Journal: The Journal of Biological Chemistry

Article Title: Biochemical characterization of the first step in sulfonolipid biosynthesis in Alistipes finegoldii

doi: 10.1016/j.jbc.2022.102195

Figure Lengend Snippet: Identification and purification of cysteate acyl-ACP transferase. A , TLC separation of 3-keto[ 14 C]capnine (3[ 14 C]KC) arising from SulA activity using cellular lysates from Escherichia coli cells expressing nothing (empty vector control), alfi_1224 (SulA), or alfi_1465 . The thin-layer plate is representative of four independent experiments. B , purified SulA migrates as a single 85 kDa species ( V e = 76.6 ml) based on a calibrated HiLoad 16/600 Superdex 200 column (98.1 kDa theoretical homodimer mass). Left inset , molecular weight calculation using calibration standards described under . Right inset , SDS gel electrophoresis shows SulA purity (∼48 kDa) along with molecular weight standards. C , the sedimentation velocity profile (fringe displacement) was fit to a continuous sedimentation coefficient distribution model c(S). SulA sedimented as a 98.9 kDa homodimer with a sedimentation coefficient of 5.11 S. ACP, acyl carrier protein; SulA, cysteate acyl-ACP transferase.

Article Snippet: E. coli strain BL21(DE3) cells harboring the pET-SulA or pET-SulA(K281A) expression plasmids were grown in 2 l LB medium with 50 μg/ml kanamycin (Gold Biotechnology) at 37 °C and 200 rpm shaking to A 600 = 0.6.

Techniques: Purification, Activity Assay, Expressing, Plasmid Preparation, Molecular Weight, SDS-Gel, Electrophoresis, Sedimentation

Journal: Nature chemical biology

Article Title: Monitoring methionine sulfoxide with stereospecific mechanism-based fluorescent sensors

doi: 10.1038/nchembio.1787

Figure Lengend Snippet: E. coli cells expressing MetSOx ( a ) or MetROx ( b ) were incubated with MetO for ~30 min, and then rinsed to remove the oxidant. Arrows indicate the addition (↓) of MetO and rinsing (↑) the cells for two representative experiments (Assay 1 and Assay 2). MetSOx ( c ) and MetROx ( d ) oxidized fractions calculated from (n = 3), using and , respectively, described in the “ online methods ” section.

Article Snippet: SoluBL21 TM E. coli (Gelantis, San Diego, CA) cells were transformed with an indicated expression vector and grown in Luria-Bertani containing ampicillin (50 μg.ml −1 ) at 37°C.

Techniques: Expressing, Incubation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The actin regulatory protein HS1 is required for antigen uptake and presentation by dendritic cells 5

doi: 10.4049/jimmunol.1100870

Figure Lengend Snippet: (A–B) BMDCs cultured from WT and HS1−/− on the Balb/c background were pulsed with the class II-restricted OVA323–339 peptide (A) or with the whole ovalbumin protein (B) at the indicated doses for 4h. They were then co-cultured with OVA-specific DO-11.10 T cells and 24h later, IL-2 levels in the culture supernatants were measured by ELISA. (C) BMDCs cultured from WT and HS1−/− mice on the C57Bl/6 background were incubated in the presence or absence of mannan to block mannose receptors, and then ovalbumin protein was added, so that any residual uptake would be by macropinocytosis. Antigen presentation to OTII T cells was then assayed as in panels A and B. In this experiment (representing 2 of 4 replicates), mannan completely blocked the response, indicating that even very high doses of ovalbumin are taken up almost exclusively by receptor-mediated endocytosis. , WT DCs without mannan; ■, HS1−/− DCs without mannan; Δ, WT DCs with mannan; ◆, HS1−/− DCs with mannan. Inset, data from a separate experiment (representing 2 of 4 replicates) where T cell responses to mannan-blocked DCs were measurable at high ovalbumin doses. Open bars, WT DCs with mannan, filled bars, HS1−/− DCs with mannan; differences between WT and HS1−/− DCs in the presence of mannan were not statistically significant at any dose of ovalbumin. (D) WT and HS1−/− BMDCs (C57Bl/6 background) were allowed to phagocytose latex beads coated with ovalbumin and serum albumin in the indicated ratios, holding total protein and bead number constant. OTII T cell responses were then measured. (E–F) WT and HS1−/− BMDCs were pulsed with free VSV8 peptide (E) or VSV8 peptide pre-bound to GRP94 (F) at the indicated doses for 4h. VSV8-specific N15 T hybridoma cells were incubated with antigen-pulsed BMDCs for 24h and IL-2 levels in the supernatants were measured by ELISA. Data represent means +/− StDev from replicate wells of one representative experiment.

Article Snippet: Biochemical analysis of receptor-mediated endocytosis 75–100 μg of GRP94 N1–355, mouse transferrin or ovalbumin (US Biological) were each labeled with 1.0 mCi of 125 I-Iodine (Amersham) in Tris buffered saline, pH 7.2, using IODOBEADS (Amersham) according to the manufacturer’s recommendations.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Incubation, Blocking Assay, Immunopeptidomics

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The actin regulatory protein HS1 is required for antigen uptake and presentation by dendritic cells 5

doi: 10.4049/jimmunol.1100870

Figure Lengend Snippet: WT and HS1−/− BMDCs were incubated with 125I-Transferrin (A), 125I-Ovalbumin (B) or 125I-GRP94 (C) at 4°C for 1h. Cells were then washed and warmed to 37°C for the indicated times. At each time point, residual surface-bound proteins were removed by acid wash (transferrin) or pronase treatment (GRP94 and ovalbumin). Radioactivity in the cell pellets was then determined and uptake calculated. Data represent means +/− StDev from independent experiments, each done in duplicate or triplicate. The number of binding sites for each protein did not differ between WT and HS1−/− BMDCs within experimental error. We calculate 6000–8500 binding sites for transferrin per cell, 20,000–40,000 sites for GRP94, and approximately 80,000 sites for ovalbumin.

Article Snippet: Biochemical analysis of receptor-mediated endocytosis 75–100 μg of GRP94 N1–355, mouse transferrin or ovalbumin (US Biological) were each labeled with 1.0 mCi of 125 I-Iodine (Amersham) in Tris buffered saline, pH 7.2, using IODOBEADS (Amersham) according to the manufacturer’s recommendations.

Techniques: Incubation, Radioactivity, Binding Assay