Journal: The Journal of Biological Chemistry

Article Title: Elucidation of the functional roles of the Q and I motifs in the human chromatin-remodeling enzyme BRG1

doi: 10.1074/jbc.RA118.005685

Figure Lengend Snippet: Western blotting, immunocytochemical, and cell cycle analysis of H1299 cells expressing wtBRG1 and mutant proteins. A, 48 h after transient transfection with the indicated plasmids, 40 μg of whole cell extracts of H1299 cells were loaded on 6.5% SDS gels, which were subsequently transferred to PVDF membranes via semidry blotting. The PVDF membrane was cut into three pieces, which were incubated with the indicated antibodies. Tubulin served as a loading control. B, 48 h after transient transfection with the indicated plasmids, H1299 cells were fixed in 4% PFA, Triton X-100–permeabilized, and immunostained with an “anti-FLAG” primary antibody in combination with an Alexa 488–labeled secondary antibody. The nuclear DNA was colored via DAPI. The cells were finally mounted in PBS/glycerol, and pictures for the indicated fluorophores were taken at the fluorescence microscope Axiovert 200M. Scale bar, 20 μm. C, 24 h after transient transfection with the indicated plasmids, H1299 cells were seeded (subconfluent) for a period of 6 days on P150 plates. Six days after transfection, the cells were labeled with Hoechst, trypsinized, filtered, and finally analyzed with a CyFLOW space flow cytometer (Partec). The ratio between the cell numbers between the G1 and G2 populations (see “Experimental procedures”) from five or six biological replicates was calculated (G1/G2 ratio) and plotted as box-plot diagrams, using the software Origin 2017 (vc, vector control = empty mCherry vector). The median is shown as a line, and the average is shown as a green box and is displayed right to the box. The statistical significance was calculated in Origin 2017 software, performing a t test with two samples, mean 1 − mean 2 <> 0, significance-niveau 0.05, assuming heteroscedastic variance (Welch Korrektur) with *, p < 0.05; **, p < 0.01; and ***, p < 0.001 and not significant (n.s.).

Article Snippet: Materials and methods The following reagents were used: mCherry antibody (orb66657 biorybt); rat anti-DYKDDDDK antibody (200474, Agilent Technologies); anti-BRG1 antibody (ab1110641, Abcam); anti–α-tubulin (rabbit) antibody (600-401-880S, Rockland); anti-H2B (07-371, Upstate-Millipore); mouse anti-p53 antibody (sc-126, Santa Cruz (DO-1)); peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H+L) (111-035-144, Jackson ImmunoResearch); peroxidase-conjugated AffiniPure goat anti-mouse IgG (H+L) (111-035-146, Jackson ImmunoResearch); Alexa Fluor® 488 goat anti-rat IgG (H+L) (A-11006, Invitrogen or Thermo Fisher Scientific); streptavidin, immobilized on agarose CL-4B (Fluka/Sigma–Aldrich); ATP ultrapure (Jena Bioscience); ADP ultrapure (Cell Technologies); ADPγS (Jena Bioscience); [γ- 35 S]ATP (PerkinElmer); [γ- 32 P]ATP (Hartmann Analytic); NuPAGE 4–12% Bis-Tris gels (Thermo Fisher Scientific); anti-FLAG M2-agarose beads (Sigma–Aldrich); DAPI (Sigma–Aldrich); Amicon Ultra-4 centrifugal filter units 500 μl of 10,000 molecular mass cutoff (Merck Millipore); FuGENE® HD transfection kit (Promega); complete EDTA-free protease inhibitor mixture (Roche); benzonase (E1014-5KU, Sigma); glass coverslips (Ø = 12 mm) (Roth); 24-mm × 60-mm cover glasses (Roth); 76-mm × 26-mm glass microscope slides (Roth); PEI-cellulose F plates (Merck Millipore); Amersham Biosciences Protran premium nitrocellulose blotting membrane 0.45-μm pore size (GE Healthcare); PVDF Immobilon-P transfer membrane 0.45-μm pore size (Merck Millipore); Super Signal West Dura Extended Duration Substrate (Thermo Fisher Scientific), NativePAGE 4–16% Bis-Tris protein gels (Thermo Fisher Scientific); Sf-900 TM II SFM (1×) medium (Thermo Fisher); Dulbecco's modified Eagle's medium, low glucose [1 g/L], GlutaMAX TM -I, medium (Thermo Fisher); penicillin/streptomycin (10,000 units/ml) (Gibco–Thermo Fisher Scientific); 0.5% trypsin-EDTA (10×) (Gibco– Thermo Fisher Scientific); fetal calf serum (Gibco); DNase I (Roche); Hoechst 33342 (Sigma–Aldrich); G418 (Sigma–Aldrich); 1× PBS (Ca 2+ and Mg 2+ free) (Gibco); 1 m HEPES (Gibco); fetal bovine serum (dialyzed) (Thermo Fisher); Partec CellTrics® filter (50 μm) (Partec); polystyrene round-bottomed tubes (3.5 ml, 55 mm × 12 mm) (Sarstedt); polypropylene tubes (5 ml; 75 mm × 12 mm) (Fisher Scientific, part of Thermo Fisher Scientific); 8W10E+ PET microelectrode arrays (Applied BioPhysics); 30-μm preseparation filter (MACS; Miltenyi Biotec).

Techniques: Western Blot, Cell Cycle Assay, Expressing, Mutagenesis, Transfection, Incubation, Labeling, Fluorescence, Microscopy, Flow Cytometry, Software, Plasmid Preparation