Journal: Communications Biology

Article Title: Comprehensive analysis of Enterobacteriaceae IncX plasmids reveals robust conjugation regulators PrfaH, H-NS, and conjugation-fitness tradeoff

doi: 10.1038/s42003-025-07782-w

Figure Lengend Snippet: a EMSA analysis of the interaction between the 5’ UTR of prfaH and ∆14-PrfaH protein. In the EMSA experiment, each ssDNA (~0.4 μM) was incubated with different concentrations (0, 0.5, and 1.3 μM) of ∆14-PrfaH protein, respectively. b Precise analysis of the key nucleotides affecting PrfaH function. The core target sequence (the −42 to −25 region, relative to prfaH ATG) is marked as +1 to +18, and the mutated bases are indicated in red font. WT represents pHNYX644 plasmid. T1A represents a point mutation at T1A in the 5’ UTR of prfaH on the pHNYX644 plasmid. Similarly, C2A, G3A, …, G18A represent point mutations at C2A, G3A, …, G18A, respectively, in the 5’ UTR of prfaH on the pHNYX644 plasmid. The conjugation assays were carried out with n = 3 biological replicates. The asterisk denotes that the transfer rate fell beneath the detectable threshold(~1 × 10 −18 ). The conjugation rate of each mutant was compared with that of the WT using t -test with Welch’s correction. The DNA secondary structure of the core sequence was predicted by RNAfold web server ( http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi ).

Article Snippet: The EMSA experiments were performed using the Electrophoretic Mobility-Shift Assay (EMSA) kit according to the manufacturer’s instructions (Thermo Fisher Scientific, USA).

Techniques: Incubation, Sequencing, Plasmid Preparation, Mutagenesis, Conjugation Assay

Journal: Nucleic Acids Research

Article Title: Single-molecule analysis reveals that IPMK enhances the DNA-binding activity of the transcription factor SRF

doi: 10.1093/nar/gkae1281

Figure Lengend Snippet: Single-molecule characterization of SRF binding to SRE in the absence or presence of IPMK. ( A ) EMSA using 1 nM of 5′ biotinylated c-fos DNA probe (SRE) and recombinant His-SRF (25 nM), GST-His-Elk1 (25, 50, 100 nM) and IPMK (25, 50, 100 nM). ( B ) Schematic of single-molecule binding assay of Cy5-SRF to Cy3-SRE. ( C ) smFRET histogram of binding of SRF at 15 nM to SRE after 10-min incubation in the absence (top) or presence (bottom) of IPMK at 100 nM. ( D ) Binding fraction of ( C ) with mean ± standard error of the mean (SEM) (n = 3). One-tailed unpaired t -test, P = 0.03.

Article Snippet: LightShiftTM Chemiluminescent electrophoretic mobility shift assay (EMSA) Kit (Thermo Fisher Scientific, Waltham, MA, USA, #20 148) was used to conduct nonradioactive EMSA with biotinylated DNA probes.

Techniques: Binding Assay, Recombinant, Incubation, One-tailed Test

Journal: Nucleic Acids Research

Article Title: Single-molecule analysis reveals that IPMK enhances the DNA-binding activity of the transcription factor SRF

doi: 10.1093/nar/gkae1281

Figure Lengend Snippet: In the presence of IPMK, enhanced binding to the SRE is not observed in the SRF deletion mutant (SRF Δ220–236 ). ( A ) EMSA using 1 nM of 5′ biotinylated c-fos DNA probe and recombinant His-SRF WT , His-SRF Δ220–236 (25 nM) and IPMK (50, 100 nM). ( B ) Quantification of the EMSA results (mean values ± SEM of four independent experiments). Error bars reflect standard deviations, calculated as Student's t -test, * P -value < 0.05; ** P -value < 0.01; n.s., not significant). ( C ) Schematic of single-molecule binding assay of Cy5-SRF Δ220–236 to Cy3-SRE. ( D ) smFRET histogram of binding of Cy5-SRF Δ220–236 at 15 nM to SRE after 10-min incubation in the absence (top) or presence (bottom) of IPMK at 100 nM. ( E ) Binding fraction of ( D ) with mean ± SEM (n > 3). Student t -test calculated that the difference was not significant (N.S). ( F ) Schematic of single-molecule PIFE assay using Cy3-SRE with SRF Δ220–236 . ( G ) τ on1 (black) and τ on2 (red) of SRF Δ220–236 as a function of IPMK concentration (top). And, τ off (blue) (bottom). ( H ) IPMK-concentration-dependence of K d1 (top) and K d2 (bottom) of SRF Δ220–236 , obtained from ( G ).

Article Snippet: LightShiftTM Chemiluminescent electrophoretic mobility shift assay (EMSA) Kit (Thermo Fisher Scientific, Waltham, MA, USA, #20 148) was used to conduct nonradioactive EMSA with biotinylated DNA probes.

Techniques: Binding Assay, Mutagenesis, Recombinant, Incubation, Concentration Assay