Journal: The Journal of Biological Chemistry

Article Title: Repression of Retrotransposal Elements in Mouse Embryonic Stem Cells Is Primarily Mediated by a DNA Methylation-independent Mechanism *

doi: 10.1074/jbc.M110.125674

Figure Lengend Snippet: IAP protein is only detected in lysates of E18.5 Dnmt1−/− mutant brain. A, depiction of protein structural domains of the full-length IAP clone MIA14. A deletion series of the gag peptide domain, which most closely corresponds to the p73 peptide used to generate the original IAP antibody (45), was cloned to create GST fusion peptides. B, immunoblotting revealing IAP expression in control (Con) and E18.5 Nestin-Cre; Dnmt1 conditional mutant brain lysate (Mut) of the original p73 serum before purification (anti-p73, left blot) and after column purification using the IAP2-GST fusion peptide (anti-p73, middle blot). Our IAP2 polyclonal antibody also strongly recognizes the 73-kDa gag protein (anti-IAP2, right blot) from mutant brain lysate (32). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: IAP2 Fluorescent in Situ Hybridization (FISH) and Northern Analysis IAP2 (amino acids 250–700) sequence was PCR-amplified from the MIA14 plasmid gel purified (Promega).

Techniques: Mutagenesis, Clone Assay, Western Blot, Expressing, Control, Purification

Journal: The Journal of Biological Chemistry

Article Title: Repression of Retrotransposal Elements in Mouse Embryonic Stem Cells Is Primarily Mediated by a DNA Methylation-independent Mechanism *

doi: 10.1074/jbc.M110.125674

Figure Lengend Snippet: Detection of IAP protein expression in demethylated somatic cells. A, left, immunoblotting using anti-IAP2 shows IAP protein in control and E18.5 Nestin-Cre; Dnmt1 conditional mutant brain lysates. Right, IAP immunoreactivity in E18.5 dorsal cortex of E18.5 Nestin-Cre; Dnmt1 mutant mice (29). B, left, Western blot analysis of IAP protein expression in control and Emx1-Cre; Dnmt1 cortical lysate from neonate P0 to 1.5 years old. Right, IAP immunoreactivity in the dorsal cortex (ctx) of 6-month-old Emx1-Cre; Dnmt1 mutant mice (9). C, immunocytochemistry for IAP protein in cultured N2a neuroblastoma cell lines. Western blot showing IAP expression in N2a cells but not in other types of neuroblastoma cells, including B104 and Hs 683 from ATCC. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: IAP2 Fluorescent in Situ Hybridization (FISH) and Northern Analysis IAP2 (amino acids 250–700) sequence was PCR-amplified from the MIA14 plasmid gel purified (Promega).

Techniques: Expressing, Western Blot, Control, Mutagenesis, Immunocytochemistry, Cell Culture