Journal: Nature Communications

Article Title: Discovery of a proteolytic flagellin family in diverse bacterial phyla that assembles enzymatically active flagella

doi: 10.1038/s41467-017-00599-0

Figure Lengend Snippet: Peptidase activity assays of purified recombinant FliA(H)-hypervariable region and flagellar filaments from C. haemolyticum . a 12% SDS-PAGE of purified FliA(H)-hypervariable region (band “i”), electrophoresing close to that predicted for recombinant thioredoxin-tagged flagellin hypervariable region (50.14 kDa). b Amino acid occurrence heat map based on FliA(H)-hypervariable region protease cleaved peptide alignments of 391 cleavage sites identified in a tryptic E. coli K12 library. c IceLogos of FliA(H)-hypervariable region protease cleaved peptide specificity profiles showing percent differences compared to natural amino acid abundance, with significantly over-represented amino acids shown above the x -axis and under-represented residues below the x -axis. Amino acids that have not been identified are depicted in pink . d Fluorometric peptidase assays for FliA(H)-hypervariable region protease against three different peptidic substrates (ALG↓L, PLG↓L, PLG↓V) and + /– EDTA as indicated. e 10% SDS-PAGE of purified flagellar sheared filaments from C. haemolyticum . Band “ii” and “iii” were identified by mass spectrometry as FliA(H) (proteolytic flagellin) and the non-protease-containing structural flagellin (WP_039229459), respectively (Supplementary Fig. 7 ). f Amino acid occurrence heat map and g IceLogo based on cleaved peptide alignments of 269 cleavage sites identified in a tryptic E. coli K12 library using purified flagellar filaments. h Fluorometric peptidase assays for flagellar filaments using the ALG↓L peptide in three assay conditions including EDTA-free HALT protease inhibitors

Article Snippet: Purified recombinant FliA(H)-hypervariable region protease was incubated at a final concentration of 0.5 µM with 10 µM QF-peptide substrate in 100 µl of 150 mM NaCl, 10 mM CaCl2 , 50 mM HEPES, pH 7.5, in the presence of HALT protease inhibitor cocktail (Life Technologies) plus/minus 20 mM EDTA at 37 °C.

Techniques: Activity Assay, Purification, Recombinant, SDS Page, Mass Spectrometry

Journal: Scientific Reports

Article Title: Intestinal helminth infection enhances bacteria-induced recruitment of neutrophils to the airspace

doi: 10.1038/s41598-019-51991-3

Figure Lengend Snippet: Coinfection with H. polygyrus increases neutrophil transepithelial migration into the P. aeruginosa infected airspace of the lung. FACS analysis using Ly6G and CD11b markers to isolate the neutrophil population in the ( a ) BAL, ( b ) lung tissue and ( c ) peripheral blood (PB), data are shown as mean ± SD, and are representative of three independent experiments. * P

Article Snippet: For protein lysate samples, one of the 0.5 mL BAL aliquots was treated with 10 μL protease inhibitor cocktail set III, EDTA-free (Calbiochem, MA) and 20 μl of 10% Triton X-100 (Sigma, MA) and mixed by inversion for 20 minutes at 4 °C.

Techniques: Migration, Infection, FACS

Journal: Scientific Reports

Article Title: Intestinal helminth infection enhances bacteria-induced recruitment of neutrophils to the airspace

doi: 10.1038/s41598-019-51991-3

Figure Lengend Snippet: Helminth-induced increase in neutrophil migration during P. aeruginosa infection is correlated with increased MPO and ELA2 levels within the airspace. Protein extracts were prepared from BAL samples and analyzed for ( a ) neutrophil myeloperoxidase (MPO) and ( b ) elastase (ELA2). ( c ) LDH levels were assessed in the cell-free supernatant of the BAL to assess cytotoxicity. Data are shown as mean ± SD and are representative of three independent experiments. * P

Article Snippet: For protein lysate samples, one of the 0.5 mL BAL aliquots was treated with 10 μL protease inhibitor cocktail set III, EDTA-free (Calbiochem, MA) and 20 μl of 10% Triton X-100 (Sigma, MA) and mixed by inversion for 20 minutes at 4 °C.

Techniques: Migration, Infection

Journal: Scientific Reports

Article Title: Intestinal helminth infection enhances bacteria-induced recruitment of neutrophils to the airspace

doi: 10.1038/s41598-019-51991-3

Figure Lengend Snippet: Coinfection with H. polygyrus does not impact macrophage levels within the airspace, lung tissue or circulation during P. aeruginosa infection of the airspace. FACS analysis using F4/80 and CD11b markers to isolate the macrophage populations in the ( a ) BAL, ( b ) lung tissue and ( c ) monocytes in peripheral blood. Data are shown as mean ± SD and are representative of three independent experiments. * P

Article Snippet: For protein lysate samples, one of the 0.5 mL BAL aliquots was treated with 10 μL protease inhibitor cocktail set III, EDTA-free (Calbiochem, MA) and 20 μl of 10% Triton X-100 (Sigma, MA) and mixed by inversion for 20 minutes at 4 °C.

Techniques: Infection, FACS

Journal: Scientific Reports

Article Title: Intestinal helminth infection enhances bacteria-induced recruitment of neutrophils to the airspace

doi: 10.1038/s41598-019-51991-3

Figure Lengend Snippet: Coinfection with H. polygyrus increases cellular transepithelial migration into the lung airspace. After 2 weeks of helminth infection, the mice were infected with/without P. aeruginosa through intranasal inoculation. Mice were placed in the cage for 6 hours, and after incubation mice were sacrificed, BAL and lung tissue were obtained. ( a ) The result of BAL cell count. Data are shown as mean ± SD and are representative of three independent experiments. ** P

Article Snippet: For protein lysate samples, one of the 0.5 mL BAL aliquots was treated with 10 μL protease inhibitor cocktail set III, EDTA-free (Calbiochem, MA) and 20 μl of 10% Triton X-100 (Sigma, MA) and mixed by inversion for 20 minutes at 4 °C.

Techniques: Migration, Infection, Mouse Assay, Incubation, Cell Counting

Journal: Scientific Reports

Article Title: Intestinal helminth infection enhances bacteria-induced recruitment of neutrophils to the airspace

doi: 10.1038/s41598-019-51991-3

Figure Lengend Snippet: Coinfection with H. polygyrus during P. aeruginosa acute pneumonia increases the CD4 + T-cells population in the airspace and lung tissues. FACS analysis using CD4 marker to isolate the CD4 + T-cell population in the ( a ) BAL, ( b ) lung tissue and ( c ) peripheral blood. Data are shown as mean ± SD and are representative of three independent experiments. * P

Article Snippet: For protein lysate samples, one of the 0.5 mL BAL aliquots was treated with 10 μL protease inhibitor cocktail set III, EDTA-free (Calbiochem, MA) and 20 μl of 10% Triton X-100 (Sigma, MA) and mixed by inversion for 20 minutes at 4 °C.

Techniques: FACS, Marker

Journal: Aging (Albany NY)

Article Title: SIRT4 interacts with OPA1 and regulates mitochondrial quality control and mitophagy

doi: 10.18632/aging.101307

Figure Lengend Snippet: Aging-associated SIRT4 upregulation leads to a shifted L-OPA1 to S-OPA1 ratio in two different fibroblast senescence models Primary human dermal fibroblasts were either transfected with miR-15b inhibitors (or control oligonucleotides) or subjected to γ-irradiation (γIR; 20 Gy) (both in the presence or absence of siRNA duplexes against SIRT4) [ 35 ] followed by analysis of OPA1-L and OPA1-S expression by immunoblotting after four days ( A ) As a control for complete proteolytic processing of L-OPA1 to S-OPA1, fibroblasts were treated with CCCP (10 μM) for two hours. The ratio between the expression levels of L-OPA1 and S-OPA1 was determined by ImageJ-based densitometric analysis in miR-15b inhibitor transfected fibroblasts ( B ) and cells subjected toγIR ( C ). To evaluate statistical significance (compared to control), two-way ANOVA followed by Tukey's test was performed (*p

Article Snippet: Preparation of total cell lysates for immunoblot analysis Cleared cell lysates were generated using lysis buffer containing 0.3% CHAPS (3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate), 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM Na3 VO4 , 10 mM NaF, 1 mM EDTA, 1 mM EGTA, 2.5 mM Na4 O7 P2 , 1 μM DTT, 1x cOmplete™ protease inhibitor cocktail (CO-RO, Sigma-Aldrich).

Techniques: Transfection, Irradiation, Expressing

Journal: Aging (Albany NY)

Article Title: SIRT4 interacts with OPA1 and regulates mitochondrial quality control and mitophagy

doi: 10.18632/aging.101307

Figure Lengend Snippet: SIRT4-eGFP interacts with OPA1 ( A ) HEK293 cells stably expressing SIRT4-eGFP, SIRT4(H161Y)-eGFP, or SIRT4(D28N)-eGFP were either untreated or treated with CCCP (10 μM, 2h) and thereafter subjected to OPA1 co-immunoprecipitation (IP) analysis using sepharose beads coupled anti-GFP single-domain-antibodies (nanobodies). Total cell lysates were loaded as input control (5%). CCCP treatment caused a complete proteolytic processing of L-OPA1 to S-OPA1. TACC3 was detected using specific antibodies and served as a representative negative co-immunoprecipitation control. ( B ) SIRT4 enzymatic activity is required for efficient interaction of SIRT4 with L-OPA1. The amount of L-OPA1 co-immunoprecipitated with SIRT4-eGFP, SIRT4(H161Y)-eGFP, or SIRT4(D28N)-eGFP was determined in relation to the protein input and subjected to ImageJ-based densitometric analysis. To evaluate statistical significance, two-way ANOVA followed by Tukey's tests was performed (*p

Article Snippet: Preparation of total cell lysates for immunoblot analysis Cleared cell lysates were generated using lysis buffer containing 0.3% CHAPS (3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate), 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM Na3 VO4 , 10 mM NaF, 1 mM EDTA, 1 mM EGTA, 2.5 mM Na4 O7 P2 , 1 μM DTT, 1x cOmplete™ protease inhibitor cocktail (CO-RO, Sigma-Aldrich).

Techniques: Stable Transfection, Expressing, Immunoprecipitation, Activity Assay

Journal: Aging (Albany NY)

Article Title: SIRT4 interacts with OPA1 and regulates mitochondrial quality control and mitophagy

doi: 10.18632/aging.101307

Figure Lengend Snippet: SIRT4-eGFP expression stabilizes the mitochondrial fusion regulator L-OPA1 ( A ) The expression of L-OPA1 vs . S-OPA1 was analyzed by immunoblotting in HEK293 cells stably expressing eGFP, SIRT4-eGFP, SIRT4(H161Y)-eGFP, or SIRT4(D28N)-eGFP. As a control for complete proteolytic processing of L-OPA1 to S-OPA1 eGFP-expressing control cells were treated with CCCP (10 μM) for two hours. ( B ) The ratio between the expression levels of L-OPA1 and S-OPA1 was determined by ImageJ-based densitometric analysis. To evaluate statistical significance (compared to eGFP), two-way ANOVA followed by Tukey's tests was performed (**p

Article Snippet: Preparation of total cell lysates for immunoblot analysis Cleared cell lysates were generated using lysis buffer containing 0.3% CHAPS (3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate), 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM Na3 VO4 , 10 mM NaF, 1 mM EDTA, 1 mM EGTA, 2.5 mM Na4 O7 P2 , 1 μM DTT, 1x cOmplete™ protease inhibitor cocktail (CO-RO, Sigma-Aldrich).

Techniques: Expressing, Stable Transfection