Journal: bioRxiv

Article Title: Primer- and template-independent RNA polymerization by terminal nucleotidyltransferase TENT4B

doi: 10.64898/2026.03.05.709691

Figure Lengend Snippet: A, Extension of a 20-mer RNA substrate oligo (oligo 1) using rTENT4B and ATP analogs demonstrating differential incorporation. (AMP: Adenosine-5’-monophosphate; ADP: Adenosine-5’-diphosphate; ATP: Adenosine-5’-triphosphate. n=3 replicates, one representative shown. B, Utilization of nucleotide diphosphates (NDPs) by rTENT4B. rTENT4B along with a 20-mer RNA-substrate oligo (oligo 2) was incubated in the presence of four canonical NDPs, in RNA-substrate extension assays, and reaction products were resolved using denaturing TBE/Urea-acrylamide gel followed by Sybr gold staining. C , rTENT4B was incubated with four canonical NDPs plus 5’-Cy5-labeled ATP as substrate. NDP incorporation was confirmed via detecting generation of Cy5-labeled RNA chains after 30 min in no RNA-substrate added reactions. D , De novo ADP polymerization using rTENT4B. rTENT4B along with increasing concentrations of ADP was subjected to RNA-substrate extension assay in the absence of an RNA-substrate, and generation of de novo synthesized RNA products were detected using denaturing TBE/Urea-acrylamide gel followed by Sybr gold staining. E , Consumption of ADP by rTENT4B via analysis of Pi yield, in the presence/ absence of inorganic pyrophosphatase (PPA) as a measure of ATP contamination (ADP + Pi), in the presence/absence of an RNA-substrate oligo, compared to that of rTENT4B alone versus rTENT4B plus ATP. n=4 biological replicates. Mean + S.D. shown, 2-way ANOVA, ns- not significant, ****p<0.001.

Article Snippet: ATP- Adenosine-5’-triphosphate (N0450, New England Biolabs) ADP- Adenosine-5’-diphosphate (NU-1198, Jena Bioscience) AMP- Adenosine-5’-monophosphate (A1752, Sigma) GTP- Guanosine-5’-triphosphate (N0450, New England Biolabs) GDP- Guanosine-5’-diphosphate (G7127, Sigma) CTP- Cytidine-5’-triphosphate (N0450, New England Biolabs) CDP- Cytidine-5’-diphosphate (C9755, Sigma) UTP- Uridine-5’-triphosphate (N0450, New England Biolabs) UDP- Uridine-5’-diphosphate (94330, Sigma) Cy5-ATP- γ-(6-Aminohexyl)-ATP-Cy5- (NU-833-CY5, Jena Bioscience) Cy5-GTP- γ-(6-Aminohexyl)-GTP-Cy5- (NU-834-CY5, Jena Bioscience)

Techniques: Incubation, Acrylamide Gel Assay, Staining, Labeling, Synthesized

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Differential expression of adenosine A3 receptors controls adenosine A2A receptor-mediated inhibition of TLR responses in microglia.

doi: 10.4049/jimmunol.0803383

Figure Lengend Snippet: FIGURE 4. The enhanced inhibitory effect of NECA is mediated by the enhanced contribution of A2AR-mediated signaling and the decreased contribution of A3R-mediated signaling. Unstimulated and activated microglia were exposed to 100 ng LPS/ml in the presence or absence of 10 M NECA in combination with A1, A2A, A2B, and A3 receptor antagonists. After 16 h, TNF- (A) and IL-12p40/p70 (B) levels were measured and expressed as relative values of exposure to 100 ng LPS/ml alone. DPCPX was used as an A1R, SCH58261 as an A2AR, alloxazine as an A2BR and VUF5574 as an A3R antagonist. All antagonists were used at 10 M concentrations. Solid lines in the graph indicate relative cytokine production levels of exposure to 100 ng LPS/ml 10 M NECA without antagonists of unstimulated microglia, the dashed lines indicate activated microglia. , p 0.05; Student’s t test. TNF- (C) and IL-12p40/p70 (D) production after exposure to 100 ng LPS/ml in the presence or absence of 10 M CGS21680, an A2AR-selective agonist, and/or of 10 M VUF5574, an A3R antagonist. Cytokine production is expressed relative to production after exposure to 100 ng LPS/ml alone for unstimulated and activated microglia. , p 0.05; Student’s t test. TNF- (E) and IL-12p40/p70 (F) production after exposure to 100 ng LPS/ml different concentrations of CGS21680, in the presence or absence of A3R antagonist, is expressed relative to production after exposure to 100 ng LPS/ml alone for unstimulated and activated microglia. All data are representative for three independent experiments using different donors.

Article Snippet: Cells were washed and incubated for 30 min with anti-adenosine A3 receptor Abs (SP055P, Acris Antibodies) or total rabbit IgG (AbD Serotec) as an isotype control.

Techniques: