Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Multiple factors regulate the expression of sufCDSUB in Streptococcus mutans

doi: 10.3389/fcimb.2024.1499476

Figure Lengend Snippet: EMSA assays of PerR- sufCDSUB promoter interactions. Inclusion of rPerR resulted in mobility retardation of biotinylated suf promoter DNA probes (Psuf, ln 1-4), and such interaction was concentration-dependent as indicated. Addition of cold-probe led to release of biotinylated probes from the rPerR-Psuf complex (Psuf, ln 5&6). Similar effects were also observed when whole cell lysate of S. mutans UA159 (WCL) was used. However, mutations in the PerR-box 1 (perRm) showed no effect under the conditions studied.

Article Snippet: The reaction mixtures were separated on a 5% polyacrylamide gel under native conditions, and DNA mobility shift was visualized via LightShift Chemiluminescent EMSA Kit by following procedures recommended by the manufacturer (Pierce, Rockford, IL) or by SYBR Gold staining (Invitrogen, CA) and a GelDoc Go Imaging system (BioRad).

Techniques: Concentration Assay

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Multiple factors regulate the expression of sufCDSUB in Streptococcus mutans

doi: 10.3389/fcimb.2024.1499476

Figure Lengend Snippet: EMSA (A) and IVT (B) analysis of CysR. Inclusion of rCysR resulted in mobility retardation of biotinylated suf promoter DNA probes (Psuf, lanes 2-4), and such interaction was concentration-dependent as indicated. Addition of cold-probe led to release of biotinylated probes from the rPerR-promoter complex (lanes 5-7). (B) For IVT assay, inclusion of rCysR led to increase of sufC transcription (indicated by an arrow) under the direction of suf promoter. No RNA polymerase served as a negative control.

Article Snippet: The reaction mixtures were separated on a 5% polyacrylamide gel under native conditions, and DNA mobility shift was visualized via LightShift Chemiluminescent EMSA Kit by following procedures recommended by the manufacturer (Pierce, Rockford, IL) or by SYBR Gold staining (Invitrogen, CA) and a GelDoc Go Imaging system (BioRad).

Techniques: Concentration Assay, Negative Control

Journal: Disease models & mechanisms

Article Title: Altered binding affinity of SIX1-Q177R correlates with enhanced WNT5A and WNT pathway effector expression in Wilms tumor.

doi: 10.1242/dmm.050208

Figure Lengend Snippet: Fig. 6. SIX1-Q177R binds the core DNA motif sequence within the WNT5A promoter ChIP-seq peak with a higher affinity than SIX1. (A,B) Top: chemiluminescence EMSA images of purified recombinant SIX1 and SIX1-Q177R protein at concentrations as indicated, together with biotin-labeled oligonucleotide probes containing the core probe DNA motif sequences (shown above), derived from wild-type WNT5A promoter ChIP-seq peak (A) and mutated WNT5A promoter ChIP-seq peak (B). Bottom: quantification of EMSA-derived DNA-protein binding data determined from signal intensities of bound and unbound probe by using ImageJ software.

Article Snippet: Electrophoretic mobility shift assays Single-stranded DNA oligonucleotide probes were synthesized by IDT and then biotin end-labeled using the Pierce 3′ Biotin end-labeling DNA kit (Thermo Scientific) following the manufacturer’s protocol with the following changes: 25 pmol oligonucleotide per reaction were labeled, reactions stopped with 1 μl 0.5M EDTA after 30 min. Complementary oligonucleotide labeling reactions were mixed prior to centrifugation at 13,000 g for 2 min. For unlabeled oligonucleotides, 50 μl H2O was mixed with 25 μl of each complementary 100 μM oligonucleotide.

Techniques: Sequencing, ChIP-sequencing, Purification, Recombinant, Labeling, Derivative Assay, Protein Binding, Software