Journal: Scientific Reports
Article Title: Keap1–MCM3 interaction is a potential coordinator of molecular machineries of antioxidant response and genomic DNA replication in metazoa
Figure Lengend Snippet: Keap1 interacts with MCM3 in mammalian cells. ( a ) Western blots with antibodies against indicated proteins either with nuclear (‘N’) or cytoplasmic (‘C’) extracts of the FLAG-MCM3 expressing CHO-EBNALT85 cells (‘input’), or in MCM3 complexes immunoprecipitated with anti-FLAG affinity beads (‘flag IP’). Histone H3 and GAPDH were used as fractionation controls. See Supplementary Fig. S2a for full-length blots. ( b ) Coomassie brilliant blue stained SDS-PAGE gels (top panels) and Western blots with antibodies against indicated proteins (bottom panels) showing distribution of FLAG-MCM3 immunoprecipitated nuclear and cytoplasmic protein complexes in the Superdex 200 size exclusion chromatography. ‘flag’ depicts the lanes with input material. Co-elution of molecular weight markers is indicated at the bottom. See Supplementary Fig. S2b for full-length gels and blots. ( c ) Proximity ligation analysis (PLA) of the Keap1 - MCM3 interaction in human primary epithelial keratinocytes (HPEK). The images of red PLA channel alone are shown in the left column, and combined with blue DAPI staining of nuclei in the right column. ‘Keap1 + MCM3’ indicates the images with interaction specific signals, other images correspond to the control experiments with single antibodies. Shown are the maximum intensity projection images of the Z stacks from confocal microscopy; white scale bar = 10 µM. ( d ) Scatter dot plot of the quantified data of nuclear and cytoplasmic Keap1 + MCM3 PLA signals (M3 + K1) compared to negative control with MCM3 antibody alone (M3). Each data point represents an average number of nuclear or cytoplasmic PLA dots per cell from one micrograph. Bars represent the mean and standard deviation of combined data from two independent PLA experiments, one slide analysed in first and two in second experiment and three different micrographs quantified from each slide. The significance values (***p
Article Snippet: Antibodies used in Western blotting Antibodies against following proteins were used: MCM2 (N19, sc-9839), MCM3 (N19, sc-9850), MCM4 (C-10, sc-48407), MCM5 (33, sc-136366), MCM6 (H-300, sc-22780), MCM6 (C-20, sc-9843), MCM7 (H-5, sc-374403), Heme Oxygenase 1 (HO1) (A-3, sc-136960), actin (I-19, sc-1616), Histone H3 (FL-136, sc-10809), GAPDH (6C5, sc-32233), Lamin B1 (A-11, sc-377000), Sp1 (sc-59), and Keap1 (E-20, sc-15246) - all from Santa Cruz Biotechnology; Keap1 (D6B12), and Nrf2 (D1Z9C) - Cell Signaling Technology; MCM-BP (HPA038481), and anti-FLAG-tag (M2, F1804) - Sigma-Aldrich; anti- basal cell cytokeratin (RCK103, ab9222) - Abcam.
Techniques: Western Blot, Expressing, Immunoprecipitation, Fractionation, Staining, SDS Page, Size-exclusion Chromatography, Co-Elution Assay, Molecular Weight, Ligation, Proximity Ligation Assay, Confocal Microscopy, Negative Control, Standard Deviation