Journal: Biomolecules

Article Title: Probenecid Inhibits NLRP3 Inflammasome Activity and Mitogen-Activated Protein Kinases (MAPKs).

doi: 10.3390/biom15040511

Figure Lengend Snippet: Figure 1. MAPK signaling in LPS-primed macrophages. J774A.1 macrophages were treated with 0.1 µM probenecid, 25 µM JNK inhibitor (SP600125), 10 µM ERK inhibitor (PD98059), or dilu- ent (0.1% DMSO) as described. (A) Samples were probed for phospho-ERK1/2 proteins using anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204), (B) phospho-JNK1/2 proteins using anti- phospho-SAPK/JNK (Thr183/Tyr185), or (C) phospho-p38 proteins using anti-phospho-p38 MAPK (Thr180/Tyr182) (Cell Signaling). The membranes were washed three times with TBS-T, and the signal developed with SuperSignal West Pico PLUS chemiluminescent substrate (Thermo Fisher). Total protein staining of transferred membranes utilizing the fluorescent ‘no-stain’ protein labeling reagent (Thermo Fisher) was used to normalize for protein loading with (n = 3) independent replicates for each condition. Immunoblot and total protein blot images were obtained using the iBright 1500 Im- ager (Thermo Fisher), representing three independent replicates per condition. Background-corrected band volumes were normalized to total protein, and the mean was calculated from three independent replicates for each condition using iBright analysis software (Thermo Fisher). The graphs depict the band volume quantification in arbitrary units (A.U.) represented as mean ± SD. Original western blots can be found at Supplementary Materials.

Article Snippet: J774A.1 murine macrophages (ATCC TIB-67; isolated in 1968 from the ascites of an adult female mouse with reticulum cell sarcoma) were propagated in DMEM (Gibco, Waltham, MA, USA) + 10% heat-inactivated FBS (Hyclone, Logan, UT, USA) at 37 ◦C.

Techniques: Staining, Labeling, Western Blot, Software

Journal: Biomolecules

Article Title: Probenecid Inhibits NLRP3 Inflammasome Activity and Mitogen-Activated Protein Kinases (MAPKs).

doi: 10.3390/biom15040511

Figure Lengend Snippet: Figure 3. MAPK signaling and NLRP3 inflammasome activity in LPS and nigericin-activated macrophages. J774A.1 macrophages were stimulated with 100 ng/mL LPS for 6 h and 0.1 µM probenecid, 25 µM JNK inhibitor (SP600125), 10 µM ERK inhibitor (PD98059), 20 µM p38 inhibitor (SB202190), or diluent (0.1% DMSO) were added for the last 2 h of LPS treatment. NLRP3 inflamma- some was activated by adding 20 µM nigericin for 1 h. LPS-primed macrophages without further treatment were activated with nigericin (LPS + nigericin) or not activated (LPS only) to serve as NLRP3 inflammasome activation controls. Culture supernatants were harvested and adjusted with 2 mM PMSF protease inhibitor. The cell-free supernatants were evaluated using a capture ELISA to measure the levels of (A) IL-1β protein, or (B) TNFα per the manufacturer’s instructions. Protein concentration was calculated from three independent replicates, represented as mean ± SD for each condition.

Article Snippet: J774A.1 murine macrophages (ATCC TIB-67; isolated in 1968 from the ascites of an adult female mouse with reticulum cell sarcoma) were propagated in DMEM (Gibco, Waltham, MA, USA) + 10% heat-inactivated FBS (Hyclone, Logan, UT, USA) at 37 ◦C.

Techniques: Activity Assay, Activation Assay, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Protein Concentration

Journal: Biomolecules

Article Title: Probenecid Inhibits NLRP3 Inflammasome Activity and Mitogen-Activated Protein Kinases (MAPKs).

doi: 10.3390/biom15040511

Figure Lengend Snippet: Figure 4. Probenecid treatment inhibits NLRP3 inflammasome activity in LPS and nigericin-activated macrophages. J774A.1 macrophage was stimulated with 100 ng/mL LPS for 6 h with the addition of 0.1 µM probenecid, 10 µM NLRP3 inhibitor (MCC950), or diluent (0.1% DMSO) for the last 2 h of LPS treatment. NLRP3 inflammasomes were activated by adding 20 µM nigericin for 1 h. LPS- primed macrophages without further treatment were activated with nigericin (LPS + nigericin) or not activated (LPS only) to serve as NLRP3 inflammasome activation controls. Culture supernatants were harvested and adjusted with 2 mM PMSF protease inhibitor. The cell-free supernatants were evaluated using a capture ELISA to measure the levels of (A) IL-1β protein, or (B) TNFα per the manufacturer’s instructions. Mean protein concentration was calculated from three independent replicates, represented as mean ± SD for each condition. (C) LDH assay for production of LDH per the manufacturer’s instructions. Data expressed as a percentage of maximal release calculated from three independent replicates per condition and represented as mean ± SD.

Article Snippet: J774A.1 murine macrophages (ATCC TIB-67; isolated in 1968 from the ascites of an adult female mouse with reticulum cell sarcoma) were propagated in DMEM (Gibco, Waltham, MA, USA) + 10% heat-inactivated FBS (Hyclone, Logan, UT, USA) at 37 ◦C.

Techniques: Activity Assay, Activation Assay, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Protein Concentration, Lactate Dehydrogenase Assay

Journal: Biomolecules

Article Title: Probenecid Inhibits NLRP3 Inflammasome Activity and Mitogen-Activated Protein Kinases (MAPKs).

doi: 10.3390/biom15040511

Figure Lengend Snippet: Figure 5. Probenecid treatment inhibits cas-1 and GASD in response to NLRP3 inflammasome activation in macrophages. J774A.1 macrophages were stimulated with 100 ng/mL LPS for 6 h with the addition of 0.1 µM probenecid, 10 µM NLRP3 inhibitor (MCC950), or diluent (0.1% DMSO) for the last 2 h of LPS treatment. The NLRP3 inflammasome was activated by adding 20 µM nigericin for 1 h. LPS-primed macrophages without further treatment were activated with nigericin (LPS + nigericin) or not activated (LPS only) to serve as NLRP3 inflammasome activation controls. Western blots were performed using equal amounts (20 µL) of a 1:1 mixture of cell lysates and culture supernatants separated on 4–20% gradient SDS-PAGE gels and then transferred to nitrocellulose membrane for immunoprobing. Membranes were detected using anti-cas-1 rabbit mAb (Cell Signaling), anti-cas-1 (p20 subunit) (Casper-1 Adipogen), anti-GASD rabbit mAb (Cell Signaling), anti-cleaved GASD (Asp276) rabbit mAb (Cell Signaling), or anti-IL-1β rabbit mAb (Cell Signaling). After three washes, the membrane signal was developed with SuperSignal West Pico PLUS chemiluminescent substrate

Article Snippet: J774A.1 murine macrophages (ATCC TIB-67; isolated in 1968 from the ascites of an adult female mouse with reticulum cell sarcoma) were propagated in DMEM (Gibco, Waltham, MA, USA) + 10% heat-inactivated FBS (Hyclone, Logan, UT, USA) at 37 ◦C.

Techniques: Activation Assay, Western Blot, SDS Page, Membrane