Fig 1: (A) Mobility shift assay showing the binding of recombinant MAZ to KRAS quadruplexes of NHE. Increasing amounts of MAZ (1, 2 and 4 µg) were incubated for 30 min in binding buffer with 10 nM radiolabeled G-quadruplex and run in 5% PAGE in TB buffer (500 V, 3 h, 20°C). The white arrows indicate the MAZ-G4 complexes. The radiolabeled quadruplexes have also been incubated with nuclear extract from Panc-1 and HeLa cells (3 µg). They form two main G4-protein complexes (P:G4). Bovine serum albumin (4 µg) was used as a control. (B) Left panel shows the level of MAZ mRNA in Panc-1 cells transfected with pCMV-MAZ; right panel shows KRAS mRNA level in the same cells. C = cells treated with an empty plasmid, T = treated cells. (C) Left panel shows the level of MAZ mRNA in Panc-1 cells in which MAZ was silenced with siRNA; right panel shows KRAS mRNA level in the same cells. C = cells treated with control siRNA, T = treated cells. Student’s t-test, **P < 0.01. (D) Filter-binding assay showing the binding of recombinant MAZ to quadruplex 32R-3n, in 50 and 100 mM KCl. Top membrane (nitrocellulose) shows MAZ [or control proteins, trypsinogen (T), ovalbumin (O)] bound to the quadruplex; bottom membrane (nylon+) shows unbound radiolabeled quadruplex. The fraction of quadruplex 32R-3n bound to MAZ is plotted against the MAZ concentration. The binding curve relative to MAZ was best-fitted to a standard binding equation. A KD of 320 ± 2 nM was obtained.
Fig 2: (A) Level of KRAS mRNA in Panc-1 cells treated with 600 nM G4-decoy and control oligonucleotides, measured 24 h after decoy transfection in the presence of jetPEI. KRAS mRNA was measured with respect to housekeeping genes ß2-microglobulin and HPRT. Ordinate reports the percentage of KRAS transcript, i.e T/C × 100 where T is (KRAS transcript)/(ß2-microglobulin and HPRT transcripts) in the decoy-treated cells, whereas C is (KRAS transcript)/(ß2-microglobulin and HPRT transcripts) in the decoy-untreated cells. (B) Western blot showing the level of p21RAS at 48 and 72 h in Panc-1 cells after transfection with decoy and control oligonucleotides, in the presence of jetPEI. The data have been normalized to ß-actin. The band intensities have been measured with ChemiDOC XRS apparatus (BioRad). Right panel shows a histogram reporting the average values of three independent western blots (12% SDS–PAGE, 1 h, 180 V).
Fig 3: (A) Sequence of the NHE present in the human KRAS promoter upstream of the transcription start site. NHE contains six guanine repeats and three quadruplex-forming motifs, named 32R (repeats 1–6), 32R-3n (repeats 2–6) and 21R (repeats 1–4); (B) CD of the quadruplex-forming sequences at 20°C and 90°C; (C) sequence homology between human and murine NHEs. Human NHE contains two binding sites [(GGG(A/C)GG] for the transcription factor MAZ at the 5'- and 3'-ends.
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