Journal: Nucleic Acids Research

Article Title: HPr prevents FruR-mediated facilitation of RNA polymerase binding to the fru promoter in Vibrio cholerae

doi: 10.1093/nar/gkad220

Figure Lengend Snippet: Effects of dephosphorylated HPr on FruR activity. ( A ) Comparison of the growth of the wild-type strains harbouring a plasmid expressing wild-type HPr or its mutant form (H15A or H15D) in M9 medium supplemented with indicated sugar. The means and standard deviations of three independent measurements are shown. EV, empty vector. ( B ) in vitro transcription assay was conducted to further confirm the HPr-mediated inhibition of FruR-dependent fruB transcription. The 418-bp template DNA spanning from −99 to +319 bp relative to the transcription start site (TSS) was incubated with V. cholerae RNAP holoenzyme (RpoA, RpoB, RpoC, RpoZ, and RpoD) in the absence or presence of FruR, F1P or HPr as indicated. The resulting RNA products were purified and annealed with HEX-labelled primer, which anneals to the region from +92 to +112 relative to the TSS and extended using reverse transcriptase. A 133-bp HEX-labelled DNA added to each reaction as a loading control is indicated by an open triangle, and the 112-bp fruB transcript is indicated by a closed triangle. Fragment sizes were determined by comparison to the internal molecular weight standards, and nucleotide positions relative to the TSS are indicated. ( C ) FruR-binding affinities for F1P in the presence or absence of HPr were determined using ITC. In ITC, 1 mM F1P is incrementally titrated into 0.1 mM FruR alone (upper panel, –HPr) or a mixture of 0.1 mM FruR and 0.2 mM HPr (upper panel, +HPr). The binding heat signals generated per mol of injected F1P are plotted as a function of molar ratio [F1P]/[FruR] (lower panel) . Curve fitting model is one set of binding. ( D ) Effect of HPr on FruR binding to the fruB promoter was assessed in the absence or presence of F1P by EMSA. The 338-bp probe covering the entire fruR – fruB intergenic region was incubated with FruR in the absence and presence of 0.5 mM F1P or 105.6 nM HPr and analysed on a 6% polyacrylamide gel.

Article Snippet: After a brief wash with buffer B, the bound proteins were eluted with buffer C and then analysed on a 4–20% gradient polyacrylamide gel (acrylamide/bisacrylamide ratio of 37.5:1) (KOMA biotech, Seoul, Korea, KG8531) in Tris-glycine buffer (25 mM Tris; 192 mM glycine) supplemented with 0.1% SDS followed by staining with Coomassie Brilliant Blue R. After the protein bands specifically bound to the His-tagged bait protein were excised from the gel, in-gel digestion and peptide mapping of the tryptic digests were performed using MALDI-TOF MS.

Techniques: Activity Assay, Comparison, Plasmid Preparation, Expressing, Mutagenesis, In Vitro, Transcription Assay, Inhibition, Incubation, Purification, Reverse Transcription, Control, Molecular Weight, Binding Assay, Generated, Injection

Journal: PLoS ONE

Article Title: Inhibition of Calpain Prevents Manganese-Induced Cell Injury and Alpha-Synuclein Oligomerization in Organotypic Brain Slice Cultures

doi: 10.1371/journal.pone.0119205

Figure Lengend Snippet: After treatment with calpain inhibitor II and Mn for 24 hours, brain slices were homogenized. Total proteins were extracted and separated using 4–20% nondenaturing polyacrylamide gradient gel electrophoresis and analyzed using immunoblotting. (A) Western blotting for oligomeric/monomeric alpha-synuclein (full-length alpha-synuclein antibody) and β-actin in the calpain inhibitor II and Mn-treated slices. (B) Semi-quantitative analyses of the expression of oligomeric alpha-synuclein using image analyzing software (FluorChem v2.0) after western blotting experiments. Expression of protein was normalized with β-actin protein. ** P <0.01 compared with control slices; # # P <0.01 compared with 400 μM Mn-treated slices.

Article Snippet: Equal amounts of protein from each fraction were mixed with 2× non-denaturing protein loading buffer [without sodium dodecyl sulfate (SDS) and DL-Dithiothreitol (DTT)], without boiling, loaded onto a 4–20% nondenaturing polyacrylamide gradient gel (Dycent Biotech Co. Ltd., China), electrophoresed and transferred to PVDF membranes (Immobilon-P SQ , Millipore).

Techniques: Nucleic Acid Electrophoresis, Western Blot, Expressing, Software, Control