peptidoglycans pgn  (InvivoGen)

Journal: Science Advances

Article Title: An evolutionarily conserved serine protease network mediates melanization and Toll activation in Drosophila

doi: 10.1126/sciadv.adk2756

Figure Lengend Snippet: ( A ) ProcSP48 activation. Purified procSP48 (1 μg) was incubated with E. coli peptidoglycan (1 μg), PGRP-SA (500 ng), GNBP1 (500 ng), proModSP* (500 ng), and buffer A (to 25 μl) for 2 hours at 37°C. The reaction mixture and controls were separated by 10% SDS-PAGE under reducing conditions and detected by immunoblotting using anti-FLAG antibody. Sizes and positions of the M r markers are indicated. The cSP48 precursor and catalytic domain are marked with a dot and an arrowhead, respectively. ( B ) ProGrass activation. Purified proGrass (1 μg) was incubated with E. coli peptidoglycan (1 μg), PGRP-SA (500 ng), GNBP1 (500 ng), proModSP* (500 ng), procSP48 (100 ng), and buffer A (to 25 μl) for 2 hours at 37°C. The reaction mixture and controls were subjected to 12% SDS-PAGE under reducing conditions and detected by immunoblotting using anti-FLAG antibody. The small amount of procSP48 added in the reaction was hardly detected by the antibody, although it has a FLAG tag at the C terminus. Sizes and positions of the M r markers are indicated. The Grass precursor and light chain produced after activation cleavage are marked with a dot and an arrowhead, respectively.

Article Snippet: Soluble DAP-type peptidoglycan from the Gram-negative bacterium E. coli K12 (Cat. tlrl-ksspgn) and insoluble Lys-type peptidoglycan from the Gram-positive bacterium S. aureus (Cat. tlrl-pgns2) were purchased from InvivoGen.

Techniques: Activation Assay, Purification, Incubation, SDS Page, Western Blot, FLAG-tag, Produced

Journal: Science Advances

Article Title: An evolutionarily conserved serine protease network mediates melanization and Toll activation in Drosophila

doi: 10.1126/sciadv.adk2756

Figure Lengend Snippet: ( A to C ) Purified proPsh (400 ng) (A) or proHayan-PA (400 ng) (B) or proHayan-PB (400 ng) (C) was incubated with active cSP48 (100 ng), proGrass (500 ng), and buffer A (to 25 μl) for 1 hour at 37°C. Active cSP48 was produced in a mixture of E. coli peptidoglycan (1 μg), PGRP-SA (500 ng), GNBP1 (500 ng), proModSP* (500 ng), procSP48 (100 ng), and buffer A (to 20 μl) for 1 hour at 37°C. ( D and E ) Purified proPsh (500 ng) or proHayan-PA (500 ng) or proHayan-PB (500 ng) was incubated with 1 μl of serially diluted subtilisin purified from B. subtilis (D) or various amounts of purified M. anisopliae Pr1A (E), and buffer A (to 25 μl) for 30 min at 29°C. The reaction mixture and controls were resolved by 10% SDS-PAGE under reducing conditions and detected by immunoblotting using anti-Myc antibody. Sizes and positions of the M r markers are indicated. The precursors and catalytic domains of Psh, Hayan-PA, and Hayan-PB are marked with dots and arrowheads, respectively.

Article Snippet: Soluble DAP-type peptidoglycan from the Gram-negative bacterium E. coli K12 (Cat. tlrl-ksspgn) and insoluble Lys-type peptidoglycan from the Gram-positive bacterium S. aureus (Cat. tlrl-pgns2) were purchased from InvivoGen.

Techniques: Purification, Incubation, Produced, SDS Page, Western Blot

Journal: Science Advances

Article Title: An evolutionarily conserved serine protease network mediates melanization and Toll activation in Drosophila

doi: 10.1126/sciadv.adk2756

Figure Lengend Snippet: Purified proSp7 (600 ng) was incubated with Grass (100 ng), proPsh (500 ng) or proHayan-PA (500 ng) ( A ) or proHayan-PB (500 ng) ( B ), and buffer A (to 25 μl) for 1 hour at 37°C. Active Grass was produced in a mixture of E. coli peptidoglycan (1 μg), PGRP-SA (500 ng), GNBP1 (500 ng), proModSP* (300 ng), procSP48 (100 ng), proGrass (100 ng), and buffer A (to 20 μl) for 1 hour at 37°C. In two other sets of reactions, purified proSp7 (600 ng) was incubated with 1 μl of 1:96,000 diluted subtilisin ( C ) or 20 ng of purified Pr1A ( D ), proPsh (500 ng) or proHayan-PA (500 ng) or proHayan-PB (500 ng), and buffer A (to 25 μl) for 30 min at 37°C. The reaction mixture and controls were subjected to 10% SDS-PAGE under reducing conditions and detected by immunoblotting using anti-V5 antibody. Sizes and positions of the M r markers are indicated. The precursor and catalytic domain of Sp7 are marked with dots and arrowheads, respectively. Products from proSp7 cleaved by subtilisin or Pr1A are marked with asterisks. ( E ) Amidase activity of Sp7 activated by Psh, Hayan-PA, and Hayan-PB. In the duplicate experiment (C), amidase activities in the reaction and control mixtures were determined in 150 μl of 50 μM IEAR p NA and plotted as mean ± SD ( n = 3). Statistical significance was calculated with one-way ANOVA with Tukey’s multiple comparisons test, *** P < 0.001.

Article Snippet: Soluble DAP-type peptidoglycan from the Gram-negative bacterium E. coli K12 (Cat. tlrl-ksspgn) and insoluble Lys-type peptidoglycan from the Gram-positive bacterium S. aureus (Cat. tlrl-pgns2) were purchased from InvivoGen.

Techniques: Purification, Incubation, Produced, SDS Page, Western Blot, Activity Assay, Control

Journal: Science Advances

Article Title: An evolutionarily conserved serine protease network mediates melanization and Toll activation in Drosophila

doi: 10.1126/sciadv.adk2756

Figure Lengend Snippet: ( A ) Purified proSpz (400 ng) was incubated with cSP48 (100 ng), proGrass (50 or 400 ng), proPsh (500 ng) or proHayan-PA (500 ng), proSPE (500 ng) or proSp7 (500 ng), and buffer A (to 25 μl) for 1 hour at 37°C. Active cSP48 was produced in a mixture of E. coli peptidoglycan (1 μg), PGRP-SA (500 ng), GNBP1 (500 ng), proModSP* (300 ng), procSP48 (100 ng), and buffer A (to 20 μl) for 1 hour at 37°C. ( B ) Purified proSpz (400 ng) was incubated with Pr1A (20 ng), proPsh (500 ng) or proHayan-PA (500 ng) or proHayan-PB (500 ng), proMP1 (500 ng) or proSer7 (500 ng), and buffer A (to 25 μl) for 1 hour at 37°C. The reaction mixture and controls were subjected to 12% SDS-PAGE under reducing conditions and immunoblot analyses using anti-Spz C106 antibody. Sizes and positions of the M r markers are indicated. The Spz precursor, partially processed Spz, and the cystine-knot domain are marked with dots, vertical lines, and arrowheads, respectively.

Article Snippet: Soluble DAP-type peptidoglycan from the Gram-negative bacterium E. coli K12 (Cat. tlrl-ksspgn) and insoluble Lys-type peptidoglycan from the Gram-positive bacterium S. aureus (Cat. tlrl-pgns2) were purchased from InvivoGen.

Techniques: Purification, Incubation, Produced, SDS Page, Western Blot