ portion of RT primer 1 is complementary with exon 1b sequences. e. Western blot analysis of E2F1 protein in proliferating MEFs with the indicated genotypes; α-tubulin was detected as a loading control. (*) indicates a non-specific band. f. Western blot analysis of E2F1 proteins in serum stimulated quiescent MEFs having the indicated genotypes; (hrs) indicates the time when cells were harvested post-stimulation. g. Western blot analysis of E2F3a and E2F3b proteins (E2F3 Sc-878) in proliferating MEFs with the indicated genotypes. (*) indicates a non-specific band. h. Body weights of six month-old E2f3a+/+, E2f3a−/−, E2f3a3bki/3bki and E2f3a1ki/1ki mice; n, number of animals measured for each genotype. Student t-test was performed for each genetic group in comparison with labeled group; (*) indicates significant p-values. i. Micrograph of one month-old E2f3a+/+, E2f1−/− E2f3a−/− and E2f1−/− E2f3a1ki/1ki mice. j. H&E staining of tissue sections of skin and bone from 21 day-old mice with the indicated genotypes. Higher magnification images of each tissue are presented in the bottom panels. (WAT), white adipose tissue; (epi), epidermis; (s mus), skeletal muscle; (gr pl), growth plate; (bm), bone marrow.