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. 2010 Feb 18;3(3):288–302.

Figure 7.

Figure 7

(A) K562 cells (300,000 cells/ml) were maintained in absence (lane +FN) and in presence of 5μM (lane +Bayll-7085 (5μM) +FN), 10μM (lane +Bayll-7085 (10μM) +FN) NF-kB inhibitor for 24 hrs prior to 20μg/ml fibronectin for 2 hrs in SFCM. Lane Marker is MMP-9/MMP-2 marker; obtained from culture supernatant of HT-1080 cells grown for 24 h in SFCM. MMPs were concentrated using gelatin sepharose 4B bead and gelatin zymography was performed as described before. (B) Control and 20μg/ml fibronectin treated K562 cells were smeared on cover-slips and allowed to air dry prior to formaldehyde fixation. Coverslips were then reacted with 0.5 % triton-100, followed by incubation with anti-NF-kB primary antibody and respective FITC coupled secondary antibody. Coverslips were then washed thoroughly and mounted on glass slides with glycerol. Nuclear extracts (D) were prepared from K562 cells (300,000 cells/ml) cultured with (lane +FN) or without (lane -FN) 20μg fibronectin for 2 hrs in SFCM. Cytoplasmic (C) extracts were also collected from both fibronectin treated (lane +FN) and untreated (lane -FN) K562 cells as described in the methods. 50μg of protein from each extracts were subjected to run on 7.5% SDS-PAGE as discussed above. Gel was transferred on nitrocellulose membrane. Membrane was blocked, followed by reaction with anti -NF-kB primary antibody and respective secondary antibody and western blot was developed keeping actin as internal control in case of cytoplasmic fraction (C) and in case of nuclear fraction (D) B23 was used as internal control. (E) K562 (300,000 cells/ml) cells were grown with (lane 3) or without (lane 2) 20μg fibronectin for 2 hrs in SFCM and nuclear extract were prepared. Oligonucleotides containing NF-kB binding sequence was end labeled with [ y-32P] ATP using T4 polynucleotide kinase and was incubated with 10 μg of nuclear extract from control (lane 2) and FN treated (lane 3) K562 cells. Then protein-DNA complexes were electrophoresed on 5% polyacrylamide gel, gel was then dried and subjected to autoradiography at − 80°C. Lane 1 represents NF-kB binding sequences without nuclear extract.