Journal: Oncology Letters

Article Title: Suppressive effect of YY1-mediated RGS22 regulation on the proliferation, migration and invasion of pancreatic ductal adenocarcinoma

doi: 10.3892/ol.2022.13577

Figure Lengend Snippet: Regulation of RGS22 expression by the binding of YY1 directly to the RGS22 promoter. (A-C) RGS22 expression in YY1-overexpressing PANC-1 cells or YY1-knockdown CFPAC-1 cells was measured by reverse transcription-quantitative PCR and western blotting. (D) Schematic diagram of the luciferase reporter construct containing the human RGS22 promoter and the mutant construct containing the RGS22 promoter in which the predicted YY1 binding site was mutated. (E) EMSA showing that YY1 binds to the RGS22 promoter. The wild-type probe was incubated without (lane 1) or with (lane 2) PANC-1-YY1 cell nuclear proteins in the absence or presence of unlabeled probes (lanes 3–6). Lanes 3 and 4 contain the wild-type probe, and lanes 5 and 6 contain the mutant probe, each at 50- and 100-fold molar excess. EMSA was performed using an anti-YY1 antibody (lane 7), and the IgG antibody was used as a negative control for the YY1 antibody (lane 8). (F) Luciferase assays demonstrated the luciferase activity of PANC-1 cells transfected with YY1-overexpression or control lentiviruses. Data are presented as the mean ± SD of three independent experiments.*P≤0.05, **P≤0.01, ***P≤0.001. EMSA, electrophoretic mobility shift assay; RGS22, Regulator of G-protein signaling 22; YY1, Yin Yang-1; OE, overexpression; NC, negative control; KD, knockdown; WT, wildtype; MT, mutant.

Article Snippet: Each transfection was performed using 1 μg luciferase reporter construct (WT pRGS22 or MT pRGS22) plus 2.5 ng Renilla luciferase reporter vector, pRL-SV40 as an internal control (Promega Corporation).

Techniques: Expressing, Binding Assay, Knockdown, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Luciferase, Construct, Mutagenesis, Incubation, Negative Control, Activity Assay, Transfection, Over Expression, Control, Electrophoretic Mobility Shift Assay

Journal: The Journal of Biological Chemistry

Article Title: A kinetic model for compound heterozygous pathogenic variants in Tyrosyl-tRNA synthetase gene YARS2 -Associated neonatal phenotype

doi: 10.1016/j.jbc.2024.108092

Figure Lengend Snippet: Aminoacylation of homodimers . A , protein expression of compound heterozygosity of the subject, showing the 1:1:2 ratio of the homodimers F185L/F185L ( green ) and ΔE264/ΔE264 ( magenta ) to the heterodimer F185L-ΔE264. B , the nascent chain of an mt-TyrRS monomer of one ribosome can associate with the nascent chain of an adjacent ribosome on the same or a different mRNA to form a homodimer . C , Michaelis-Menten plots of the three homodimers WT/WT, F185L/F185L, and ΔE264/ΔE264, showing the r 2 of each. Error bars are shown for the WT/WT but are invisible for the two variants. D , kinetic parameters of each homodimer. Values are the average ± SD, N = 3 to 6.

Article Snippet: The WT-WT heterodimer construct was made by GenScript, where the native YARS2 was covalently linked to a recoded gene and the cassette was cloned into the BamHI and EcoRV sites of pET30a.

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: A kinetic model for compound heterozygous pathogenic variants in Tyrosyl-tRNA synthetase gene YARS2 -Associated neonatal phenotype

doi: 10.1016/j.jbc.2024.108092

Figure Lengend Snippet: Aminoacylation of heterodimers. A , assembly of a heterodimer via a covalent link of two monomers as translated by a ribosome based on one copy of YARS2 in the native sequence ( green ), followed by the linker ( cyan ), and the recoded sequence ( magenta ). B , Michaelis-Menten plots of WT-WT and F185L-ΔE264, showing the r 2 of each. C , kinetic parameters of each homodimer vs. heterodimer, showing the fold-decrease in k cat / K m relative to WT/WT. Individual values are the average ± SD (N = 4–6). D , Western blot of an SDS-PAGE of homo- and heterodimers, where the markers were superimposed from the image of a pre-stained gel. E , Western blot of a native PAGE of unlinked and linked proteins, marking the YARS2 protein by an arrow, which was recognized by the α-His antibody (as shown) and the α-mt-TyrRS antibody (not shown). The lower band was recognized only by the α-His antibody, indicating that it is a co-purified contaminant. The different migration profile of F185L/F185L could be due to perturbation of the dimer interface when F185L was in an unlinked homodimer. F , Western blot analysis of limited trypsin digestion of WT/WT, WT-WT, and F185L-ΔE264, showing degradation products in red boxes .

Article Snippet: The WT-WT heterodimer construct was made by GenScript, where the native YARS2 was covalently linked to a recoded gene and the cassette was cloned into the BamHI and EcoRV sites of pET30a.

Techniques: Sequencing, Western Blot, SDS Page, Staining, Clear Native PAGE, Purification, Migration