Journal: Biology

Article Title: Light Quality-Dependent Regulation of Non-Photochemical Quenching in Tomato Plants

doi: 10.3390/biology10080721

Figure Lengend Snippet: Western Blot analyses ( a ) and densitometric analyses of PsbS ( b ), VDE ( c ), PGRL1 ( d ), and cyt f ( e ) proteins in leaves of tomato plants ( Solanum lycopersicum L. cv. Malinowy Ozarowski) grown under different light conditions (see Material and Methods for details). The bands were normalized to the appropriate β subunit of ATP synthase (ATPB) band (loading control, ) ( a ). The bar diagrams ( b – e ) represent pixel volumes (densitometric analyses) of proteins in samples. Each value represents the mean ± SD ( n = 3) considering the control sample (WL) value as 1 (100%). Different letters indicate significant differences between treatments ( p = 0.05) with a Tukey’s HSD test. MW—molecular weight.

Article Snippet: Air-dried blots were blocked with 5% non-fat dry milk blocking reagent (1 h, 25 °C) (Bio-Rad) and incubated with primary antibodies against PsbS (AS09 533; 1:1000), VDE (AS15 3091; 1:1000), PGRL1 (AS10 725/AS19 4311; 1:1000), cyt f (AS08 306; 1:5000) and ATPB (beta subunit of ATP synthase; AS05 085; 1:5000; loading control) (Agrisera, Vännäs, Sweden) overnight at 4 °C.

Techniques: Western Blot, Molecular Weight

Journal: International Journal of Molecular Sciences

Article Title: Impaired Expression of GABA Signaling Components in the Alzheimer’s Disease Middle Temporal Gyrus

doi: 10.3390/ijms21228704

Figure Lengend Snippet: Representative fluorescent immunohistochemistry images from the human Alzheimer’s disease and control middle temporal gyrus (MTG). Post-mortem human MTG sections were labelled with antibodies against GABA A R subunits and imaged with uniform settings for each subunit on a confocal microscope. GABA A R subunits are presented in red and Hoechst staining is presented in blue. A reduction in fluorescent intensity across cortical layers was apparent for the α 1 subunit (cases AZ90 and H122 presented) ( a – d ), β 2 subunit (cases AZ90 and H122 presented) ( e – h ) and β 3 subunit (cases AZ90 and H181 presented) ( i – l ). Scale bar, 200 µm.

Article Snippet: Following this, sections were washed with PBST a further three times for 10 min and treated with primary antibodies raised against the GABA A R α 1 subunit (mouse monoclonal, clone BD24, gifted by Jean-Marc Fritschy, Switzerland; 1:5000), β 2 subunit (rabbit polyclonal, Novus, NBP1-51214; 1:1000) or β 3 subunit (mouse monoclonal, Novus, NBP1-47613; 1:1000) for 72 h at 4 °C.

Techniques: Immunohistochemistry, Control, Microscopy, Staining

Journal: International Journal of Molecular Sciences

Article Title: Impaired Expression of GABA Signaling Components in the Alzheimer’s Disease Middle Temporal Gyrus

doi: 10.3390/ijms21228704

Figure Lengend Snippet: Complete list of genes, isoform accession numbers and target sequences for NanoString nCounter probes.

Article Snippet: Following this, sections were washed with PBST a further three times for 10 min and treated with primary antibodies raised against the GABA A R α 1 subunit (mouse monoclonal, clone BD24, gifted by Jean-Marc Fritschy, Switzerland; 1:5000), β 2 subunit (rabbit polyclonal, Novus, NBP1-51214; 1:1000) or β 3 subunit (mouse monoclonal, Novus, NBP1-47613; 1:1000) for 72 h at 4 °C.

Techniques:

Journal: Molecular microbiology

Article Title: The master quorum sensing regulators LuxR/HapR directly interact with the alpha subunit of RNA polymerase to drive transcription activation in Vibrio harveyi and Vibrio cholerae

doi: 10.1111/mmi.14223

Figure Lengend Snippet: (A) Elutions from IP reactions with a ΔluxR V. harveyi strain (KM669) containing plasmids expressing either FLAG-LuxR (pAP116), an empty vector control plasmid (pSLS3), or FLAG-R17C (pST012). The bands corresponding to the FLAG-LuxR protein and the light chain antibody to the FLAG epitope are indicated. (B) Western blot analyses of lysates and elutions from IP reactions with a ΔluxR V. harveyi strain (KM669) containing either a plasmid expressing FLAG-LuxR (pAP116) or an empty vector control plasmid (pSLS3). Purified FLAG-LuxR protein (1 μg) was included in lane 1 as a positive control for the FLAG western. For each strain, two IP reactions were tested with differing stringencies in the wash steps: 1) 100 mM NaCl, 0.1% Triton-X, and 2) 500 mM NaCl, 1% Triton-X. Antibodies used are indicated on the left of each panel. (C, D) SDS-PAGE (C) and western blot (D) analyses of co-IP experiments with purified FLAG-LuxR and His-RNAP holoenzyme incubated with anti-FLAG resin. Purified E. coli His-RNAP (216 pmol) and purified FLAG-LuxR (54 pmol) were loaded in lanes 2 and 3, respectively, in panel C. Samples were taken of the supernatants (S) after incubation with the anti-FLAG resin and samples of the elution (E) were collected from the resin after elution. The western blot was probed with antibodies against β’. (E) Western blot analysis of reciprocal co-IP experiments performed with FLAG-LuxR and His-RNAP incubated with nickel-NTA resin. Samples were taken of the supernatants (S) after incubation with the nickel-NTA resin and samples of the elution (E) were collected from the resin after elution. The western blot was probed with antibodies against the FLAG epitope.

Article Snippet: The membrane was washed 3 times with TBS-T rocking for 5 min and subsequently incubated with anti-FLAG-HRP primary antibody (Sigma, 1:5,000), anti-His-HRP primary antibody (Sigma, 1:3,000), primary antibodies against RNAP subunits (α 1:10,000; β, β’, σ, 1:1,000; all from Neoclone), or primary antibodies against LuxR (1:3,000) for 1 hr at RT.

Techniques: Expressing, Plasmid Preparation, Control, FLAG-tag, Western Blot, Purification, Positive Control, SDS Page, Co-Immunoprecipitation Assay, Incubation

Journal: Molecular microbiology

Article Title: The master quorum sensing regulators LuxR/HapR directly interact with the alpha subunit of RNA polymerase to drive transcription activation in Vibrio harveyi and Vibrio cholerae

doi: 10.1111/mmi.14223

Figure Lengend Snippet: (A) Diagram of LuxR peptide array layout; corresponding peptides presented in Figure S2A. (B, C, D) LuxR peptide array probed with His-RNAP (panel B, western blot against β’), E. coli His-α (panel C, western blot against α), or His-TEV protease (panel D, western blot against 6xHis epitope). (E) Crystal structure of V. vulnificus SmcR (PDB: 3KZ9, gray); image generated by PyMol. The LuxR peptides identified in the peptide array that interact with RNAP are highlighted in red. (F) GFP and mCherry expression from E. coli strains containing plasmids expressing LuxR (pJV239), LuxR R17C (pAB38), empty vector (pJV036), or LuxR with amino acid substitutions as listed (see Table S2 for all plasmid names). Relative fluorescence expression was calculated by dividing the fluorescence value by the OD600 value. The locations of amino acid substitutions in LuxR are indicated (N-terminus, α-helix 4, or α-helix 7).

Article Snippet: The membrane was washed 3 times with TBS-T rocking for 5 min and subsequently incubated with anti-FLAG-HRP primary antibody (Sigma, 1:5,000), anti-His-HRP primary antibody (Sigma, 1:3,000), primary antibodies against RNAP subunits (α 1:10,000; β, β’, σ, 1:1,000; all from Neoclone), or primary antibodies against LuxR (1:3,000) for 1 hr at RT.

Techniques: Peptide Microarray, Western Blot, Generated, Expressing, Plasmid Preparation, Fluorescence

Journal: Molecular microbiology

Article Title: The master quorum sensing regulators LuxR/HapR directly interact with the alpha subunit of RNA polymerase to drive transcription activation in Vibrio harveyi and Vibrio cholerae

doi: 10.1111/mmi.14223

Figure Lengend Snippet: LuxR binds to sites F and G in the luxCDABE promoter, which are centered at −88 and −34 relative to the primary transcription start site (small black arrow), respectively. LuxR binding facilitates recruitment of RNAP via interactions with the α subunit, both at the C-terminal domain (sites F and G) and N-terminal domain (site G). Putative sites of interaction between LuxR and RNAP are indicated by black circles. LuxR binding site G is positioned on top of the −35 site for σ70, which may result in an interaction between LuxR and σ at this site.

Article Snippet: The membrane was washed 3 times with TBS-T rocking for 5 min and subsequently incubated with anti-FLAG-HRP primary antibody (Sigma, 1:5,000), anti-His-HRP primary antibody (Sigma, 1:3,000), primary antibodies against RNAP subunits (α 1:10,000; β, β’, σ, 1:1,000; all from Neoclone), or primary antibodies against LuxR (1:3,000) for 1 hr at RT.

Techniques: Binding Assay