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: Nucleosome binding of WT BRG1 and mutants. 527 nm wtBRG1 or BRG1 mutants were incubated with streptavidin beads, with and without immobilized, biotinylated 77-NPS-77 nucleosomes (see also “Experimental procedures”). 20% of the IP reactions were used for gel analysis (lanes 9–16). The input samples (lanes 1–7) for gel analysis contained 0.21 μg of nucleosomes, 1% (final concentration) gelatin, 2 μg of BSA, or 0.96 μg of remodeler. All samples were loaded on 4–12% Bis-Tris PAA gels, whereby the upper part was stained with Coomassie and subsequently silver. The lower part of the gel was used for a Western blot with anti-H2B to verify the presence/release of nucleosomes. All IP reactions resolved in this figure originate from the same reaction.

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: Binding Assay, Incubation, Concentration Assay, Staining, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: A Soluble Fragment of the Tumor Antigen BCL2-associated Athanogene 6 (BAG-6) Is Essential and Sufficient for Inhibition of NKp30 Receptor-dependent Cytotoxicity of Natural Killer Cells *

doi: 10.1074/jbc.M113.483602

Figure Lengend Snippet: NKp30 binding to peptide arrays of BAG-6. A, domain organization of human BAG-6 (P46379-1). The domain borders are given by numbers corresponding to the amino acid positions. The caspase-3 cleavage site at position 1001 is indicated. UBL, ubiquitin-like domain; DUF, domain of unknown function; NLS, nuclear localization signal; BAG, Bcl-2-associated athanogene domain. B, overlapping peptide arrays (18-amino acid-long peptides, offset by one amino acid) of human BAG-6 were probed with soluble NKp30-IgG1-Fc fusion proteins. Bound NKp30 was detected with a human Fc-specific HRP-conjugated antibody and visualized by chemiluminescence imaging. The identified interaction hot spots (I–X) are marked with boxes. *, nonspecific spot (compare supplemental Fig. S1).

Article Snippet: Antibodies The following antibodies were used: mouse monoclonal anti-polyhistidine tag HRP-conjugate and anti-human HRP-conjugate (both Sigma-Aldrich), anti-Strep-mAb classic HRP (IBA), anti-NKp30 clone p30-15 (kindly provided by Carsten Watzl), goat anti-mouse IgG1 APC conjugate (Life Technologies), and anti-CD4 APC conjugate (eBioscience).

Techniques: Binding Assay, Imaging

Journal: The Journal of Biological Chemistry

Article Title: A Soluble Fragment of the Tumor Antigen BCL2-associated Athanogene 6 (BAG-6) Is Essential and Sufficient for Inhibition of NKp30 Receptor-dependent Cytotoxicity of Natural Killer Cells *

doi: 10.1074/jbc.M113.483602

Figure Lengend Snippet: Expression and purification of BAG-6686–936. A, soluble hexahistidine-tagged BAG-6686–936 was purified from BL21 E. coli bacteria by IMAC after isopropyl-β-d-thiogalactoside induction and sonication. Representative fractions of the purification were analyzed by Coomassie Blue-stained reducing SDS-PAGE and by Western blot with a mouse monoclonal polyhistidine tag-specific HRP-conjugated antibody prior to chemiluminescence imaging. B, after heterologous expression, secreted Strep-tag II-tagged BAG-6686–936 was purified from High Five insect cell culture supernatant via StrepTactin. Representative fractions of the purification were analyzed by Coomassie Blue-stained reducing SDS-PAGE and Western blotting with a mouse monoclonal Strep-tag-specific HRP-conjugated antibody prior to chemiluminescence imaging. C and D, aliquots of the concentrated elution fractions from IMAC (C) and StrepTactin (D) purification were analyzed by Coomassie Blue-stained nonreducing (− DTT) and reducing (+ DTT) SDS-PAGE. E and F, top, IMAC-purified (E) and StrepTactin-purified BAG-6686–936 (F) were subjected to size exclusion chromatography on a Superdex 200 column to remove minor contaminations. E and F, bottom, representative aliquots of the peak fractions were analyzed by Coomassie Blue-stained SDS-PAGE and Western blotting with a mouse monoclonal anti-polyhistidine tag- (E) or Strep-tag-specific HRP-conjugated antibody (F). CV, column volume.

Article Snippet: Antibodies The following antibodies were used: mouse monoclonal anti-polyhistidine tag HRP-conjugate and anti-human HRP-conjugate (both Sigma-Aldrich), anti-Strep-mAb classic HRP (IBA), anti-NKp30 clone p30-15 (kindly provided by Carsten Watzl), goat anti-mouse IgG1 APC conjugate (Life Technologies), and anti-CD4 APC conjugate (eBioscience).

Techniques: Expressing, Purification, Sonication, Staining, SDS Page, Western Blot, Imaging, Strep-tag, Cell Culture, Size-exclusion Chromatography