iodoacetamide Millipore Search Results


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  • 99
    Millipore iodoacetamide
    Formation of the μ1/μ1C ds bonds during the course of infection. T1L reovirus-infected cells were harvested at either 24, 48, 72, or 96 h postinfection. 50 mM <t>IAM</t> was added immediately upon resuspension of the infected cells in homogenization buffer. After purification, virions from each time point were mixed with nonreducing sample buffer (NR) and disrupted by boiling. The viral proteins were then resolved on a mini-sized SDS-8% PAGE gel. The amount of ds-bonded μ1/μ1C (% ds μ1/μ1C) relative to the total μ1, μ1C, and ds-bonded μ1/μ1C in each preparation was determined by densitometry of the Coomassie-stained gel. Immediately before harvesting, aliquots were taken from each time point and the proportion of dead/dying cells (% dead cells) was determined by trypan blue staining.
    Iodoacetamide, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 12778 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore ammonium bicarbonate
    Formation of the μ1/μ1C ds bonds during the course of infection. T1L reovirus-infected cells were harvested at either 24, 48, 72, or 96 h postinfection. 50 mM <t>IAM</t> was added immediately upon resuspension of the infected cells in homogenization buffer. After purification, virions from each time point were mixed with nonreducing sample buffer (NR) and disrupted by boiling. The viral proteins were then resolved on a mini-sized SDS-8% PAGE gel. The amount of ds-bonded μ1/μ1C (% ds μ1/μ1C) relative to the total μ1, μ1C, and ds-bonded μ1/μ1C in each preparation was determined by densitometry of the Coomassie-stained gel. Immediately before harvesting, aliquots were taken from each time point and the proportion of dead/dying cells (% dead cells) was determined by trypan blue staining.
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    99
    Millipore dtt
    Redox state of Trx1 in A549 cells under oxidative stress. A , principle of redox Western blot analysis. To prepare mobility standards, cell lysates were denatured with urea and fully reduced with <t>DTT.</t> Varying molar ratios of <t>IAA</t> to IAM were incubated with
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    99
    Millipore dithiothreitol
    Flowchart of the identification of the quantitative phosphoproteome in neuronal autophagy. According to the weights of the labeled essential amino acids, N2a cells were divided into 3 groups, including “heavy” ( 13 C 6 15 N 2 -Lysine, 13 C 6 15 N 4 -Arginine), “middle” ( 13 C 4 14 N 2 -Lysine, 13 C 6 14 N 4 -Arginine) and “light” ( 12 C 6 14 N 2 -Lysine, 12 C 6 14 N 4 -Arginine). After checking the labeling efficiency ( > 96%), the “light” and “middle” groups were treated with 15 μM Cory or Cory B for 3 h (Fig. S1), and the “heavy” group, which acted as a control group, was treated with 0.1% (v:v) DMSO. The cell lysates were mixed at a ratio of 1:1:1 and reduced with 10 mM <t>dithiothreitol</t> for 1 h at 56°C and then alkylated with 20 mM iodoacetamide for 45 min at room temperature and protected from light. Then, proteins were digested in solution with trypsin gold at a ratio of 1:50 (trypsin to protein) overnight and 1:100 (trypsin to protein) for another 4 h. After fractionation by high-pH reverse-phase HPLC, peptides were divided into 12 fractions. Finally, enriched phosphopeptides were subjected to LC-MS/MS for the identification of the quantitative phosphoproteome.
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    99
    Millipore dithiothreitol dtt
    Flowchart of the identification of the quantitative phosphoproteome in neuronal autophagy. According to the weights of the labeled essential amino acids, N2a cells were divided into 3 groups, including “heavy” ( 13 C 6 15 N 2 -Lysine, 13 C 6 15 N 4 -Arginine), “middle” ( 13 C 4 14 N 2 -Lysine, 13 C 6 14 N 4 -Arginine) and “light” ( 12 C 6 14 N 2 -Lysine, 12 C 6 14 N 4 -Arginine). After checking the labeling efficiency ( > 96%), the “light” and “middle” groups were treated with 15 μM Cory or Cory B for 3 h (Fig. S1), and the “heavy” group, which acted as a control group, was treated with 0.1% (v:v) DMSO. The cell lysates were mixed at a ratio of 1:1:1 and reduced with 10 mM <t>dithiothreitol</t> for 1 h at 56°C and then alkylated with 20 mM iodoacetamide for 45 min at room temperature and protected from light. Then, proteins were digested in solution with trypsin gold at a ratio of 1:50 (trypsin to protein) overnight and 1:100 (trypsin to protein) for another 4 h. After fractionation by high-pH reverse-phase HPLC, peptides were divided into 12 fractions. Finally, enriched phosphopeptides were subjected to LC-MS/MS for the identification of the quantitative phosphoproteome.
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    99
    Millipore bugbuster protein extraction reagent
    Expression and purification of r-Cam-dis analysis by 4–12% SDS-PAGE gel under non-reducing condition. Samples were run on 4–12% (w/v) Bis-Tris Gel using an Xcell SureLock Mini-Cell at 200V for 30min. The gel was stained with Rapid-Stain. Lane 1: SeeBlue Plus2 Markers; lane 2: soluble fraction of lysates of expressed E. coli BL21 cells by <t>BugBuster</t> reagent (150μg); lane 3: cleaved r-Cam-dis after wash with binding buffer (3μg); lane 4: purified r-Cam-dis after wash with high salt buffer (3 μg). An asterisk ( * ) represents the N-terminal amino acid sequence of purified r-Cam-dis containing the five amino acids from the vector (italicized) before the disintegrin sequence, which are shown in bold letters.
    Bugbuster Protein Extraction Reagent, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4492 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore tris
    CAT are able to cleave HIV-1 <t>Env-A244</t> and CAT degradation products are susceptible to proteasomal degradation. HIV-1 Env-A244 (panels A and B, lanes 0) was treated with CAT for 90 min or 16 hrs and then analyzed on a 4–20% gradient <t>Tris-glycine</t> polyacrylamide gel. The 90 min (lanes 90) and 16 hrs (lanes 0/N) degradation pattern with the various CAT are shown in panels A and B. Env-A244 was treated with the individual CAT for 90 min followed by proteasomes in the presence (panels C and D, lanes PI) or absence of epoxomicin (lanes P). The molecular weight markers are shown in lane M in all panels.
    Tris, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 38479 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore trypsin
    CAT are able to cleave HIV-1 <t>Env-A244</t> and CAT degradation products are susceptible to proteasomal degradation. HIV-1 Env-A244 (panels A and B, lanes 0) was treated with CAT for 90 min or 16 hrs and then analyzed on a 4–20% gradient <t>Tris-glycine</t> polyacrylamide gel. The 90 min (lanes 90) and 16 hrs (lanes 0/N) degradation pattern with the various CAT are shown in panels A and B. Env-A244 was treated with the individual CAT for 90 min followed by proteasomes in the presence (panels C and D, lanes PI) or absence of epoxomicin (lanes P). The molecular weight markers are shown in lane M in all panels.
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    99
    Millipore edta
    rFIBCD1-FReD binds A. fumigatus AIF calcium- and acetate-independently. (A) The presence and binding activity of β-1,3-glucan, galactomannan, and chitin in AIF was validated by pull down of know ligands Dectin-1 fc, rMBL, and WGA. The samples were subjected to <t>SDS-PAGE</t> and analyzed by silver staining. Color is adjusted to gray scale. (B) Western blot showing FIBCD1-FReD pull down by AIF-associated polysaccharides in the presence of calcium (5 mM CaCl 2 ), <t>EDTA</t> (10 mM), and acetate (100 mM). 2 mg of AIF, β-1,3-glucan, and chitin beads were incubated with 5 μg/mL FIBCD1-FReD and pull down was performed as described. Data represent three independent experiments. (C) Western blot showing wild type and A432V mutant FIBCD1-FReD pull down by chitin beads and AIF. Color is adjusted to gray scale. Lane 1, 3: chitin beads, Lane 2: AIF.
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    96
    Promega trypsin
    rFIBCD1-FReD binds A. fumigatus AIF calcium- and acetate-independently. (A) The presence and binding activity of β-1,3-glucan, galactomannan, and chitin in AIF was validated by pull down of know ligands Dectin-1 fc, rMBL, and WGA. The samples were subjected to <t>SDS-PAGE</t> and analyzed by silver staining. Color is adjusted to gray scale. (B) Western blot showing FIBCD1-FReD pull down by AIF-associated polysaccharides in the presence of calcium (5 mM CaCl 2 ), <t>EDTA</t> (10 mM), and acetate (100 mM). 2 mg of AIF, β-1,3-glucan, and chitin beads were incubated with 5 μg/mL FIBCD1-FReD and pull down was performed as described. Data represent three independent experiments. (C) Western blot showing wild type and A432V mutant FIBCD1-FReD pull down by chitin beads and AIF. Color is adjusted to gray scale. Lane 1, 3: chitin beads, Lane 2: AIF.
    Trypsin, supplied by Promega, used in various techniques. Bioz Stars score: 96/100, based on 39092 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore trifluoroacetic acid tfa
    rFIBCD1-FReD binds A. fumigatus AIF calcium- and acetate-independently. (A) The presence and binding activity of β-1,3-glucan, galactomannan, and chitin in AIF was validated by pull down of know ligands Dectin-1 fc, rMBL, and WGA. The samples were subjected to <t>SDS-PAGE</t> and analyzed by silver staining. Color is adjusted to gray scale. (B) Western blot showing FIBCD1-FReD pull down by AIF-associated polysaccharides in the presence of calcium (5 mM CaCl 2 ), <t>EDTA</t> (10 mM), and acetate (100 mM). 2 mg of AIF, β-1,3-glucan, and chitin beads were incubated with 5 μg/mL FIBCD1-FReD and pull down was performed as described. Data represent three independent experiments. (C) Western blot showing wild type and A432V mutant FIBCD1-FReD pull down by chitin beads and AIF. Color is adjusted to gray scale. Lane 1, 3: chitin beads, Lane 2: AIF.
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    99
    Millipore protease inhibitors cocktail
    rFIBCD1-FReD binds A. fumigatus AIF calcium- and acetate-independently. (A) The presence and binding activity of β-1,3-glucan, galactomannan, and chitin in AIF was validated by pull down of know ligands Dectin-1 fc, rMBL, and WGA. The samples were subjected to <t>SDS-PAGE</t> and analyzed by silver staining. Color is adjusted to gray scale. (B) Western blot showing FIBCD1-FReD pull down by AIF-associated polysaccharides in the presence of calcium (5 mM CaCl 2 ), <t>EDTA</t> (10 mM), and acetate (100 mM). 2 mg of AIF, β-1,3-glucan, and chitin beads were incubated with 5 μg/mL FIBCD1-FReD and pull down was performed as described. Data represent three independent experiments. (C) Western blot showing wild type and A432V mutant FIBCD1-FReD pull down by chitin beads and AIF. Color is adjusted to gray scale. Lane 1, 3: chitin beads, Lane 2: AIF.
    Protease Inhibitors Cocktail, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2907 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore tcep
    Experimental Procedure. (A) Comparison of the reduction with <t>DTT,</t> 2-ME, <t>TCEP,</t> or THPP. (B) Comparison of the alkylation with iodoacetamide, acrylamide, N-EM, or 4-VP. The reduction and alkylation were performed at the peptide level.
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    Millipore triton x 100
    The US6 gene encodes a 22-kDa glycoprotein. US6 and control-transfected Pala cells were extracted in 1% Triton X-100 and lysates separated by SDS/12.5% PAGE, transferred to Immobilon-P membranes, and probed with anti-US6 ( A ) and anti-calnexin ( B ) antibodies. ( C ) Crude membrane extracts from US6 and control-transfected cells were prepared by freeze thawing. Membranes were extracted in endo H buffer, subjected to endo H or mock digestion, separated by SDS/PAGE, and after transfer to Immobilon-P membranes probed with antibody specific for US6.
    Triton X 100, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 65173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore protease inhibitors
    The US6 gene encodes a 22-kDa glycoprotein. US6 and control-transfected Pala cells were extracted in 1% Triton X-100 and lysates separated by SDS/12.5% PAGE, transferred to Immobilon-P membranes, and probed with anti-US6 ( A ) and anti-calnexin ( B ) antibodies. ( C ) Crude membrane extracts from US6 and control-transfected cells were prepared by freeze thawing. Membranes were extracted in endo H buffer, subjected to endo H or mock digestion, separated by SDS/PAGE, and after transfer to Immobilon-P membranes probed with antibody specific for US6.
    Protease Inhibitors, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 34531 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore bovine serum albumin bsa
    Fig. 4. MALDI-TOF analysis of (a) a mixture of 100 ng <t>BSA</t> /100 ng <t>HRP</t> and (b) a mixture of 1 μg BSA /100 ng HRP. Upper panel: before BP-plate enrichment. Lower panel: after BP-plate enrichment.
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    99
    Millipore sodium deoxycholate
    Fig. 4. MALDI-TOF analysis of (a) a mixture of 100 ng <t>BSA</t> /100 ng <t>HRP</t> and (b) a mixture of 1 μg BSA /100 ng HRP. Upper panel: before BP-plate enrichment. Lower panel: after BP-plate enrichment.
    Sodium Deoxycholate, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 18429 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore pmsf
    Cysteine and serine protease inhibitors decrease mucinolytic activities. (A) Ethanol-treated secretory products were incubated with protease inhibitors and evaluated in bovine submaxillary mucin–zymograms as follows: ethanol-treated secretory products without inhibitor (lane 1), or with 10 mM pHMB (lane 2), 10 μM E-64 (lane 3), 5 mM <t>NEM</t> (lane 4), 5 μM IAA (lane 5), 5 mM <t>PMSF</t> (lane 6), aprotinin (lane 7), or 10 mM 1,10-phenanthroline (lane 8). All the zymograms were activated at pH 7, the results correspond a one gel with several lanes. (B) Densitometric analyses of > 250-kDa (white bars), 94-kDa (black bars) and 53-kDa (gray bars) protein activities using ImageJ software, graphed by GraphPad Prism and expressed as ROD values. Statistical analysis was performed by two-way ANOVA comparing the degradation without inhibitors. Bars display the mean ± SE of three independent assays. *p
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    Millipore acetonitrile acn
    Cysteine and serine protease inhibitors decrease mucinolytic activities. (A) Ethanol-treated secretory products were incubated with protease inhibitors and evaluated in bovine submaxillary mucin–zymograms as follows: ethanol-treated secretory products without inhibitor (lane 1), or with 10 mM pHMB (lane 2), 10 μM E-64 (lane 3), 5 mM <t>NEM</t> (lane 4), 5 μM IAA (lane 5), 5 mM <t>PMSF</t> (lane 6), aprotinin (lane 7), or 10 mM 1,10-phenanthroline (lane 8). All the zymograms were activated at pH 7, the results correspond a one gel with several lanes. (B) Densitometric analyses of > 250-kDa (white bars), 94-kDa (black bars) and 53-kDa (gray bars) protein activities using ImageJ software, graphed by GraphPad Prism and expressed as ROD values. Statistical analysis was performed by two-way ANOVA comparing the degradation without inhibitors. Bars display the mean ± SE of three independent assays. *p
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    99
    Millipore ammonium formate
    Cysteine and serine protease inhibitors decrease mucinolytic activities. (A) Ethanol-treated secretory products were incubated with protease inhibitors and evaluated in bovine submaxillary mucin–zymograms as follows: ethanol-treated secretory products without inhibitor (lane 1), or with 10 mM pHMB (lane 2), 10 μM E-64 (lane 3), 5 mM <t>NEM</t> (lane 4), 5 μM IAA (lane 5), 5 mM <t>PMSF</t> (lane 6), aprotinin (lane 7), or 10 mM 1,10-phenanthroline (lane 8). All the zymograms were activated at pH 7, the results correspond a one gel with several lanes. (B) Densitometric analyses of > 250-kDa (white bars), 94-kDa (black bars) and 53-kDa (gray bars) protein activities using ImageJ software, graphed by GraphPad Prism and expressed as ROD values. Statistical analysis was performed by two-way ANOVA comparing the degradation without inhibitors. Bars display the mean ± SE of three independent assays. *p
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    Image Search Results


    Formation of the μ1/μ1C ds bonds during the course of infection. T1L reovirus-infected cells were harvested at either 24, 48, 72, or 96 h postinfection. 50 mM IAM was added immediately upon resuspension of the infected cells in homogenization buffer. After purification, virions from each time point were mixed with nonreducing sample buffer (NR) and disrupted by boiling. The viral proteins were then resolved on a mini-sized SDS-8% PAGE gel. The amount of ds-bonded μ1/μ1C (% ds μ1/μ1C) relative to the total μ1, μ1C, and ds-bonded μ1/μ1C in each preparation was determined by densitometry of the Coomassie-stained gel. Immediately before harvesting, aliquots were taken from each time point and the proportion of dead/dying cells (% dead cells) was determined by trypan blue staining.

    Journal: Journal of Virology

    Article Title: Disulfide Bonding among ?1 Trimers in Mammalian Reovirus Outer Capsid: a Late and Reversible Step in Virion Morphogenesis

    doi: 10.1128/JVI.77.9.5389-5400.2003

    Figure Lengend Snippet: Formation of the μ1/μ1C ds bonds during the course of infection. T1L reovirus-infected cells were harvested at either 24, 48, 72, or 96 h postinfection. 50 mM IAM was added immediately upon resuspension of the infected cells in homogenization buffer. After purification, virions from each time point were mixed with nonreducing sample buffer (NR) and disrupted by boiling. The viral proteins were then resolved on a mini-sized SDS-8% PAGE gel. The amount of ds-bonded μ1/μ1C (% ds μ1/μ1C) relative to the total μ1, μ1C, and ds-bonded μ1/μ1C in each preparation was determined by densitometry of the Coomassie-stained gel. Immediately before harvesting, aliquots were taken from each time point and the proportion of dead/dying cells (% dead cells) was determined by trypan blue staining.

    Article Snippet: Iodoacetamide at a concentration of 50 mM (IAM; Sigma) was added during the course of some purifications as indicated in the legend to Fig. .

    Techniques: Infection, Homogenization, Purification, Polyacrylamide Gel Electrophoresis, Staining

    Analyses for ds bond formation with μ1 mutant C679S. (A) Recoated cores containing T1L wt σ3 and either T1L wt μ1 or the T1L μ1 mutant C679S were generated and purified, and particle concentrations were determined by densitometry. Equal amounts of the wt (lanes 2 and 5) or C679S (lanes 3 and 6) recoated cores were mixed with reducing (R) or nonreducing (NR) sample buffer, disrupted by boiling, and resolved on a mini-sized SDS-PAGE (8% acrylamide) gel. Virions disrupted under reducing (lane 1) or nonreducing (lane 4) conditions were included for comparison. Viral proteins were visualized by Coomassie staining. (B) Purified μ1-σ3 heterohexamers stored frozen in buffer with 10 mM DTT were thawed, and a small amount of each was diluted into nonreducing sample buffer and analyzed on a 4 to 15% acrylamide gradient native gel (Amersham Pharmacia Biotech) (lanes 1 to 3; 0 days). The remainder of each sample was passed through a PD-10 column to remove DTT and then stored at 4°C for 8 days at ambient conditions. At the end of that time, a small amount of each was diluted into sample buffer and analyzed on another 4 to 15% native gel (lanes 4 to 6). Three types of μ1-σ3 preparations were included in this analysis: complexes containing wt T1L μ1 and σ3 (lanes 1 and 4), complexes containing wt T1L μ1 and σ3 that were treated with 10 mM IAM to block free cysteines before storage (lanes 2 and 5), and complexes containing T1L C679S μ1 and wt T1L σ3 (lanes 3 and 6). The position of the native μ1-σ3 heterohexamer is indicated to the left. The positions of higher- M r forms specific to the complexes containing wt T1L μ1 and σ3 (lanes 1) are indicated (§). Positions of native gel markers (Amersham Pharmacia Biotech) are indicated in kilodaltons.

    Journal: Journal of Virology

    Article Title: Disulfide Bonding among ?1 Trimers in Mammalian Reovirus Outer Capsid: a Late and Reversible Step in Virion Morphogenesis

    doi: 10.1128/JVI.77.9.5389-5400.2003

    Figure Lengend Snippet: Analyses for ds bond formation with μ1 mutant C679S. (A) Recoated cores containing T1L wt σ3 and either T1L wt μ1 or the T1L μ1 mutant C679S were generated and purified, and particle concentrations were determined by densitometry. Equal amounts of the wt (lanes 2 and 5) or C679S (lanes 3 and 6) recoated cores were mixed with reducing (R) or nonreducing (NR) sample buffer, disrupted by boiling, and resolved on a mini-sized SDS-PAGE (8% acrylamide) gel. Virions disrupted under reducing (lane 1) or nonreducing (lane 4) conditions were included for comparison. Viral proteins were visualized by Coomassie staining. (B) Purified μ1-σ3 heterohexamers stored frozen in buffer with 10 mM DTT were thawed, and a small amount of each was diluted into nonreducing sample buffer and analyzed on a 4 to 15% acrylamide gradient native gel (Amersham Pharmacia Biotech) (lanes 1 to 3; 0 days). The remainder of each sample was passed through a PD-10 column to remove DTT and then stored at 4°C for 8 days at ambient conditions. At the end of that time, a small amount of each was diluted into sample buffer and analyzed on another 4 to 15% native gel (lanes 4 to 6). Three types of μ1-σ3 preparations were included in this analysis: complexes containing wt T1L μ1 and σ3 (lanes 1 and 4), complexes containing wt T1L μ1 and σ3 that were treated with 10 mM IAM to block free cysteines before storage (lanes 2 and 5), and complexes containing T1L C679S μ1 and wt T1L σ3 (lanes 3 and 6). The position of the native μ1-σ3 heterohexamer is indicated to the left. The positions of higher- M r forms specific to the complexes containing wt T1L μ1 and σ3 (lanes 1) are indicated (§). Positions of native gel markers (Amersham Pharmacia Biotech) are indicated in kilodaltons.

    Article Snippet: Iodoacetamide at a concentration of 50 mM (IAM; Sigma) was added during the course of some purifications as indicated in the legend to Fig. .

    Techniques: Mutagenesis, Generated, Purification, SDS Page, Acrylamide Gel Assay, Staining, Blocking Assay

    Effects of IAM addition on the migration of reovirus μ1 and μ1C proteins during SDS-PAGE. (A and B) Purified virions of reovirus T1L were used in these experiments. Viral proteins were resolved on a mini-sized SDS-PAGE [10% (A) or 8% (B) acrylamide] gel and visualized by Coomassie staining. The major new high- M r band observed after nonreducing disruption is indicated (*). (A) Virions were disrupted in sample buffer that contained no reducing agent (NR), and IAM was either not added (−) or added at different times after disruption (0 to 10 min) as indicated. (B) Virions were disrupted in pH 6.8 or 8.0 sample buffer that contained no reducing agent but to which 50 mM IAM was added either before (b) or immediately after (a) disruption as indicated. A sample of virions disrupted in reducing sample buffer (R; 5 mM DTT) was analyzed on the same gel (lane 1). Positions of molecular mass markers also resolved on the gel are indicated in kilodaltons. (C) Purified [ 35 S]methionine-cysteine-labeled virions of reovirus T3D were used in this experiment. The Coomassie-stained protein bands excised from the first gel (not shown; μ1C was obtained from samples of reduced virions and the high- M r band [*] was obtained from samples of IAM-treated nonreduced virions) were subjected to reduction in the gel fragments and otherwise treated as described in the text. The resulting proteins and protein fragments were resolved on a second gel and visualized by fluorography. The position of the full-length μ1C monomer is indicated. The amount of chymotrypsin added to each gel piece atop the second gel is also indicated. (D) Purified [ 35 S]methionine-cysteine- or [ 3 H]myristate-labeled virions of reovirus T3D were mixed with reducing or nonreducing sample buffer and disrupted in a boiling water bath. The viral proteins were then resolved on a full-sized 5 to 20% acrylamide gradient SDS-PAGE gel and visualized by fluorography. The major (*) and minor (**) new high- M r bands observed after nonreducing disruption of each sample are indicated.

    Journal: Journal of Virology

    Article Title: Disulfide Bonding among ?1 Trimers in Mammalian Reovirus Outer Capsid: a Late and Reversible Step in Virion Morphogenesis

    doi: 10.1128/JVI.77.9.5389-5400.2003

    Figure Lengend Snippet: Effects of IAM addition on the migration of reovirus μ1 and μ1C proteins during SDS-PAGE. (A and B) Purified virions of reovirus T1L were used in these experiments. Viral proteins were resolved on a mini-sized SDS-PAGE [10% (A) or 8% (B) acrylamide] gel and visualized by Coomassie staining. The major new high- M r band observed after nonreducing disruption is indicated (*). (A) Virions were disrupted in sample buffer that contained no reducing agent (NR), and IAM was either not added (−) or added at different times after disruption (0 to 10 min) as indicated. (B) Virions were disrupted in pH 6.8 or 8.0 sample buffer that contained no reducing agent but to which 50 mM IAM was added either before (b) or immediately after (a) disruption as indicated. A sample of virions disrupted in reducing sample buffer (R; 5 mM DTT) was analyzed on the same gel (lane 1). Positions of molecular mass markers also resolved on the gel are indicated in kilodaltons. (C) Purified [ 35 S]methionine-cysteine-labeled virions of reovirus T3D were used in this experiment. The Coomassie-stained protein bands excised from the first gel (not shown; μ1C was obtained from samples of reduced virions and the high- M r band [*] was obtained from samples of IAM-treated nonreduced virions) were subjected to reduction in the gel fragments and otherwise treated as described in the text. The resulting proteins and protein fragments were resolved on a second gel and visualized by fluorography. The position of the full-length μ1C monomer is indicated. The amount of chymotrypsin added to each gel piece atop the second gel is also indicated. (D) Purified [ 35 S]methionine-cysteine- or [ 3 H]myristate-labeled virions of reovirus T3D were mixed with reducing or nonreducing sample buffer and disrupted in a boiling water bath. The viral proteins were then resolved on a full-sized 5 to 20% acrylamide gradient SDS-PAGE gel and visualized by fluorography. The major (*) and minor (**) new high- M r bands observed after nonreducing disruption of each sample are indicated.

    Article Snippet: Iodoacetamide at a concentration of 50 mM (IAM; Sigma) was added during the course of some purifications as indicated in the legend to Fig. .

    Techniques: Migration, SDS Page, Purification, Acrylamide Gel Assay, Staining, Labeling

    Reversibility of the virion-associated μ1/μ1C ds bonds. For each experiment, aliquots were removed from the reaction mixture at the indicated intervals, and the reaction was quenched with 50 mM IAM. When all samples had been collected for each experiment, they were mixed with nonreducing sample buffer (NR) and disrupted by boiling. Viral proteins were resolved on mini-sized SDS-8% PAGE gels and visualized by Coomassie staining. (A) DTT at a concentration of 5 mM was added to purified T1L virions and allowed to incubate at room temperature to effect in situ reduction of the ds bonds. The 0-min aliquot (lane 1) was removed before the addition of DTT. Positions of molecular mass markers also resolved on the gel are indicated to the left. (B) The ds bonds in a sample of T1L virions were reduced by DTT as in panel A, lane 6, after which the DTT was removed by dialysis. Cystine at a concentration of 5 mM was then added to promote in situ reformation of the ds bonds. The 0-min aliquot (lane 1) was removed before the addition of cystine. A sample to which cysteine was never added was also analyzed (lane 9).

    Journal: Journal of Virology

    Article Title: Disulfide Bonding among ?1 Trimers in Mammalian Reovirus Outer Capsid: a Late and Reversible Step in Virion Morphogenesis

    doi: 10.1128/JVI.77.9.5389-5400.2003

    Figure Lengend Snippet: Reversibility of the virion-associated μ1/μ1C ds bonds. For each experiment, aliquots were removed from the reaction mixture at the indicated intervals, and the reaction was quenched with 50 mM IAM. When all samples had been collected for each experiment, they were mixed with nonreducing sample buffer (NR) and disrupted by boiling. Viral proteins were resolved on mini-sized SDS-8% PAGE gels and visualized by Coomassie staining. (A) DTT at a concentration of 5 mM was added to purified T1L virions and allowed to incubate at room temperature to effect in situ reduction of the ds bonds. The 0-min aliquot (lane 1) was removed before the addition of DTT. Positions of molecular mass markers also resolved on the gel are indicated to the left. (B) The ds bonds in a sample of T1L virions were reduced by DTT as in panel A, lane 6, after which the DTT was removed by dialysis. Cystine at a concentration of 5 mM was then added to promote in situ reformation of the ds bonds. The 0-min aliquot (lane 1) was removed before the addition of cystine. A sample to which cysteine was never added was also analyzed (lane 9).

    Article Snippet: Iodoacetamide at a concentration of 50 mM (IAM; Sigma) was added during the course of some purifications as indicated in the legend to Fig. .

    Techniques: Polyacrylamide Gel Electrophoresis, Staining, Concentration Assay, Purification, In Situ

    Specific activity of AgTG3. A , conjugation of FITC-CAD to Plugin-C in solution by AgTG3 (9.6 μg/ml, 30 °C). B , HRP-streptavidin assay (TMB, A 405 ) for conjugation of biotin-TQQVEL to polylysine plates by 1.5 units mg −1 guinea pig TG2 (□) or AgTG3 (○). C , fluorescence ( I 510 ) for conjugation of FITC-CAD to Plugin-coated plates by 1.5 units mg −1 guinea pig TG2 (□) or AgTG3 (○). D , inhibition of AgTG3 by iodoacetamide ( IA ) versus negative control ( NC ) in Plugin/FITC-CAD assay.

    Journal: The Journal of Biological Chemistry

    Article Title: Characterization of Anopheles gambiae Transglutaminase 3 (AgTG3) and Its Native Substrate Plugin *

    doi: 10.1074/jbc.M112.435347

    Figure Lengend Snippet: Specific activity of AgTG3. A , conjugation of FITC-CAD to Plugin-C in solution by AgTG3 (9.6 μg/ml, 30 °C). B , HRP-streptavidin assay (TMB, A 405 ) for conjugation of biotin-TQQVEL to polylysine plates by 1.5 units mg −1 guinea pig TG2 (□) or AgTG3 (○). C , fluorescence ( I 510 ) for conjugation of FITC-CAD to Plugin-coated plates by 1.5 units mg −1 guinea pig TG2 (□) or AgTG3 (○). D , inhibition of AgTG3 by iodoacetamide ( IA ) versus negative control ( NC ) in Plugin/FITC-CAD assay.

    Article Snippet: Inhibition was performed by 30-min preincubation with 50 m m iodoacetamide (Sigma A3221).

    Techniques: Activity Assay, Conjugation Assay, Fluorescence, Inhibition, IA, Negative Control

    Sensitivity of auto-ADP-ribosylation of TIPARP to different treatment conditions. ( A ) GST-TIPARP was incubated with different concentrations of MIBG for 5 min and auto-ribosylation was initiated with the addition of 32 P-NAD + as described in the Materials and Methods. ( B ) GST-TIPARP was incubated with 32 P-NAD + for 20 min at room temperature and then treated with 1 M neutral hydroxylamine (NH 2 OH) or NaCl for 1 or 2 h. ( C ) GST-TIPARP was incubated with different concentrations of IAM or 2 mM NaCl for 30 min at 37°C and the reaction was initiated with the addition of 32 P-NAD + as described in Materials and Methods. ADP-ribosylation was detected by autoradiography after SDS–PAGE followed by transfer onto a PVDF membrane. GCB staining shows levels of proteins loaded into SDS–PAGE gel. 3-ABA, 3-aminobemzamide. The data are representative of at least two independent experiments.

    Journal: Biochemical Journal

    Article Title: Characterization of TCDD-inducible poly-ADP-ribose polymerase (TIPARP/ARTD14) catalytic activity

    doi: 10.1042/BCJ20180347

    Figure Lengend Snippet: Sensitivity of auto-ADP-ribosylation of TIPARP to different treatment conditions. ( A ) GST-TIPARP was incubated with different concentrations of MIBG for 5 min and auto-ribosylation was initiated with the addition of 32 P-NAD + as described in the Materials and Methods. ( B ) GST-TIPARP was incubated with 32 P-NAD + for 20 min at room temperature and then treated with 1 M neutral hydroxylamine (NH 2 OH) or NaCl for 1 or 2 h. ( C ) GST-TIPARP was incubated with different concentrations of IAM or 2 mM NaCl for 30 min at 37°C and the reaction was initiated with the addition of 32 P-NAD + as described in Materials and Methods. ADP-ribosylation was detected by autoradiography after SDS–PAGE followed by transfer onto a PVDF membrane. GCB staining shows levels of proteins loaded into SDS–PAGE gel. 3-ABA, 3-aminobemzamide. The data are representative of at least two independent experiments.

    Article Snippet: 3-Aminobenzoamide (3-ABA), ( N -(6-oxo-5,6-dihydrophenanthridin-2-yl)( N , N -dimethylamino) acetamide) (PJ34), meta-iodobenzylguanidine (MIBG), iodoacetamide (IAM), and NH2 OH (hydroxylamine) were purchased from Sigma-Aldrich (Oakville, Ontario).

    Techniques: Incubation, Autoradiography, SDS Page, Staining

    Redox state of Trx1 in A549 cells under oxidative stress. A , principle of redox Western blot analysis. To prepare mobility standards, cell lysates were denatured with urea and fully reduced with DTT. Varying molar ratios of IAA to IAM were incubated with

    Journal: The Journal of Biological Chemistry

    Article Title: Thioredoxin 1 Is Inactivated Due to Oxidation Induced by Peroxiredoxin under Oxidative Stress and Reactivated by the Glutaredoxin System *

    doi: 10.1074/jbc.M113.495150

    Figure Lengend Snippet: Redox state of Trx1 in A549 cells under oxidative stress. A , principle of redox Western blot analysis. To prepare mobility standards, cell lysates were denatured with urea and fully reduced with DTT. Varying molar ratios of IAA to IAM were incubated with

    Article Snippet: Buthionine sulfoximine (BSO), DTT, GSH, iodoacetic acid (IAA), iodoacetamide (IAM), insulin, and glutathione reductase were purchased from Sigma-Aldrich.

    Techniques: Western Blot, Incubation

    Flowchart of the identification of the quantitative phosphoproteome in neuronal autophagy. According to the weights of the labeled essential amino acids, N2a cells were divided into 3 groups, including “heavy” ( 13 C 6 15 N 2 -Lysine, 13 C 6 15 N 4 -Arginine), “middle” ( 13 C 4 14 N 2 -Lysine, 13 C 6 14 N 4 -Arginine) and “light” ( 12 C 6 14 N 2 -Lysine, 12 C 6 14 N 4 -Arginine). After checking the labeling efficiency ( > 96%), the “light” and “middle” groups were treated with 15 μM Cory or Cory B for 3 h (Fig. S1), and the “heavy” group, which acted as a control group, was treated with 0.1% (v:v) DMSO. The cell lysates were mixed at a ratio of 1:1:1 and reduced with 10 mM dithiothreitol for 1 h at 56°C and then alkylated with 20 mM iodoacetamide for 45 min at room temperature and protected from light. Then, proteins were digested in solution with trypsin gold at a ratio of 1:50 (trypsin to protein) overnight and 1:100 (trypsin to protein) for another 4 h. After fractionation by high-pH reverse-phase HPLC, peptides were divided into 12 fractions. Finally, enriched phosphopeptides were subjected to LC-MS/MS for the identification of the quantitative phosphoproteome.

    Journal: Autophagy

    Article Title: Phosphoproteome-based kinase activity profiling reveals the critical role of MAP2K2 and PLK1 in neuronal autophagy

    doi: 10.1080/15548627.2017.1371393

    Figure Lengend Snippet: Flowchart of the identification of the quantitative phosphoproteome in neuronal autophagy. According to the weights of the labeled essential amino acids, N2a cells were divided into 3 groups, including “heavy” ( 13 C 6 15 N 2 -Lysine, 13 C 6 15 N 4 -Arginine), “middle” ( 13 C 4 14 N 2 -Lysine, 13 C 6 14 N 4 -Arginine) and “light” ( 12 C 6 14 N 2 -Lysine, 12 C 6 14 N 4 -Arginine). After checking the labeling efficiency ( > 96%), the “light” and “middle” groups were treated with 15 μM Cory or Cory B for 3 h (Fig. S1), and the “heavy” group, which acted as a control group, was treated with 0.1% (v:v) DMSO. The cell lysates were mixed at a ratio of 1:1:1 and reduced with 10 mM dithiothreitol for 1 h at 56°C and then alkylated with 20 mM iodoacetamide for 45 min at room temperature and protected from light. Then, proteins were digested in solution with trypsin gold at a ratio of 1:50 (trypsin to protein) overnight and 1:100 (trypsin to protein) for another 4 h. After fractionation by high-pH reverse-phase HPLC, peptides were divided into 12 fractions. Finally, enriched phosphopeptides were subjected to LC-MS/MS for the identification of the quantitative phosphoproteome.

    Article Snippet: H2 O was purchased from Thermo (10977023); ACN (acetonitrile; A998–1) and fa (ethyl alcohol; A995–4) was purchased from Fisher Chemical; formic acid (FA; 5.43804) was purchased from Fluka; DMEM medium (11965118) and fetal bovine serum (FBS; 16000044) were purchased from Invitrogen; SILAC™ Protein Identification and Quantitation Media Kit was purchased from Thermo (SP10001); Sequencing Grade Modified Trypsin was purchased from Promega (V5280); trifluoroacetic acid (TFA; 574732), iodoacetamide (A3221) and dithiothreitol (43817) were purchased from Sigma; 2-D Quant kit was purchased from GE Healthcare (80–6483–56); IMAC beads was purchased from Dalian Institute of Chemical Physics.

    Techniques: Labeling, Fractionation, High Performance Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Expression and purification of r-Cam-dis analysis by 4–12% SDS-PAGE gel under non-reducing condition. Samples were run on 4–12% (w/v) Bis-Tris Gel using an Xcell SureLock Mini-Cell at 200V for 30min. The gel was stained with Rapid-Stain. Lane 1: SeeBlue Plus2 Markers; lane 2: soluble fraction of lysates of expressed E. coli BL21 cells by BugBuster reagent (150μg); lane 3: cleaved r-Cam-dis after wash with binding buffer (3μg); lane 4: purified r-Cam-dis after wash with high salt buffer (3 μg). An asterisk ( * ) represents the N-terminal amino acid sequence of purified r-Cam-dis containing the five amino acids from the vector (italicized) before the disintegrin sequence, which are shown in bold letters.

    Journal: Journal of Venom Research

    Article Title: Recombinant disintegrin (r-Cam-dis) from Crotalus adamanteus inhibits adhesion of human pancreatic cancer cell lines to laminin-1 and vitronectin

    doi:

    Figure Lengend Snippet: Expression and purification of r-Cam-dis analysis by 4–12% SDS-PAGE gel under non-reducing condition. Samples were run on 4–12% (w/v) Bis-Tris Gel using an Xcell SureLock Mini-Cell at 200V for 30min. The gel was stained with Rapid-Stain. Lane 1: SeeBlue Plus2 Markers; lane 2: soluble fraction of lysates of expressed E. coli BL21 cells by BugBuster reagent (150μg); lane 3: cleaved r-Cam-dis after wash with binding buffer (3μg); lane 4: purified r-Cam-dis after wash with high salt buffer (3 μg). An asterisk ( * ) represents the N-terminal amino acid sequence of purified r-Cam-dis containing the five amino acids from the vector (italicized) before the disintegrin sequence, which are shown in bold letters.

    Article Snippet: Bacterial cells were collected by centrifugation at 10000xg for 10min and resuspended in 1x BugBuster Protein Extraction reagent (Novagen CA, USA) by gentle vortexing, using 5ml reagent per gram of wet cell paste.

    Techniques: Expressing, Purification, Chick Chorioallantoic Membrane Assay, SDS Page, Staining, Binding Assay, Sequencing, Plasmid Preparation

    CAT are able to cleave HIV-1 Env-A244 and CAT degradation products are susceptible to proteasomal degradation. HIV-1 Env-A244 (panels A and B, lanes 0) was treated with CAT for 90 min or 16 hrs and then analyzed on a 4–20% gradient Tris-glycine polyacrylamide gel. The 90 min (lanes 90) and 16 hrs (lanes 0/N) degradation pattern with the various CAT are shown in panels A and B. Env-A244 was treated with the individual CAT for 90 min followed by proteasomes in the presence (panels C and D, lanes PI) or absence of epoxomicin (lanes P). The molecular weight markers are shown in lane M in all panels.

    Journal: PLoS ONE

    Article Title: HIV-1 Envelope Resistance to Proteasomal Cleavage: Implications for Vaccine Induced Immune Responses

    doi: 10.1371/journal.pone.0042579

    Figure Lengend Snippet: CAT are able to cleave HIV-1 Env-A244 and CAT degradation products are susceptible to proteasomal degradation. HIV-1 Env-A244 (panels A and B, lanes 0) was treated with CAT for 90 min or 16 hrs and then analyzed on a 4–20% gradient Tris-glycine polyacrylamide gel. The 90 min (lanes 90) and 16 hrs (lanes 0/N) degradation pattern with the various CAT are shown in panels A and B. Env-A244 was treated with the individual CAT for 90 min followed by proteasomes in the presence (panels C and D, lanes PI) or absence of epoxomicin (lanes P). The molecular weight markers are shown in lane M in all panels.

    Article Snippet: Reduction and alklyation of Env-A244 and RNaseB Env-A244 and RNaseB (150 µg each) were each diluted in 20 mM TRIS, pH 7.2 containing 20 mM DTT (Sigma-Aldrich) and heated for 30 min at 56°C.

    Techniques: Molecular Weight

    Proteasomes are unable to cleave HIV-1 Env proteins. Env-A244 (CRF01_AE) derived from CHO cells and gp140 (clade B) derived from H9 cells (panel A, lanes C) were treated with purified proteasomes isolated from activated CD4 + T-cells in the absence (lanes, P) or presence of epoxomicin, an irreversible proteasome inhibitor (lanes PI). Proteasomes were unable to cleave the Env proteins from two different HIV-1 clades. In panel B, RNaseB, a folded-glycosylated protein (lane C) was treated as in panel A with purified proteasomes in the absence (lane P) or presence of epoxomicin (lane PI). The proteasomes were also unable to cleave RNaseB. The functional activity and the specificity of the proteasomes were demonstrated by the proteolytic cleavage of Gag-p24 (panel C, lane C) in the absence (lane P) or presence of epoxomicin (lane PI). Env-A244 and RNaseB (panels D and E, lanes C) were subjected to the same treatment as in panels A and B. Env-A244 and RNaseB were treated with Endo-F and Endo-H followed by incubation with purified proteasomes in the absence (lanes P) or presence of epoxomicin (lanes PI). Proteasomes were unable to cleave Env-A244 and RNaseB following treatment with either Endo-F or Endo-H. Env-A244 and RNaseB (panel F, lanes C) were reduced and alkylated (IAA) by treatment with iodioacetamine and DTT and then treated with purified proteasomes in the absence (lanes P) or presence of epoxomicin (lanes PI). Env-A244 remained resistant while RNaseB became susceptible to proteasomal cleavage. Samples were run on a 4–20% gradient Tris-glycine polyacrylamide gel. The molecular weight markers are shown in lane M in panels A, B, D, E, and F.

    Journal: PLoS ONE

    Article Title: HIV-1 Envelope Resistance to Proteasomal Cleavage: Implications for Vaccine Induced Immune Responses

    doi: 10.1371/journal.pone.0042579

    Figure Lengend Snippet: Proteasomes are unable to cleave HIV-1 Env proteins. Env-A244 (CRF01_AE) derived from CHO cells and gp140 (clade B) derived from H9 cells (panel A, lanes C) were treated with purified proteasomes isolated from activated CD4 + T-cells in the absence (lanes, P) or presence of epoxomicin, an irreversible proteasome inhibitor (lanes PI). Proteasomes were unable to cleave the Env proteins from two different HIV-1 clades. In panel B, RNaseB, a folded-glycosylated protein (lane C) was treated as in panel A with purified proteasomes in the absence (lane P) or presence of epoxomicin (lane PI). The proteasomes were also unable to cleave RNaseB. The functional activity and the specificity of the proteasomes were demonstrated by the proteolytic cleavage of Gag-p24 (panel C, lane C) in the absence (lane P) or presence of epoxomicin (lane PI). Env-A244 and RNaseB (panels D and E, lanes C) were subjected to the same treatment as in panels A and B. Env-A244 and RNaseB were treated with Endo-F and Endo-H followed by incubation with purified proteasomes in the absence (lanes P) or presence of epoxomicin (lanes PI). Proteasomes were unable to cleave Env-A244 and RNaseB following treatment with either Endo-F or Endo-H. Env-A244 and RNaseB (panel F, lanes C) were reduced and alkylated (IAA) by treatment with iodioacetamine and DTT and then treated with purified proteasomes in the absence (lanes P) or presence of epoxomicin (lanes PI). Env-A244 remained resistant while RNaseB became susceptible to proteasomal cleavage. Samples were run on a 4–20% gradient Tris-glycine polyacrylamide gel. The molecular weight markers are shown in lane M in panels A, B, D, E, and F.

    Article Snippet: Reduction and alklyation of Env-A244 and RNaseB Env-A244 and RNaseB (150 µg each) were each diluted in 20 mM TRIS, pH 7.2 containing 20 mM DTT (Sigma-Aldrich) and heated for 30 min at 56°C.

    Techniques: Derivative Assay, Purification, Isolation, Functional Assay, Activity Assay, Incubation, Molecular Weight

    rFIBCD1-FReD binds A. fumigatus AIF calcium- and acetate-independently. (A) The presence and binding activity of β-1,3-glucan, galactomannan, and chitin in AIF was validated by pull down of know ligands Dectin-1 fc, rMBL, and WGA. The samples were subjected to SDS-PAGE and analyzed by silver staining. Color is adjusted to gray scale. (B) Western blot showing FIBCD1-FReD pull down by AIF-associated polysaccharides in the presence of calcium (5 mM CaCl 2 ), EDTA (10 mM), and acetate (100 mM). 2 mg of AIF, β-1,3-glucan, and chitin beads were incubated with 5 μg/mL FIBCD1-FReD and pull down was performed as described. Data represent three independent experiments. (C) Western blot showing wild type and A432V mutant FIBCD1-FReD pull down by chitin beads and AIF. Color is adjusted to gray scale. Lane 1, 3: chitin beads, Lane 2: AIF.

    Journal: Frontiers in Immunology

    Article Title: FIBCD1 Binds Aspergillus fumigatus and Regulates Lung Epithelial Response to Cell Wall Components

    doi: 10.3389/fimmu.2018.01967

    Figure Lengend Snippet: rFIBCD1-FReD binds A. fumigatus AIF calcium- and acetate-independently. (A) The presence and binding activity of β-1,3-glucan, galactomannan, and chitin in AIF was validated by pull down of know ligands Dectin-1 fc, rMBL, and WGA. The samples were subjected to SDS-PAGE and analyzed by silver staining. Color is adjusted to gray scale. (B) Western blot showing FIBCD1-FReD pull down by AIF-associated polysaccharides in the presence of calcium (5 mM CaCl 2 ), EDTA (10 mM), and acetate (100 mM). 2 mg of AIF, β-1,3-glucan, and chitin beads were incubated with 5 μg/mL FIBCD1-FReD and pull down was performed as described. Data represent three independent experiments. (C) Western blot showing wild type and A432V mutant FIBCD1-FReD pull down by chitin beads and AIF. Color is adjusted to gray scale. Lane 1, 3: chitin beads, Lane 2: AIF.

    Article Snippet: Acetylated BSA (acBSA), BSA, citric acid, EDTA, glycine, glycerol, silver nitrate (AgNO3 ), SDS, sodium azide (NaN3 ), Tris-base, Triton X-100, H2 SO4 , iodoacetamide (IAA), DTT, CaCl2 , sodium acetate, NaHCO3 , sterile PBS, curdlan (β-1,3-glucan, from Alcaligenes faecalis ), RNase ZAP®, M-MLV reverse transcriptase, and oligo-dT primers were ordered from Sigma-Aldrich Co., St Louis, MO, United States.

    Techniques: Binding Assay, Activity Assay, Whole Genome Amplification, SDS Page, Silver Staining, Western Blot, Incubation, Mutagenesis

    Experimental Procedure. (A) Comparison of the reduction with DTT, 2-ME, TCEP, or THPP. (B) Comparison of the alkylation with iodoacetamide, acrylamide, N-EM, or 4-VP. The reduction and alkylation were performed at the peptide level.

    Journal: Molecular bioSystems

    Article Title: Evaluation and optimization of reduction and alkylation methods to maximize peptide identification with MS-based proteomics

    doi: 10.1039/c7mb00393e

    Figure Lengend Snippet: Experimental Procedure. (A) Comparison of the reduction with DTT, 2-ME, TCEP, or THPP. (B) Comparison of the alkylation with iodoacetamide, acrylamide, N-EM, or 4-VP. The reduction and alkylation were performed at the peptide level.

    Article Snippet: Four reducing reagents, DTT (Sigma-Aldrich), 2-ME (Sigma-Aldrich), TCEP (Calbiochem), and THPP (Santa Cruz Biotechnology), were compared at the same concentration of 5 mM.

    Techniques:

    The US6 gene encodes a 22-kDa glycoprotein. US6 and control-transfected Pala cells were extracted in 1% Triton X-100 and lysates separated by SDS/12.5% PAGE, transferred to Immobilon-P membranes, and probed with anti-US6 ( A ) and anti-calnexin ( B ) antibodies. ( C ) Crude membrane extracts from US6 and control-transfected cells were prepared by freeze thawing. Membranes were extracted in endo H buffer, subjected to endo H or mock digestion, separated by SDS/PAGE, and after transfer to Immobilon-P membranes probed with antibody specific for US6.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: The human cytomegalovirus US6 glycoprotein inhibits transporter associated with antigen processing-dependent peptide translocation

    doi:

    Figure Lengend Snippet: The US6 gene encodes a 22-kDa glycoprotein. US6 and control-transfected Pala cells were extracted in 1% Triton X-100 and lysates separated by SDS/12.5% PAGE, transferred to Immobilon-P membranes, and probed with anti-US6 ( A ) and anti-calnexin ( B ) antibodies. ( C ) Crude membrane extracts from US6 and control-transfected cells were prepared by freeze thawing. Membranes were extracted in endo H buffer, subjected to endo H or mock digestion, separated by SDS/PAGE, and after transfer to Immobilon-P membranes probed with antibody specific for US6.

    Article Snippet: Cells were extracted for 30 min on ice at 4 × 106 cells/ml in 1% digitonin (Wako Biochemicals, Osaka) or 1% Triton X-100 (Sigma) in Tris-buffered saline (10 mM Tris, 150 mM NaCl, pH 7.4), containing 0.5 mM phenylmethylsulfonyl fluoride (Sigma), 0.1 mM N -α-tosyl- l -lysyl-chloromethyl ketone (Sigma), and 5.0 mM iodoacetamide (Sigma).

    Techniques: Transfection, Polyacrylamide Gel Electrophoresis, SDS Page

    US6 associates with the TAP/tapasin/class I complex. Digitonin lysates (1%) from US6 and control-transfected PaLa cells were immunoprecipitated with TAP1-specific (148.3) and control (κ immunoglobulin light chain-specific) mAb, coupled to A-15 m Sepharose beads. Precipitated proteins were eluted in 0.1% SDS/0.05% Triton X-100 buffer, separated by 12.5% SDS/PAGE under nonreducing conditions, transferred to Immobilon P membrane, and probed with TAP-specific, R.RING 4C ( A ), tapasin-specific, R.tapasinN ( B ), class I heavy chain-specific, 3B10.7 ( C ), and anti-US6 ( D ) antibodies.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: The human cytomegalovirus US6 glycoprotein inhibits transporter associated with antigen processing-dependent peptide translocation

    doi:

    Figure Lengend Snippet: US6 associates with the TAP/tapasin/class I complex. Digitonin lysates (1%) from US6 and control-transfected PaLa cells were immunoprecipitated with TAP1-specific (148.3) and control (κ immunoglobulin light chain-specific) mAb, coupled to A-15 m Sepharose beads. Precipitated proteins were eluted in 0.1% SDS/0.05% Triton X-100 buffer, separated by 12.5% SDS/PAGE under nonreducing conditions, transferred to Immobilon P membrane, and probed with TAP-specific, R.RING 4C ( A ), tapasin-specific, R.tapasinN ( B ), class I heavy chain-specific, 3B10.7 ( C ), and anti-US6 ( D ) antibodies.

    Article Snippet: Cells were extracted for 30 min on ice at 4 × 106 cells/ml in 1% digitonin (Wako Biochemicals, Osaka) or 1% Triton X-100 (Sigma) in Tris-buffered saline (10 mM Tris, 150 mM NaCl, pH 7.4), containing 0.5 mM phenylmethylsulfonyl fluoride (Sigma), 0.1 mM N -α-tosyl- l -lysyl-chloromethyl ketone (Sigma), and 5.0 mM iodoacetamide (Sigma).

    Techniques: Transfection, Immunoprecipitation, SDS Page

    Transport of class I heavy chains is inhibited in US6-transfected cells. US6 and vector control-transfected HeLaM cells were radiolabeled with [ 35 S]methionine and [ 35 S]cysteine for 15 min and chased for the indicated time periods. Triton X-100 lysates (1%) were immunoprecipitated with the mAb w6/32, digested or mock-digested with endo H, and subjected to 10% SDS/PAGE ( A ), and the ratio of endo H-resistant vs. endo H-sensitive class I heavy chains present at each time point quantitated by image analysis ( B ).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: The human cytomegalovirus US6 glycoprotein inhibits transporter associated with antigen processing-dependent peptide translocation

    doi:

    Figure Lengend Snippet: Transport of class I heavy chains is inhibited in US6-transfected cells. US6 and vector control-transfected HeLaM cells were radiolabeled with [ 35 S]methionine and [ 35 S]cysteine for 15 min and chased for the indicated time periods. Triton X-100 lysates (1%) were immunoprecipitated with the mAb w6/32, digested or mock-digested with endo H, and subjected to 10% SDS/PAGE ( A ), and the ratio of endo H-resistant vs. endo H-sensitive class I heavy chains present at each time point quantitated by image analysis ( B ).

    Article Snippet: Cells were extracted for 30 min on ice at 4 × 106 cells/ml in 1% digitonin (Wako Biochemicals, Osaka) or 1% Triton X-100 (Sigma) in Tris-buffered saline (10 mM Tris, 150 mM NaCl, pH 7.4), containing 0.5 mM phenylmethylsulfonyl fluoride (Sigma), 0.1 mM N -α-tosyl- l -lysyl-chloromethyl ketone (Sigma), and 5.0 mM iodoacetamide (Sigma).

    Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, SDS Page

    TAP-mediated peptide translocation is inhibited in US6-transfected cells and can be overcome with IFN-γ. Streptolysin-O (Murex)-permeabilized US6 and control-transfected Pala cells ( A ), HeLaM ( B ), and HeLaM cells that had been pretreated with IFN-γ (200 unit/ml) for 48 hr ( C ) were incubated with an iodinated reporter peptide at 37°C for the indicated time period, and the reaction was stopped by lysis with 3% Triton X-100, as described in  Materials and Methods . Translocation into the ER was assessed by binding of the glycosylated reporter peptide to concanavalin A-Sepharose beads and counting on a γ-counter. HeLaM, US6-transfected HeLaM, and US6-transfected HeLaM cells that had been pretreated with IFN-γ were analyzed for class I surface expression by flow cytometry, using the mAb w6/32 and fluorescein-conjugated rabbit anti-mouse IgG ( D ). The control was a nonspecific isotype-matched monoclonal antibody.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: The human cytomegalovirus US6 glycoprotein inhibits transporter associated with antigen processing-dependent peptide translocation

    doi:

    Figure Lengend Snippet: TAP-mediated peptide translocation is inhibited in US6-transfected cells and can be overcome with IFN-γ. Streptolysin-O (Murex)-permeabilized US6 and control-transfected Pala cells ( A ), HeLaM ( B ), and HeLaM cells that had been pretreated with IFN-γ (200 unit/ml) for 48 hr ( C ) were incubated with an iodinated reporter peptide at 37°C for the indicated time period, and the reaction was stopped by lysis with 3% Triton X-100, as described in Materials and Methods . Translocation into the ER was assessed by binding of the glycosylated reporter peptide to concanavalin A-Sepharose beads and counting on a γ-counter. HeLaM, US6-transfected HeLaM, and US6-transfected HeLaM cells that had been pretreated with IFN-γ were analyzed for class I surface expression by flow cytometry, using the mAb w6/32 and fluorescein-conjugated rabbit anti-mouse IgG ( D ). The control was a nonspecific isotype-matched monoclonal antibody.

    Article Snippet: Cells were extracted for 30 min on ice at 4 × 106 cells/ml in 1% digitonin (Wako Biochemicals, Osaka) or 1% Triton X-100 (Sigma) in Tris-buffered saline (10 mM Tris, 150 mM NaCl, pH 7.4), containing 0.5 mM phenylmethylsulfonyl fluoride (Sigma), 0.1 mM N -α-tosyl- l -lysyl-chloromethyl ketone (Sigma), and 5.0 mM iodoacetamide (Sigma).

    Techniques: Translocation Assay, Transfection, Incubation, Lysis, Binding Assay, Expressing, Flow Cytometry, Cytometry

    Fig. 4. MALDI-TOF analysis of (a) a mixture of 100 ng BSA /100 ng HRP and (b) a mixture of 1 μg BSA /100 ng HRP. Upper panel: before BP-plate enrichment. Lower panel: after BP-plate enrichment.

    Journal: Mass Spectrometry

    Article Title: A Rapid Approach for Fabricating Boronic Acid-Functionalized Plates for On-Probe Detection of Glycoprotein and Glycopeptide

    doi: 10.5702/massspectrometry.S0063

    Figure Lengend Snippet: Fig. 4. MALDI-TOF analysis of (a) a mixture of 100 ng BSA /100 ng HRP and (b) a mixture of 1 μg BSA /100 ng HRP. Upper panel: before BP-plate enrichment. Lower panel: after BP-plate enrichment.

    Article Snippet: Dithiothreitol (DTT), iodoacetamide (IAA), horseradish peroxidase (HRP), bovine serum albumin (BSA), and trifluoroacetic acid (TFA) were from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques:

    Cysteine and serine protease inhibitors decrease mucinolytic activities. (A) Ethanol-treated secretory products were incubated with protease inhibitors and evaluated in bovine submaxillary mucin–zymograms as follows: ethanol-treated secretory products without inhibitor (lane 1), or with 10 mM pHMB (lane 2), 10 μM E-64 (lane 3), 5 mM NEM (lane 4), 5 μM IAA (lane 5), 5 mM PMSF (lane 6), aprotinin (lane 7), or 10 mM 1,10-phenanthroline (lane 8). All the zymograms were activated at pH 7, the results correspond a one gel with several lanes. (B) Densitometric analyses of > 250-kDa (white bars), 94-kDa (black bars) and 53-kDa (gray bars) protein activities using ImageJ software, graphed by GraphPad Prism and expressed as ROD values. Statistical analysis was performed by two-way ANOVA comparing the degradation without inhibitors. Bars display the mean ± SE of three independent assays. *p

    Journal: Future Microbiology

    Article Title: Nf-GH, a glycosidase secreted by Naegleria fowleri, causes mucin degradation: an in vitro and in vivo study

    doi: 10.2217/fmb-2016-0230

    Figure Lengend Snippet: Cysteine and serine protease inhibitors decrease mucinolytic activities. (A) Ethanol-treated secretory products were incubated with protease inhibitors and evaluated in bovine submaxillary mucin–zymograms as follows: ethanol-treated secretory products without inhibitor (lane 1), or with 10 mM pHMB (lane 2), 10 μM E-64 (lane 3), 5 mM NEM (lane 4), 5 μM IAA (lane 5), 5 mM PMSF (lane 6), aprotinin (lane 7), or 10 mM 1,10-phenanthroline (lane 8). All the zymograms were activated at pH 7, the results correspond a one gel with several lanes. (B) Densitometric analyses of > 250-kDa (white bars), 94-kDa (black bars) and 53-kDa (gray bars) protein activities using ImageJ software, graphed by GraphPad Prism and expressed as ROD values. Statistical analysis was performed by two-way ANOVA comparing the degradation without inhibitors. Bars display the mean ± SE of three independent assays. *p

    Article Snippet: The following final concentrations and types of protease inhibitors were used: for cysteine proteases, 10-mM pHMB, 10-μM (1-{ N -[(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]amino}-4-guanidinobutane [E-64]), 5-mM n-ethylmaleimide (NEM) and 5-μM iodoacetamide (IAA); for serine proteases, 5 mM PMSF and 1 mM aprotinin; for metalloproteases, 10-mM 1,10-phenanthroline [ ] (Sigma-Aldrich).

    Techniques: Incubation, Software