nlrp3 antibody  (MedChemExpress)

Journal: The Journal of Experimental Medicine

Article Title: FIP200 regulates plasma B cell differentiation via mitochondrial and heme homeostasis

doi: 10.1084/jem.20250535

Figure Lengend Snippet: Dysfunctional mitochondria accumulate in Fip200 −/− B cells. (A) Mitochondrial mass in naïve (left, WT, n = 3; and Fip200 −/− , n = 3) and day 3 IL4+LPS-activated (right, WT, n = 6; and Fip200 −/− , n = 3) B cells stained with MitoTracker Deep Red, ****P < 0.0001 (unpaired t test). Representative data of at least two experimental replicates shown. (B) OCR measured by Seahorse XF analyzer for activated B cells (four samples per group) at day 0 (left), day 1 (middle), and day 2 (right). FCCP is a mitochondrial uncoupling agent. Oligo., oligomycin; R/A, rotenone/antimycin. From left to right: day 0, *P = 0.0325, **P = 0.0014, ***P = 0.0007,**P = 0.0022, **P = 0.0083, **P = 0.0026, **P = 0.0057; day 1, **P = 0.0027, **P = 0.0041, **P = 0.0051, **P = 0.0058, ***P = 0.0002, ***P = 0.0003, ***P = 0.0004; day 2, ****P < 0.0001, ****P < 0.0001, ****P < 0.0001, ****P < 0.0001 (unpaired t test). (C) Splenocytes from WT ( n = 6) and B- Fip200 −/− ( n = 5) mice were stained with MitoSOX Red and naïve splenic B cells (left) and B220 − cells (right) detected by FACS, **P = 0.0022 (unpaired t test). (D and E) WT and Fip200 −/− B cells were stimulated by IL4+LPS and stained with MitoSOX Red and gated on CD138 + population. Representative plots (D) and the corresponding quantifications (E) of total live cells (left) or plasma cells (right) of WT and Fip200 −/− B cells. Data are representative of at least two independent experiments run with two mice per group. Representative data from one experiment are shown. Left to right: ****P < 0.0001, ****P < 0.0001 (unpaired t test). (F and G) BM cells from WT ( n = 5) and B- Fip200 −/− ( n = 6) mice were stained with MitoSOX and gated on IgD hi cells (DUMP − IgD hi ) and plasma cells (DUMP − IgD − Sca-1 + CD138 + ). Representative plots (F) and the corresponding quantifications (G) of IgD hi cells (top left), mROS of IgD hi cells (top right), plasma cells (bottom left), or mROS of plasma (bottom right) of WT and Fip200 −/− B cells. Two independent experiments were performed; representative data from one experiment are shown. Top left to right: *P = 0.0173, *P = 0.0173; bottom left to right: *P = 0.0173, **P = 0.0043 (unpaired t test). (H) Expression level of NLRP3 in WT and Fip200 −/− B cells and cleaved caspase-1 in the supernatant upon IL4+LPS stimulation at day 2. This experiment was performed twice, with results from one independent run shown; each lane represents one mouse. Significant (α = 0.05) P values were determined by an unpaired t test. *P = 0.0182. Source data are available for this figure: .

Article Snippet: Membranes were blocked by incubation in WB blocking buffer (5% milk in TBS, 0.1% Tween) at RT for 1 h and probed with specific Abs diluted in WB blocking buffer or 5% BSA in TBS-T. Proteins were detected with antibodies against FIP200 (D10D11, AB_2797913), P62 (D1Q5S, AB_2799160), LC3B (D11, AB_2137707), cleaved caspase-3 (Asp175, AB_2341188), NLRP3 (D4D8T, AB_2722591), cleaved caspase-1 (Asp296) (E2G2I, AB_2923067) (Cell Signaling Technology), and TAX1BP1 (14424-1-AP) (AB_2198921; Proteintech) using the secondary HRP-conjugated anti-rabbit or anti-mouse antibodies (AB_2307391, AB_2338506; Jackson ImmunoResearch) or anti-β-actin−peroxidase antibody (AC-15) (AB_262011; MilliporeSigma).

Techniques: Staining, Clinical Proteomics, Expressing

Journal: iScience

Article Title: Size-dependent pyroptosis induction by calcium oxalate monohydrate crystals in HK-2 cells

doi: 10.1016/j.isci.2025.114459

Figure Lengend Snippet: Differences in pyroptosis induced by COM crystals of different sizes (A) Caspase-1/PI double staining flow cytometry quantitative analysis. (B) Caspase-1/PI/Hoechst 33342 triple staining confocal observation. Scale bars, 50 μm. (C) NLRP3 immunofluorescence map. Scale bars, 100 μm. (D) Quantitative histogram of pyroptosis; n = 3; mean ± SEM. (E) Quantitative bar graph of NLRP3; n = 3; mean ± SEM. NC: normal control. COM crystals were incubated with HK-2 cells for 48 h. Compared with NC group, ∗ p < 0.05; ∗∗ p < 0.01. (A) flow graph is divided into four regions (Q1, Q2, Q3, and Q4), of which Q1 is PI high signal area and Caspase-1 low signal area, representing apoptotic necrotic cells. Q2 is the PI high signal region and Caspase-1 high signal region, which represents the late pyroptosis cells. Q3 is the PI low signal area and Caspase-1 high signal area, which represents the early pyroptosis cells (Caspase-1 is actively expressed). Q4 is the PI hypointense region and Caspase-1 hypointense region, representing normal cells. Q1+ Q2 refers to dead cells, and Q2+ Q3 refers to pyroptosis cells. (B) shows the presence of four types of cells: the first type is the normal cell (indicated by the orange arrow), which corresponds to the Q4 region cells in (A); the second type was apoptotic or necrotic cells (indicated by white arrow), namely Q1 region cells. The third type was the late pyroptosis cells (yellow arrow), which were Q2 cells. The fourth type is the cells in the early stage of pyroptosis (purple arrow), which is the Q3 region cells. Orange arrows refer to normal cells, white arrows to apoptotic necrotic cells, yellow arrows to late pyroptosis cells, and purple arrows to early pyroptosis cells.

Article Snippet: After reaching the incubation time, the culture medium was removed, washed twice with PBS, fixed with paraformaldehyde for 15 min, blocked with sheep serum for 20 min, and then incubated with NLRP3 primary antibody (1:100) (Proteintech, Cat No. 30109-1-AP) at 4°C overnight.

Techniques: Double Staining, Flow Cytometry, Staining, Immunofluorescence, Control, Incubation

Journal: iScience

Article Title: Size-dependent pyroptosis induction by calcium oxalate monohydrate crystals in HK-2 cells

doi: 10.1016/j.isci.2025.114459

Figure Lengend Snippet: Level of proteins in pyroptosis signaling pathway induced by COM crystals of different sizes (A) protein bands of NLRP3, cleaved caspase-1 p20, IL-18, and IL-1β. (B–E) Quantitative analysis of NLRP3, cleaved caspase-1 p20, IL-18, and IL-1β, respectively. n = 3; mean ± SEM. NC: normal control. COM crystals were incubated with HK-2 cells for 48 h.∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: After reaching the incubation time, the culture medium was removed, washed twice with PBS, fixed with paraformaldehyde for 15 min, blocked with sheep serum for 20 min, and then incubated with NLRP3 primary antibody (1:100) (Proteintech, Cat No. 30109-1-AP) at 4°C overnight.

Techniques: Control, Incubation