pbluescript plasmids (1 mg)  (Promega)

Journal: PLoS ONE

Article Title: Caspase-1 and IL-1β Processing in a Teleost Fish

doi: 10.1371/journal.pone.0050450

Figure Lengend Snippet: The same volume of in vitro synthesized proIL1β and proIL1β[D 100 A] were used and loaded on the gel. Fragments 1, 2 and 3 are labeled. Putative mature in vitro synthesized sea bass IL1β (MS 101 -Q 261 ) has been loaded as control. Numbers on the left indicate the mass of the molecular weight markers in kDa. The double bands corresponding to the highest molecular weight form (fragment 1) suggested cleavage at the C-terminal end of proIL-1β. This interpretation was supported by N-terminal sequencing of fragment 1′ , which revealed that this fragment has the N-terminal sequence of proIL-1β (M 1 ESEMKC). Furthermore, mutation of D 252 results in a proIL-1β protein that no longer yields fragment 1 upon incubation with caspase-1 .

Article Snippet: HSpCMV-XL5proIL1β[D 116 A], HSpCMV-XL5proIL1β[D 128 A] and HSpCMV-XL5proIL1β[D 116 A/D 128 A], for producing mutated forms of human proIL-1β in D 116 , D 128 or D 116 /D 128 : each aspartate was mutated to alanine by site-directed mutagenesis using HSpCMV-XL5proIL1β (IL-1β Human cDNA clone (NM_000576.2) in pCMV-XL5 plasmid from Origene (#SC122566)) as template and the primers listed in .

Techniques: In Vitro, Synthesized, Labeling, Molecular Weight, Sequencing, Mutagenesis, Incubation

Journal: PLoS ONE

Article Title: Caspase-1 and IL-1β Processing in a Teleost Fish

doi: 10.1371/journal.pone.0050450

Figure Lengend Snippet: ( A ) Processing of in vitro translated chicken proIL-1β and mutants by sea bass caspase-1. The same volume of in vitro translated chicken proIL-1β, proIL1β[D 77 A], proIL1β[D 80 A] and proIL1β[D 82 A] were loaded on the gel. In vitro translated putative mature chicken IL-1β forms (MI 119 -R 267 , MI 122 -R 267 and MS 81 -R 267 ) were loaded as controls. ( B ) Processing of in vitro translated human proIL-1β and mutants by sea bass caspase-1. ( C ) Processing of in vitro translated human proIL-1β and mutants by human caspase-1. The same volume of in vitro tranlsated proIL-1β , proIL1β[D 116 A], proIL1β[D 128 A] and proIL1β[D 116 A/D 128 A] was loaded on the gel. In vitro translated mature human IL-1β (MA 117 -S 269 ) and human IL-1β form (MS 129 -S 269 ) starting at S 129 , homologue to sea bass S 101 , were loaded as controls. Mature forms are highlighted by arrow heads. Numbers on the left indicate the mass of the molecular weight markers in kDa.

Article Snippet: HSpCMV-XL5proIL1β[D 116 A], HSpCMV-XL5proIL1β[D 128 A] and HSpCMV-XL5proIL1β[D 116 A/D 128 A], for producing mutated forms of human proIL-1β in D 116 , D 128 or D 116 /D 128 : each aspartate was mutated to alanine by site-directed mutagenesis using HSpCMV-XL5proIL1β (IL-1β Human cDNA clone (NM_000576.2) in pCMV-XL5 plasmid from Origene (#SC122566)) as template and the primers listed in .

Techniques: In Vitro, Molecular Weight

Journal: Science immunology

Article Title: Effector Memory CD4 + T cells induce damaging innate inflammation and auto-immune pathology by engaging CD40 and TNFR on myeloid cells

doi: 10.1126/sciimmunol.abk0182

Figure Lengend Snippet: (A) IL-1β, (B) IL-6, and IL-12p40 were measured by ELISA in the supernatants of WT BMDCs cultured with WT TH0 TEM in the presence or absence of anti-CD3 (200ng/mL) for 18hr. (C) IL-6 and IL-12p40 were measured by ELISA in the supernatants of WT BMDMs or CD11c+ splenic DCs cultured with WT TH0 TEM in the presence of anti-CD3 for 18hr. (D) IL-6 and IL-12p40 were measured by ELISA in the supernatants of WT or TLR2/4/5xUnc93b13d/3d BMDCs cultured with WT TH0 TEM in the presence anti-CD3 for 18hr. (E) Quantification of intracellular proIL-1b or IL12p40 production measured by flow cytometry of CD11c+CD11b+ or CD90.2+ cells following culture of WT BMDCs and WT TH0 TEM in the presence of anti-CD3 for 6hr. Representative flow plots shown in Fig. S1A. (F) IL-6 was measured by ELISA in the supernatants of WT or Il6−/− BMDCs cultured with WT Th0 TEM in the presence of anti-CD3 for 6hr. (G) IL-6 was measured by ELISA in the supernatants of WT BMDCs cultured with TH0 or polarized Th1, TH2, or TH17 TEM in the presence of anti-CD3 for 6hr. (H) IL-6 and IL-12p40 were measured by ELISA in the supernatants of WT BMDCs cultured with OT-II TH0 in the presence or absence of 10uM OVA peptide (323-339) (OVAp) and (I) anti-IA/IE antibody (20ug/mL) for 18hr. (J) IL-6 and IL-12p40 levels in the serum of WT mice were quantified by ELISA 6hr following injection with PBS or anti-CD3 (50ug, i.v.). Error bars indicate SEM. (A-D, F-H, J) n=3, (E, I) n= 4, (A-D,F-J) unpaired two-tailed t-test. (E) unpaired one-tailed t-test *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns = not significant.

Article Snippet: Cells were immediately harvested on ice, stained with fixable Zombie Yellow (Biolegend), then fixed with Foxp3 Transcription Factor Staining Set (Invitrogen) and stained with anti-mouse proIL-1b (eBioscience, NJTEN3) or anti-mouse IL-12/23 p40 (Biolegend, C15.6) according to manufacturer protocol.

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Flow Cytometry, Injection, Two Tailed Test, One-tailed Test