Studies on Multifunctional Effect of All-Trans Retinoic Acid (ATRA) on Matrix Metalloproteinase-2 (MMP-2) and Its Regulatory Molecules in Human Breast Cancer Cells (MCF-7)
Figure 7
(a) Western blot of NF-kB: NF-kB expression in control (lane C) and ATRA treated (lane E) MCF-7 cells was observed by western blot analysis as before using anti-NF-B primary antibody. Quantitative measurements of immunoblots were done by using Image J Launcher (version 1.4.3.67). (C) represents the expression of respective proteins in control cells whereas (E) represents expression in 30 M ATRA treated cells. (b), (c), and (d) Immunoblot assay of ERK, p-ERK, and VEGF: MCF-7 cells (300,000 cells/1.5 mL) were grown in absence (lane C) and presence of 30 M ATRA (lane E) for 24 hours in SFCM. Cells were collected and extracted in cell extraction buffer. ERK and PI-3K were immunoprecipitated from 150 g of protein from both control and ATRA-treated cell extract with anti-ERK & protein G Agarose bead (Figures 7(b) and 7(c)) and with anti-VEGF & protein G Agarose bead (Figure 7(d)), keeping the samples for overnight at 4°C with shaking. In each case the resultant immune complex was washed thrice with PBS and the respective protein bound with antibody were eluted from the agarose bead using 1X sample buffer. Samples were then incubated in -mercaptoethanol for 10 minutes at 90°C. Samples were subjected to electrophoresis on 7.5% SDS-PAGE. The proteins were transferred to nitrocellulose membrane by Western Blot. The membranes were incubated with anti-ERK (Figure 7(b)), anti-phospho ERK (Figure 7(c)), and anti-VEGF antibody (Figure 7(d)), respectively. The immunoblots were then incubated with alkaline phosphatase-coupled secondary antibodies and bands were visualized by NBT/BCIP substrate. Ig-G (Figure 7(e)) was used to confirm equal loading. Quantitative measurements of immunoblots were done by using Image J Launcher (version 1.4.3.67). (C) represents the expression of respective proteins in control cells whereas (E) represents expression in 30 M ATRA treated cells.
