Journal of Immunology Research / 2020 / Article / Fig 3

Research Article

Adenosine-Induced NLRP11 in B Lymphoblasts Suppresses Human CD4+ T Helper Cell Responses

Figure 3

Adenosine-induced endogenous NLRP11 interacts with the ASC adaptor protein but does not lead to the activation of caspase-1 enzyme and secretion of IL-1β. (a) Left panel: proteins were extracted, immunoprecipitated (IP) via incubation with anti-ASC antibody, and followed by precipitation with protein-A/G-magnetic beads. Blots were probed with an anti-NLRP11 antibody and visualized using ECL after incubation with an anti-rat HRP-conjugated antibody. Middle panels: controls. Right lane: cell lysates were separated by SDS-PAGE and immunoblotted (IB). (b) Caspase enzyme activity: cells were treated with either 50 μM adenosine or PBS for 4 hours. YVAD-AFC substrate was added and incubated for 2 hours at 37°C. Fluorescent intensity was measured on a fluorescent plate reader (excitation wavelength: 400 nm; emission wavelength: 505 nm). (c) Adenosine-treated cell lysates were separated by SDS-PAGE, and intracellular IL-1β, ASC, and caspase-1 levels were measured by immunoblotting. (d) Differential mRNA expressions of pro-IL-1β and pro-IL-18 were measured by QPCR, and data was normalized to HPRT and depicted as fold changes in the axis. Student’s -test shows the significant difference between stimulated and nonstimulated cells. indicates , , and .
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