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1.
FIGURE 7.

FIGURE 7. From: Inflammation in Mice Ectopically Expressing Human Pyogenic Arthritis, Pyoderma Gangrenosum, and Acne (PAPA) Syndrome-associated PSTPIP1 A230T Mutant Proteins.

No arthritis or inflammatory skin lesions found in mice ectopically expressing A230T mutant Pstpip1 proteins. Hematoxylin and eosin-stained tissue sections of knee and skin showed no signs of inflammation (A). Magnetic Resonance Imaging of brains of Rosa-26-PSTPIP1 WT and A230TSTOP del/+ mice (B). H&E staining of brain sections from Rosa-26-PSTPIP1 WT and A230TSTOP del/+ mice (C).

Donghai Wang, et al. J Biol Chem. 2013 February 15;288(7):4594-4601.
2.
FIGURE 4.

FIGURE 4. From: Inflammation in Mice Ectopically Expressing Human Pyogenic Arthritis, Pyoderma Gangrenosum, and Acne (PAPA) Syndrome-associated PSTPIP1 A230T Mutant Proteins.

Turpentine-induced inflammatory response is not affected in Pstpip1 knock-out mice. Sex- and age-matched wild type (n = 10) and Pstpip1-deficient mice (n = 10) were injected s.c. with 100 μl of turpentine. Body weight was measured every day for 13 days after injection. Data are expressed as mean ± S. D. (A). Blood was drawn right before, 8 and 24 h post-turpentine injection. Circulating IL-6 levels were determined by ELISA. Data are expressed as mean ± S.D. (B).

Donghai Wang, et al. J Biol Chem. 2013 February 15;288(7):4594-4601.
3.
FIGURE 1.

FIGURE 1. From: Inflammation in Mice Ectopically Expressing Human Pyogenic Arthritis, Pyoderma Gangrenosum, and Acne (PAPA) Syndrome-associated PSTPIP1 A230T Mutant Proteins.

Ectopic expression of mutant Pstpip1 proteins in 293 cells induces Pyrin dependent processing of murine pro-IL-1β. MSCV retroviral vectors harboring cDNAs encoding human wild type or mutant PSTPIP1, human ASC, human and mouse PYRIN and pCI vectors harboring pro-caspase-1 and murine pro-IL-1β fused with gaussia luciferase reporter gene were transfected into HEK 293 cells along with a Renilla luciferase reporter plasmid. Luciferase activity of cell lysates was measured 48 h after transfection.

Donghai Wang, et al. J Biol Chem. 2013 February 15;288(7):4594-4601.
4.
FIGURE 5.

FIGURE 5. From: Inflammation in Mice Ectopically Expressing Human Pyogenic Arthritis, Pyoderma Gangrenosum, and Acne (PAPA) Syndrome-associated PSTPIP1 A230T Mutant Proteins.

Generation of Rosa-26-Pstpip1-expressing conditional allele. cDNAs encoding wild type or A230T or E250Q mutant human PSTPIP1 proteins were inserted into Rosa-26 locus through homologous recombination in mouse ES cells. These targeted cDNAs were preceded by a transcriptional and translational loxP-flanked STOP signal. In this system, cre-mediated deletion of the STOP signal and subsequent expression of the conditional alleles can be monitored by the concomitant expression of the tailless human CD2 protein inserted downstream of the Pstpip1 cDNA controlled by an IRES (A). Treatment of targeted ES cells with TAT-Cre confirmed the ectopic expression of PSTPIP1 proteins (B). ●, Frt sites; -▵-, loxP site.

Donghai Wang, et al. J Biol Chem. 2013 February 15;288(7):4594-4601.
5.
FIGURE 3.

FIGURE 3. From: Inflammation in Mice Ectopically Expressing Human Pyogenic Arthritis, Pyoderma Gangrenosum, and Acne (PAPA) Syndrome-associated PSTPIP1 A230T Mutant Proteins.

Activation of conventional inflammasomes is not impaired in the absence of Pstpip1 proteins. Bone marrow-derived macrophages were primed with 200 ng/ml pure LPS for 2 h followed by stimulation with stimulants that activate Nlrp3 (ATP 5 mm, Nigericin 5 μm, silica 500 μg/ml, MSU 500 μg/ml, Gourp B streptococcus (GBS) MOI 10), Aim2 (poly dA:dT 1.5 μg, Mouse Cytomega Virus (MCMV) MOI 10, Vaccina Virus MOI 5, L. monocytogenes MOI 5), or Nlrc4 (S. typhymurium MOI 5) inflammasomes, respectively, for additional 6 h. IL-1β in the culture supernatant was measured by ELISA (A), or supernatants were precipitated with methanol chloroform, and the presence of mature IL-1β was determined by Western blot.

Donghai Wang, et al. J Biol Chem. 2013 February 15;288(7):4594-4601.
6.
FIGURE 2.

FIGURE 2. From: Inflammation in Mice Ectopically Expressing Human Pyogenic Arthritis, Pyoderma Gangrenosum, and Acne (PAPA) Syndrome-associated PSTPIP1 A230T Mutant Proteins.

Generation of a conditional knock-out allele of Pstpip1. A, conditional mouse allele of Pstpip1 gene were established, in which exons 4–11 were flanked by two loxP sites through homologous recombination in C57BL6 mouse ES cells. This established allele will be hereafter referred to as the floxed (flanked by loxP sites) Pstpip1 allele (Pstpip1 f/). ES cell clones carrying such floxed Pstpip1 alleles were subsequently injected into C57BL6 blastocysts. Independent mouse strains were derived from the injection and germline transmission of the targeted Pstpip1 allele was confirmed by Southern blotting analysis of tail DNA (B). Mice carrying the Pstpip1 f/allele were crossed to Cre deleter mice to generate the Pstpip1-deleted allele and were brought to homozygosity to generated Pstip1-deficient mice. The absence of Pstpip1 proteins was confirmed by Western blot of total cell lysate of lymphoid organs (C).

Donghai Wang, et al. J Biol Chem. 2013 February 15;288(7):4594-4601.
7.
FIGURE 8.

FIGURE 8. From: Inflammation in Mice Ectopically Expressing Human Pyogenic Arthritis, Pyoderma Gangrenosum, and Acne (PAPA) Syndrome-associated PSTPIP1 A230T Mutant Proteins.

The runty phenotype in Rosa-26-PSTPIP1 A230TSTOP del/+ mutant protein-expressing mice is not caused by mutant PSTPIP1 proteins in hematopoietic cells. Body weight of mice ectopically expressing PSTPIP1 proteins specifically in hematopoietic cells was measured every 3 days. Data are expressed as mean ± S.D. (A). Ectopic expression of PSTPIP1 proteins was confirmed by Western blot and hCD2 expression on total splenocytes (B). Bone marrow- derived macrophage from mice ectopically expressing wild type or A230T mutant PSTPIP1 proteins primed with LPS (200 ng/ml) followed by stimulation with nigericin (5 μm,1 h), silica (500 μg/ml), poly dA:dT (3 μg), or GBS (MOI 10) for additional 6 h, IL-1β in culture supernatant were measured by ELISA (C). Blood was drawn from wild type C57BL/6, Rosa-PSTPIP1-WTSTOP del/+; Rosa-26-PSTPIP1 A230TSTOP del/+; Rosa-26-PSTPIP1 WTSTOP floxed/+, VaviCre+; Rosa-26-PSTPIP1 A230TSTOP floxed/+, VaviCre+ mice. Circulating IL-1α, IL-1β, and TNFα were determined by ELISA (D).

Donghai Wang, et al. J Biol Chem. 2013 February 15;288(7):4594-4601.
8.
FIGURE 6.

FIGURE 6. From: Inflammation in Mice Ectopically Expressing Human Pyogenic Arthritis, Pyoderma Gangrenosum, and Acne (PAPA) Syndrome-associated PSTPIP1 A230T Mutant Proteins.

A runty phenotype, elevated inflammatory cytokines, and normal hematopoietic cell populations in mice ectopically expressing Pstpip1 A230T mutant proteins. Rosa-26 Pstpip1 A230TSTOP del/+ mice were not born in accordance to Mendelian ratio (A). Ectopic expression of human PSTPIP1 proteins were confirmed by Western blot of total cell lysate of splenocytes from Rosa-26 transgenic mice and by flow cytometry analysis of hCD2 expression (B). Body weight of mice ectopically expressing wild type or A230T mutant PSTPIP1 proteins was measured every other day or every 3 days 2 weeks after birth (C). Blood was drawn from mice ectopically expressing wild type or A230T mutant PSTPIP1 mutant proteins at the age of 6 months old, and circulating levels of IL-1α, IL-1β, and TNF were determined by ELISA (D).

Donghai Wang, et al. J Biol Chem. 2013 February 15;288(7):4594-4601.

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