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. 2010 Dec 17;300(3):L441–L452. doi: 10.1152/ajplung.00435.2009

Fig. 5.

Fig. 5.

EMSA analysis of C/EBP and FOXA1 binding sites of mouse Scgb1a1 gene promoter. Nuclear extracts (NE) prepared from COS-1 cells transfected with C/EBPα (A, left), C/EBPδ (A, right), or FOXA1 (B) expression plasmid were subjected to EMSA with various additives as follows. A: C/EBP-D or C/EBP-P site-containing probe with nuclear extract prepared from control vector-transfected COS-1 cells (lane 1; NE−), or nuclear extract prepared from C/EBPα or C/EBPδ expression plasmid-transfected COS-1 cells (lanes 2–6; NE+) in the presence of specific antibody (lane 3; Ab), nonrelated antibody (lane 4; nrAb), cognate oligonucleotide as competitor (lane 5; C), or mutated oligonucleotide as competitor (lane 6; mC). Mutated sequences for C/EBP-D and C/EBP-P binding sites are shown at the bottom. B: FOXA1-D or FOXA1-P site-containing probe with nuclear extract prepared from control vector-transfected COS-1 cells (lane 1; NE−), or nuclear extract prepared from FOXA1 expression plasmid-transfected COS-1 cells (lanes 2–5; NE+) in the presence of specific antibody (lane 3; Ab), cognate oligonucleotide as competitor (lane 4; C), or mutated oligonucleotide as competitor (lane 5; mC). Sequences for the mutated oligonucleotides for FOXA1-D and FOXA1-P are shown in Fig. 4D. C: EMSA was carried out with use of nuclear extracts prepared from transfected COS− cells with control vector (lane 1; NE−) or various combinations of expression plasmids (lanes 2–7; NE+) as indicated. D: EMSA was carried out with use of FOXA1-P site-containing probe and a nuclear extract prepared from C/EBPα expression plasmid-transfected COS-1 cells in the absence (lane 1) or the presence of anti-C/EBPα antibody (lane 2) or nonrelated antibody (lane 3). Black arrowheads indicate a C/EBP-specific DNA-protein shifted band; gray arrows indicate a FOXA1-specific DNA-protein shifted band. Asterisks show antibody supershifted bands. Antibodies used for supershift analysis were as follows: anti-C/EBPα (sc-61, Santa Cruz Biotechnology), anti-C/EBPδ (sc-151, Santa Cruz Biotechnology), anti-FOXA1 (Seven Hills Bioreagents) and normal rabbit IgG as nonrelated antibody (sc-2027, Santa Cruz Biotechnology).