Journal: American journal of reproductive immunology (New York, N.Y. : 1989)

Article Title: Haplotype-dependent Differential Activation of the Human IL-10 Gene Promoter in Macrophages and Trophoblasts: Implications for Placental IL-10 Deficiency and Pregnancy Complications

doi: 10.1111/j.1600-0897.2010.00854.x

Figure Lengend Snippet: EMSA with nuclear extracts from primary trophoblasts and THP-1 cells using probes corresponding to sequences encompassing −1082 (G or A), −592 (C or A), or Sp1 consensus motif. Three panels, free probe, primary trophoblast, and THP-1 represent the −1082 (lanes 1 and 2, 6 and 7, 11 and 12), the −592 (lanes 3 and 4, 8 and 9, 13 and 14), and the Sp1 consensus (lanes 5, 10, 15) sequences. The −1082 and −592 lanes represent G (lanes 1, 6, 11) or A (lanes 2, 7, 12) and C (lanes 3, 8, 13) or A (lanes 4, 9, 14) genotypes, respectively. The free probe is not visible because the panels only represent DNA–protein complexes. Note the EMSA differences between primary trophoblasts and THP-1 cells. There are two critical bands that represent specific DNA–protein complexes. Nuclear extract from primary term cytotrophoblasts, the faster migrating complex is prominent, whereas the slower migrating complex is prominent with nuclear extract from THP1 cells. The −592 sequences are the binding site with nuclear extract from trophoblast cells, whereas THP1 nuclear extract gives a similar profile with both the −1082 and −592 sequences. The DNA–protein complex profile with the consensus Sp1 binding sequences also differs between primary trophoblasts and THP1 cells.

Article Snippet: Nuclear protein extracts from primary placental cells and THP-1 cells were also tested with radiolabeled Sp1 consensus sequence (Promega, Madison, WI, USA).

Techniques: Binding Assay

Journal: American journal of reproductive immunology (New York, N.Y. : 1989)

Article Title: Haplotype-dependent Differential Activation of the Human IL-10 Gene Promoter in Macrophages and Trophoblasts: Implications for Placental IL-10 Deficiency and Pregnancy Complications

doi: 10.1111/j.1600-0897.2010.00854.x

Figure Lengend Snippet: The −1082 site DNA–protein complex does not involve participation of Sp1 in primary trophoblasts as demonstrated by lack of supershift by Sp1 antibody. EMSA and supershift were performed as described in Materials and Methods. Both the −1082 A (lanes 1–4) and −1082 G (lanes 5–8) probes were used. Lanes 1 and 5 represent free probes. Nuclear extracts were isolated from LPS untreated and treated primary trophoblasts. Lanes 2 and 6 represent nuclear extract from untreated cells, whereas lanes 3 and 7 contained nuclear extract from LPS-treated cells. Supershift with Sp1 antibody was performed with nuclear extract from LPS-treated cells (lanes 4 and 8). Note no supershifted bands were observed in the presence of Sp1 antibody.

Article Snippet: Nuclear protein extracts from primary placental cells and THP-1 cells were also tested with radiolabeled Sp1 consensus sequence (Promega, Madison, WI, USA).

Techniques: Isolation

Journal: American journal of reproductive immunology (New York, N.Y. : 1989)

Article Title: Haplotype-dependent Differential Activation of the Human IL-10 Gene Promoter in Macrophages and Trophoblasts: Implications for Placental IL-10 Deficiency and Pregnancy Complications

doi: 10.1111/j.1600-0897.2010.00854.x

Figure Lengend Snippet: LPS treatment of primary trophoblasts induces Sp1 involvement in DNA–protein complexes at the −592A sequences, not at the −592C sequences. EMSA and supershift were performed as described in legend to Fig, 4. Panels a and b represent EMSA with −592A and −592C probes (see Methods and Materials). Lanes 1–5 represent free probes (lane 1), nuclear extract from untreated cells (lane 2), nuclear extract from LPS-treated cells (lane 3), nuclear extract LPS + 1 μL of Sp1 antibody (lane 4), and nuclear extract LPS + 5 μL of Sp1 antibody. Note that Sp1 antibody abolished the faster moving band observed with the −592A probe.

Article Snippet: Nuclear protein extracts from primary placental cells and THP-1 cells were also tested with radiolabeled Sp1 consensus sequence (Promega, Madison, WI, USA).

Techniques: