Journal: bioRxiv

Article Title: Cullin1 represses systematic inflammasome activation by binding and catalyzing NLRP3 ubiquitination

doi: 10.1101/289637

Figure Lengend Snippet: A The lysates of PMA-differentiated THP-1 macrophages were immunoprecipitated with anti-Cullin1 antibody and then immunoblotted with indicated antibodies. B HEK293T cells were transfected with pCAGGS-HA-NLRP3 and pcDNA3.1(+)-3xFlag-Cullin1, pcDNA3.1(+)-3xFlag-ROC1, pcDNA3.1(+)-3xFlag-SKP1-α and pcDNA3.1(+)-3xFlag-SKP1-β, respectively. C HEK293T cells were transfected with pcDNA3.1(+)-NLRP3 and pcDNA3.1(+)-3XFlag-Cullin1. D HEK293T cells were transfected with pCAGGS-HA-NLRP3 and pcDNA3.1(+)-3xFlag-Cullin1, pcDNA3.1(+)-3xFlag-ROC1, pcDNA3.1(+)-3xFlag-Cullin1, and pcDNA3.1(+)-3xFlag-ROC1. E HEK293T cells were transfected with pcDNA3.1(+)-3xFlag-Cullin1 and pCAGGS-HA-NLRP3, pcDNA3.1(+)-3xFlag-ROC1 and pcDNA3.1(+)-3xFlag-SKP1, pcDNA3.1(+)-3XFlag-SKP1 and pCAGGS-HA-NLRP3, or pcDNA3.1(+)-3xFlag-ROC1 and pCAGGS-HA-NLRP3, respectively. F HEK293T cells were transfected with pCAGGS-HA-NLRP3 and pcDNA3.1(+)-3xFlag-Cullin1 and pcDNA3.1(+)-3xFlag-ROC1 at different concentrations. G HEK293T cells were transfected with pcDNA3.1(+)-3XFlag-Cullin1 or pcDNA3.1(+)-3XFlag-ROC1 and pCAGGS-HA-NLRP3 at different concentrations. H HEK293T cells were transfected with pcDNA3.1(+)-3xFlag-Cullin1 and pSilencer2.1-U6-sh-Cullin1#1 or pSilencer2.1-U6-sh-Cullin1#2. I HEK293T cells were transfected with pcDNA3.1(+)-3xFlag-Cullin1 or pcDNA3.1(+)-3xFlag-NLRP3 and pSilencer2.1-U6-shROC1#2. Data information: In ( B–I ), the cell lysates were immunoprecipitated with anti-Flag antibody (B), anti-NLRP3 antibody (C, E), anti-HA antibody (D, F), anti-Cullin1 antibody (G–I) and then immunoblotted with indicated antibodies.

Article Snippet: Anti-Human/Mouse/Rat ROC1 (Cat# 14895-1-AP) antibody was obtained from Proteintech (Rosemont, IL, USA).

Techniques: Immunoprecipitation, Transfection

Journal: bioRxiv

Article Title: Cullin1 represses systematic inflammasome activation by binding and catalyzing NLRP3 ubiquitination

doi: 10.1101/289637

Figure Lengend Snippet: A HEK293T cells were transfected with pcDNA3.1(+)-NLRP3 and pCAGGS-HA-Ub at different concentrations. B HEK293T cells were transfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-HA-Ub, and/or pcDNA3.1(+)-3Flag-Cullin1, respectively. C HEK293T cells were transfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-HA-Ub, pcDNA3.1(+)-3Flag-Cullin1 or pcDNA3.1(+)-3Flag-ROC1, as indicated. D Daigrams of full-length NLRP3 and its point mutants. The potential ubiquitination sites in NLRP3 among the Lys residues were predicated by using UbPred software. One Lys (Lys689) is high confidence and six Lys (Lys93, Lys192, Lys194, Lys324, Lys430, and Lys696) are medium confidence. Seven mutations of CUL1 (K93R, K192R, K194R, K324R, K430R, K689R, and K696R), in which the K residues were replaced by R residues. E HEK293T cells were co-transfected with pcDNA3.1(+)-HA-Ub and pcDNA3.1(+)-Cullin1, along with pcDNA3.1(+)-3xFlag-NLRP3 or its mutants, respectively. F HEK293T cells were co-transfected with pcDNA3.1(+)-NLRP3 and/or pcDNA3.1(+)-HA-Ub, pcDNA3.1(+)-3Flag-Cullin1, pcDNA3.1(+)-HA-K48 or pcDNA3.1(+)-HA-K63, as indicated. G PMA-differentiated THP-1 macrophages stably expressing sh-NC and sh-Cullin1#2 were infected with IAV H3N2. H HEK293T cells were transfected with pcDNA3.1(+)-3XFlag-NLRP3, pcDNA3.1(+)-3XFlag-ASC, pcDNA3.1(+)-3XFlag-pro-caspase-1, pcDNA3.1(+)-3XFlag-pro-IL-1β, or pcDNA3.1(+)-3xFlag-Cullin1, as indicated. I HEK293T cells were transfected with pCAGGS-HA-NLRP3 and/or pcDNA3.1(+)-3XFlag-Cullin1, or pcDNA3.1(+)-3XFlag-ROC1. Data information: In ( A–C ), the lysates were immunoprecipitated with anti-NLRP3 antibody and immunoblotted with indicated antibodies. In ( E–I ), the lysates were immunoprecipitated with anti-Flag antibody (E) or anti-NLRP3 antibody (F, G) and then immunoblotted with indicated antibodies. The lysates were immunoblotted with the indicated antibodies (H, I).

Article Snippet: Anti-Human/Mouse/Rat ROC1 (Cat# 14895-1-AP) antibody was obtained from Proteintech (Rosemont, IL, USA).

Techniques: Transfection, Software, Stable Transfection, Expressing, Infection, Immunoprecipitation

Journal: PLoS ONE

Article Title: Abnormal Behaviors and Developmental Disorder of Hippocampus in Zinc Finger Protein 521 (ZFP521) Mutant Mice

doi: 10.1371/journal.pone.0092848

Figure Lengend Snippet: (A) Immunohistochemical detection of neuroectodermal and neural stem cells. Sections of the dentate gyrus (a–d) and cerebellum (e–h) from five-week-old ZFP521 +/+ mouse (a, c, e, g) and ZFP521 Δ/Δ mouse (b, d, f, h) were stained with anti-Sox1 antibody. Note that the number of Sox1-positive cells in dentate gyrus and cerebellum of ZFP521 Δ/Δ mice appears smaller than that in ZFP521 +/+ mice. Bars = 100 μm (a, b, e, f) and 50 μm (c, d, g, h). (B) Immunoblot detection. Immunoblots of lysates from the cerebellum of five-week-old ZFP521 +/+ , ZFP521 +/Δ , and ZFP521 Δ/Δ mice were probed with anti-Sox1 antibody (upper panel). Anti-β-actin antibody (bottom panel) was used as control for protein loading. The experiment was performed at least three times, and all results were similar. A representative result is shown. Note that Sox1 expression was decreased in the cerebellum of ZFP521 Δ/Δ mice. (C) Densitometric quantification of Sox1 protein in the cerebellum of five-week-old mice. The density of the Sox1 band shown above was quantified using Image-J software, and normalized by β-actin. The vertical axis indicates the quantified density of Sox1 protein relative to that in ZFP521 +/+ mice. Error bar indicates SD. n = 6. *, P<0.001, compared to ZFP521 +/+ mice. Note that Sox1 expression was significantly decreased in the cerebellum of ZFP521 Δ/Δ mice compared with ZFP521 +/+ mice.

Article Snippet: Paraffin-embedded sections were stained with antibodies against a neuronal marker NeuN (Chemicon, Temecula, CA, USA), astroglial marker glial fibrillary acidic protein (GFAP) (Cell Signaling Technology, Beverly, MD, USA), or neuroectodermal marker sex determining region Y-box 1 (Sox1) (Cell Signaling Technology) using the ABC kit according to the manufacturer's protocol (Vector Laboratories, Burlingame, CA, USA) .

Techniques: Immunohistochemical staining, Staining, Western Blot, Control, Expressing, Software