Journal:

Article Title: A fragment of anthrax lethal factor delivers proteins to the cytosol without requiring protective antigen

doi: 10.1073/pnas.1131930100

Figure Lengend Snippet: HeLa cells internalize LFn-GFP in the absence of PA, but do not internalize GFP. HeLa cells were incubated with Texas red-conjugated transferrin and GFP (a and c) or LFn-GFP (b and d) for 1 hr (a and b) or 2 hr (c and d) and imaged by confocal microscopy. (Right) Overlay of red and green staining. (Center) Uptake of transferrin in red. (Left) Internalized LFn-GFP or GFP in green. Similar results were found for COS cells (data not shown).

Article Snippet: HeLa cells (American Type Culture Collection), grown on collagen-treated chamber slides (BD Science) to reach ≈80% confluence, were incubated with 40 μg/ml purified GFP or LFn-GFP at 37°C for 1 or 2 h. Some incubations were performed in the presence of 100 μg/ml Texas red-conjugated transferrin (Molecular Probes) as a marker for the endocytic pathway or with 10 μg/ml PA. For the transferrin experiments, cells were washed four times with cold DMEM and then fixed for 15 min in 4% paraformaldehyde in cold PBS.

Techniques: Incubation, Confocal Microscopy, Staining

Journal:

Article Title: A fragment of anthrax lethal factor delivers proteins to the cytosol without requiring protective antigen

doi: 10.1073/pnas.1131930100

Figure Lengend Snippet: Internalized LFn-GFP does not completely localize with the endocytic or secretory pathways. HeLa cells incubated for 1 hr with LFn-GFP in the absence of PA were stained with markers for early endosomes (EEA-1; a), late endosomes (Lamp-1; b), lysosomes (Lamp-2; c), and the Golgi apparatus (Ab-1; d) and visualized by confocal microscopy. (Right) Overlay of red and green staining. (Center) Red organelle antibody staining. (Left) Green fluorescence of LFn-GFP.

Article Snippet: HeLa cells (American Type Culture Collection), grown on collagen-treated chamber slides (BD Science) to reach ≈80% confluence, were incubated with 40 μg/ml purified GFP or LFn-GFP at 37°C for 1 or 2 h. Some incubations were performed in the presence of 100 μg/ml Texas red-conjugated transferrin (Molecular Probes) as a marker for the endocytic pathway or with 10 μg/ml PA. For the transferrin experiments, cells were washed four times with cold DMEM and then fixed for 15 min in 4% paraformaldehyde in cold PBS.

Techniques: Incubation, Staining, Confocal Microscopy, Fluorescence

Journal:

Article Title: A fragment of anthrax lethal factor delivers proteins to the cytosol without requiring protective antigen

doi: 10.1073/pnas.1131930100

Figure Lengend Snippet: Colocalization of LFn-GFP with the proteosome 20s subunit. HeLa cells were stained with the mixture of an antibody to the α-subunit and the other to the β-subunit of the 20s proteosome 2 hr after incubation with LFn-GFP. Similar results were also found after 1 hr incubation (see Fig. 1) and with a single antibody to the α-subunit or β-subunit of the proteosome, respectively (data not shown). (Top) Green fluorescence of LFn-GFP. (Middle) Red fluorescence of the proteosome antibody. (Bottom) Overlay of both channels.

Article Snippet: HeLa cells (American Type Culture Collection), grown on collagen-treated chamber slides (BD Science) to reach ≈80% confluence, were incubated with 40 μg/ml purified GFP or LFn-GFP at 37°C for 1 or 2 h. Some incubations were performed in the presence of 100 μg/ml Texas red-conjugated transferrin (Molecular Probes) as a marker for the endocytic pathway or with 10 μg/ml PA. For the transferrin experiments, cells were washed four times with cold DMEM and then fixed for 15 min in 4% paraformaldehyde in cold PBS.

Techniques: Staining, Incubation, Fluorescence

Journal:

Article Title: A fragment of anthrax lethal factor delivers proteins to the cytosol without requiring protective antigen

doi: 10.1073/pnas.1131930100

Figure Lengend Snippet: Internalization and colocalization of LFn-GFP with the proteosome 20s subunit is more efficient in the absence of PA. HeLa cells were stained for the proteosome 20s α-subunit 1 hr (a and b)or2hr(c and d) after incubation with LFn-GFP in the absence (a and c) or presence (b and d) of PA. (Right) Overlay. (Center) Red fluorescent staining for the proteosome. (Left) Green fluorescence of LFn-GFP.

Article Snippet: HeLa cells (American Type Culture Collection), grown on collagen-treated chamber slides (BD Science) to reach ≈80% confluence, were incubated with 40 μg/ml purified GFP or LFn-GFP at 37°C for 1 or 2 h. Some incubations were performed in the presence of 100 μg/ml Texas red-conjugated transferrin (Molecular Probes) as a marker for the endocytic pathway or with 10 μg/ml PA. For the transferrin experiments, cells were washed four times with cold DMEM and then fixed for 15 min in 4% paraformaldehyde in cold PBS.

Techniques: Staining, Incubation, Fluorescence

Journal: Cell Discovery

Article Title: Sequence determinants of specific pattern-recognition of bacterial ligands by the NAIP–NLRC4 inflammasome

doi: 10.1038/s41421-018-0018-1

Figure Lengend Snippet: a Schematic domain of NAIPs predicted by the NCBI Conserved Domain Database. The NBD, HD1, WHD, and HD2 domains were annotated according to homology with NLRC4. The full-length NAIP protein is divided into fine fragments and illustrated by dash lines. Chimeras were constructed by exchange each fragments of NAIP2 with corresponding ones in NAIP5. The three critical amino acids that determines rod protein binding to NAIP2 are indicated by arrows on the top of the NAIP2 scheme, whereas below are sites for three different truncations. b Yeast two-hybrid assays of interactions between B. thailandensis rod protein BsaK and NAIP2/5 chimeras in which indicated NAIP2 region is substituted by NAIP5. c BsaK induced activation of the NAIP Chimera/NLRC4 inflammasome in reconstituted 293T cells. Lysates from 293T cells transfected with indicated plasmid combinations and stimulated with LFn-BsaK were analyzed for mature IL-1β (p17) by immunoblotting. d Yeast two-hybrid assays of interactions between BsaK and NAIP2 truncations indicated in a . e Blue Native PAGE assay to monitor the formation of the NAIP/NLRC4 inflammasome. 293T cells were transfected with NLRC4 and the indicated NAIPs or their truncation and 6-Myc tagged PrgJ or flagellin. After 36 h, cells were harvest and lysed in Native lysis buffer and subjected to Blue Native PAGE analysis. A fraction of the cell lysates were also subjected to denatured SDS–PAGE for protein expression and loading. f Yeast two-hybrid interaction assays of interactions between BsaK and different NAIP2 mutants. g Blue Native PAGE assay to test the ability of the NAIP2 mutants in forming inflammasome. h BsaK induced activation of the NAIP2 mutant/NLRC4 inflammasome in reconstituted 293T cells. Expression of transfected inflammasome components for c , e , g and h is in Supplementary Figure

Article Snippet: Recombinant LFn-flagellin or BsaK were cloned into pET28a-LFn vector (Addgene) for recombinant expression in E. coli BL21 (DE3) as described previously , , .

Techniques: Construct, Protein Binding, Activation Assay, Transfection, Plasmid Preparation, Western Blot, Blue Native PAGE, Lysis, SDS Page, Expressing, Mutagenesis

Journal: Cell Discovery

Article Title: Sequence determinants of specific pattern-recognition of bacterial ligands by the NAIP–NLRC4 inflammasome

doi: 10.1038/s41421-018-0018-1

Figure Lengend Snippet: a Schematic of predicted NAIP domains in NAIP2, NAIP5, and NAIP2/5 chimeras. The sites of amino acids substitution or truncation are labeled by arrow. b Yeast two-hybrid assays of interactions between BsaK or L. pneumophila flagellin FlaA and NAIP Chimera A. c Yeast two-hybrid interaction assays of interactions between flagellin FlaA and different NAIP5 mutants. d Yeast two-hybrid interaction assays of interactions between BsaK or flagellin FlaA and different NAIP chimeras or NAIP5 truncations indicated in a . e Co-immunoprecipitation assay of interactions between BsaK or flagellins FlaA and NAIP2, NAIP5, or NAIP2/5 chameras in 293T cells. f Reconstitution of flagellin FlaA activation of the NAIP chimera/NLRC4 inflammasome in 293T cells. Expression of transfected inflammasome components for f is in Supplementary Figure

Article Snippet: Recombinant LFn-flagellin or BsaK were cloned into pET28a-LFn vector (Addgene) for recombinant expression in E. coli BL21 (DE3) as described previously , , .

Techniques: Labeling, Co-Immunoprecipitation Assay, Activation Assay, Expressing, Transfection

Journal: Cell Discovery

Article Title: Sequence determinants of specific pattern-recognition of bacterial ligands by the NAIP–NLRC4 inflammasome

doi: 10.1038/s41421-018-0018-1

Figure Lengend Snippet: a Caspase-1 activation detected 1 h after transfection of 100 nM flagellins in LPS pretreated BMMs. b IL-1β secretion detected 20 h after transfection of 100 nM flagellins in LPS pretreated BMMs. c Co-immunoprecipitation assay of flagellin of E. coli K12 strain (KF), flagellin of Salmonella typhi (SF) and C35 deletion variant of SF with NAIP5 in 293T cells. d Co-immunoprecipitation assay of flagellins KF, SF, and C35 replacement variant of SF with NAIP5 in 293T cells. Shown are immunoblots of anti-Flag immunoprecipitates (IP: Flag) and total cell lysates (Input)

Article Snippet: Recombinant LFn-flagellin or BsaK were cloned into pET28a-LFn vector (Addgene) for recombinant expression in E. coli BL21 (DE3) as described previously , , .

Techniques: Activation Assay, Transfection, Co-Immunoprecipitation Assay, Variant Assay, Western Blot

Journal: Cell Discovery

Article Title: Sequence determinants of specific pattern-recognition of bacterial ligands by the NAIP–NLRC4 inflammasome

doi: 10.1038/s41421-018-0018-1

Figure Lengend Snippet: a The alignment of C-terminal 35 amino acids in flagellin of E. coli K12 strain (KF) and flagellin of Salmonella typhi (SF). b Co-immunoprecipitation assays of flagellin SF or its mutants with NAIP5 in 293T cells transfected by plasmid pCS2-Flag-NAIP5 with Myc-flagellin. c Reconstitution of flagellin activation of the NLRC4 inflammasome in non-macrophage cells. Lysates from 293T cells transfected with indicated plasmid combinations and stimulated with 5 ng/ml LFn-Flagellins were analyzed for mature IL-1β (p17) by immunoblotting. d Alignment of C-terminal sequence in flagellins with or without NAIP5-binding ability according to the yeast two-hybrid experiments . Amino acids critical for the low binding ability of KF with NAIP5 are outlined by wireframes. e Co-immunoprecipitation assays of the wild-type flagellins FlaA (L.p), FliC (type b) (P.a), FliC2 (Y.e), and Flagellin (P.l) or their correspondent mutants of SF483, 488, or 506 with NAIP5. f Alignment of C-terminal amino-acid sequence of flagellins derived from flagellated commensal bacteria in gut from wild-type C57BL/6 mouse . Expression of transfected inflammasome components for c is in Supplementary Figure

Article Snippet: Recombinant LFn-flagellin or BsaK were cloned into pET28a-LFn vector (Addgene) for recombinant expression in E. coli BL21 (DE3) as described previously , , .

Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Activation Assay, Western Blot, Sequencing, Binding Assay, Derivative Assay, Expressing

Journal: Cell Discovery

Article Title: Sequence determinants of specific pattern-recognition of bacterial ligands by the NAIP–NLRC4 inflammasome

doi: 10.1038/s41421-018-0018-1

Figure Lengend Snippet: a Diagram of flagellin of E. coli K12 strain (KF), flagellin of Salmonella typhi (SF) and the chimeric flagellins with hyper-variable region substitutions. b IL-1β secretion 20 h after transfection with 100 nM flagellins in BMMs. c and d Co-immunoprecipitation assays of KF, SF, and their chimeric with NAIP5 in 293T cells. e Co-immunoprecipitation assays of flagellins KF or its mutations with NAIP5 in 293T cells. f Reconstitution of flagellin activation of the NLRC4 inflammasome in non-macrophage cells. Lysates from 293T cells transfected with indicated plasmid combinations and stimulated with LFn-Flagellins were analyzed for mature IL-1β (p17) by immunoblotting. Expression of transfected inflammasome components for f is in Supplementary Figure

Article Snippet: Recombinant LFn-flagellin or BsaK were cloned into pET28a-LFn vector (Addgene) for recombinant expression in E. coli BL21 (DE3) as described previously , , .

Techniques: Transfection, Immunoprecipitation, Activation Assay, Plasmid Preparation, Western Blot, Expressing