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. 2014 Jul 23;111(31):11443–11448. doi: 10.1073/pnas.1412099111

Fig. 5.

Fig. 5.

The expression of GAPDH in rat mammary glands. (A and B) A representative immunoblot of GAPDH, β-tubulin, and RNA polymerase II (Pol II) in cytoplasmic extract (A) and GAPDH, β-tubulin, and Pol II in the nuclear extract of mammary glands from rats fed 20% or 30% dietary protein (B). (C and D) Western blot densitometric analysis of cytoplasmic GAPDH/tubulin (C) and nuclear GAPDH/RNA polymerase II of the mammary glands from rats fed 20% or 30% dietary protein (D). The results are the mean ± SD of three different blots. (E) GAPDH immunohistochemistry in rat mammary gland during gestation on day 20. (F and G) Negative control (F) and GAPDH (G). The arrows indicate the presence of GAPDH in the nucleus of mammary epithelium cells. [Scale bars: 50 µm (E and F); 10 µm (G).] (H) ChIP showing GAPDH binding to the SNAT2 ERE. ChIP assays were conducted on mammary glands from virgin control rats and 5-, 14-, and 20-d pregnant rats. Soluble chromatin was immunoprecipitated with 4 µg of anti-GAPDH antibody or negative control IgG and subjected to PCR with a specific primer to the SNAT2 ERE site (Table S1). Results shown are representative of three independent experiments (n = 5 rats per group). Different letters differ: a > b > c. P < 0.05. (I) A representative immunoblot of GAPDH precipitation in SNAT2 ERE and SNAT2 ERE mutated sequences using biotinylated oligos incubated with ER-α transfected nuclear extracts from HeLa cells.