Structured Review

Roche np40
Effect of C-terminal E domains and prM protein on the recognition of E protein by different mouse anti-E mAbs. (A) Binding specificity of five mouse anti-E mAbs including GR (4G2 and DEN2-12), CR (DEN3-3), and DENV4 TS (1H10-6-7 and 1H10-5-7) mAbs. Western blot analysis was performed by using cell lysates derived from C6/36 cells infected with each of the four DENV serotypes, WNV or JEV. The size of molecular weight markers is shown in kDa. (B,C) Dot blot binding assay using these five mAbs to recognize WT E protein (expressed by prME), E protein alone and mutant E proteins containing C-terminal truncations (expressed by prME- or E-based constructs) in 1% <t>NP40</t> lysis buffer (NP40). Layout of the dot blot assay and the binding by mixed mAbs are shown in (B). Decreasing amount of native WT E protein in 1% NP40 lysis buffer (column B) as well as mixtures containing decreasing amount of native WT E protein in 1% NP40 lysis buffer and increasing amount of denatured WT E protein in reducing (R) buffer (column A) were also included to control for exposure and sensitivity of the assay signal. Relative intensities of each dot in columns A (black bars) and B (white bars) were shown below each membrane. Recognition indices of each mAb to mutant E protein = [intensity of mutant E dot/intensity of WT E dot] (recognized by a mAb) divided by [intensity of mutant E dot/intensity of WT E dot] (recognized by mixed mAbs) were shown in blue bars (column C, in the presence of prM protein) and yellow bars (column D, in the absence of prM protein) below each membrane [33] . Data are mean and standard errors from two experiments.
Np40, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 201 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/np40/product/Roche
Average 99 stars, based on 201 article reviews
Price from $9.99 to $1999.99
np40 - by Bioz Stars, 2020-03
99/100 stars

Images

1) Product Images from "C-Terminal Helical Domains of Dengue Virus Type 4 E Protein Affect the Expression/Stability of prM Protein and Conformation of prM and E Proteins"

Article Title: C-Terminal Helical Domains of Dengue Virus Type 4 E Protein Affect the Expression/Stability of prM Protein and Conformation of prM and E Proteins

Journal: PLoS ONE

doi: 10.1371/journal.pone.0052600

Effect of C-terminal E domains and prM protein on the recognition of E protein by different mouse anti-E mAbs. (A) Binding specificity of five mouse anti-E mAbs including GR (4G2 and DEN2-12), CR (DEN3-3), and DENV4 TS (1H10-6-7 and 1H10-5-7) mAbs. Western blot analysis was performed by using cell lysates derived from C6/36 cells infected with each of the four DENV serotypes, WNV or JEV. The size of molecular weight markers is shown in kDa. (B,C) Dot blot binding assay using these five mAbs to recognize WT E protein (expressed by prME), E protein alone and mutant E proteins containing C-terminal truncations (expressed by prME- or E-based constructs) in 1% NP40 lysis buffer (NP40). Layout of the dot blot assay and the binding by mixed mAbs are shown in (B). Decreasing amount of native WT E protein in 1% NP40 lysis buffer (column B) as well as mixtures containing decreasing amount of native WT E protein in 1% NP40 lysis buffer and increasing amount of denatured WT E protein in reducing (R) buffer (column A) were also included to control for exposure and sensitivity of the assay signal. Relative intensities of each dot in columns A (black bars) and B (white bars) were shown below each membrane. Recognition indices of each mAb to mutant E protein = [intensity of mutant E dot/intensity of WT E dot] (recognized by a mAb) divided by [intensity of mutant E dot/intensity of WT E dot] (recognized by mixed mAbs) were shown in blue bars (column C, in the presence of prM protein) and yellow bars (column D, in the absence of prM protein) below each membrane [33] . Data are mean and standard errors from two experiments.
Figure Legend Snippet: Effect of C-terminal E domains and prM protein on the recognition of E protein by different mouse anti-E mAbs. (A) Binding specificity of five mouse anti-E mAbs including GR (4G2 and DEN2-12), CR (DEN3-3), and DENV4 TS (1H10-6-7 and 1H10-5-7) mAbs. Western blot analysis was performed by using cell lysates derived from C6/36 cells infected with each of the four DENV serotypes, WNV or JEV. The size of molecular weight markers is shown in kDa. (B,C) Dot blot binding assay using these five mAbs to recognize WT E protein (expressed by prME), E protein alone and mutant E proteins containing C-terminal truncations (expressed by prME- or E-based constructs) in 1% NP40 lysis buffer (NP40). Layout of the dot blot assay and the binding by mixed mAbs are shown in (B). Decreasing amount of native WT E protein in 1% NP40 lysis buffer (column B) as well as mixtures containing decreasing amount of native WT E protein in 1% NP40 lysis buffer and increasing amount of denatured WT E protein in reducing (R) buffer (column A) were also included to control for exposure and sensitivity of the assay signal. Relative intensities of each dot in columns A (black bars) and B (white bars) were shown below each membrane. Recognition indices of each mAb to mutant E protein = [intensity of mutant E dot/intensity of WT E dot] (recognized by a mAb) divided by [intensity of mutant E dot/intensity of WT E dot] (recognized by mixed mAbs) were shown in blue bars (column C, in the presence of prM protein) and yellow bars (column D, in the absence of prM protein) below each membrane [33] . Data are mean and standard errors from two experiments.

Techniques Used: Binding Assay, Western Blot, Derivative Assay, Infection, Molecular Weight, Dot Blot, Mutagenesis, Construct, Lysis

Effect of E protein on the recognition of prM protein by human anti-prM mAbs. (A) Binding specificity of 4 human anti-prM mAbs including 2 CR (DVB59.3 and DVB18.5) and 2 sCR (DVB65.5 and DVB32.4) mAbs was determined as in Figure 5 . (B) Dot blot binding assay using these 4 mAbs to recognize prM protein in the presence (expressed by prME) or absence (expressed by prM) of E protein in 1% NP40 lysis buffer (NP40). Decreasing amount of prM protein (expressed by prME) in 1% NP40 lysis buffer (column B, rows 3 to 7) and mixtures containing decreasing amount of prM protein in 1% NP40 lysis buffer and increasing amount of denatured prM protein in reducing (R) buffer (column A) were included to control for exposure and sensitivity of the assay signal, respectively. Twenty times more cell lysates derived from transfection of prM alone were loaded. Relative intensities of each dot in column A (black bars) and column B (blue bars) were shown below each membrane. Data are mean and standard errors from two experiments.
Figure Legend Snippet: Effect of E protein on the recognition of prM protein by human anti-prM mAbs. (A) Binding specificity of 4 human anti-prM mAbs including 2 CR (DVB59.3 and DVB18.5) and 2 sCR (DVB65.5 and DVB32.4) mAbs was determined as in Figure 5 . (B) Dot blot binding assay using these 4 mAbs to recognize prM protein in the presence (expressed by prME) or absence (expressed by prM) of E protein in 1% NP40 lysis buffer (NP40). Decreasing amount of prM protein (expressed by prME) in 1% NP40 lysis buffer (column B, rows 3 to 7) and mixtures containing decreasing amount of prM protein in 1% NP40 lysis buffer and increasing amount of denatured prM protein in reducing (R) buffer (column A) were included to control for exposure and sensitivity of the assay signal, respectively. Twenty times more cell lysates derived from transfection of prM alone were loaded. Relative intensities of each dot in column A (black bars) and column B (blue bars) were shown below each membrane. Data are mean and standard errors from two experiments.

Techniques Used: Binding Assay, Dot Blot, Lysis, Derivative Assay, Transfection

2) Product Images from "Immuno- and Constitutive Proteasomes Do Not Differ in Their Abilities to Degrade Ubiquitinated Proteins"

Article Title: Immuno- and Constitutive Proteasomes Do Not Differ in Their Abilities to Degrade Ubiquitinated Proteins

Journal: Cell

doi: 10.1016/j.cell.2013.01.037

IFNγ Treatment Does Not Cause a Significant Accumulation of Polyubiquitin Conjugates during the Induction of Immunoproteasomes, Related to Figure 1 (A and B) Murine embryonic (A) and B8 (B) fibroblasts were stimulated with 100U/ml IFNγ for the indicated time periods. As a control for ubiquitin accumulation, cells were treated with the proteasome inhibitor lactacystin (10μM, 4hr) or vehicle (DMSO). The cells were then lysed by sonication in 25mM HEPES pH 7.4, 1mM ATP, 1mM DTT, 5mM MgCl 2 and 10% glycerol. Lysates were subsequently centrifuged for one hour at 100,000 x g. Protein concentrations of the lysates were determined using the DC Protein Assay (Biorad) and equal amounts of protein were separated by SDS-PAGE and analyzed by western blotting. α-tubulin served as a loading control. One representative experiment out of three independent experiments with similar outcomes is shown. The induction of LMP7 by IFNγ stimulation was confirmed by immunoblot analysis; GAPDH served as a loading control. The ubiquitin levels were determined by densitometric analyses (ImageJ) of five different western blots; shown are the mean values ± SD obtained after normalization to the loading control and relative to the value for unstimulated cells which was set to unity. (C and D) HeLa cells were treated with 100U/ml IFNγ as described (C) or with 10 μM lactacystin for 4 hr (D). The cells were lysed with nonionic detergent according to the methods described by Seifert et al. (20 mM TRIS-HCl, pH 7.5, 10 mM EDTA, 100 mM NaCl, 1% NP40, 10 μM MG-132, 5 mM NEM and Complete Protease Inhibitor Cocktail (Roche)) and immunblotted for ubiquitin (C, left). The ubiquitin levels, (relative to the β-actin control) were determined by densitometric analyses (ImageJ) as described (C, right). The mean values ± SEM for three replicates are shown (C).
Figure Legend Snippet: IFNγ Treatment Does Not Cause a Significant Accumulation of Polyubiquitin Conjugates during the Induction of Immunoproteasomes, Related to Figure 1 (A and B) Murine embryonic (A) and B8 (B) fibroblasts were stimulated with 100U/ml IFNγ for the indicated time periods. As a control for ubiquitin accumulation, cells were treated with the proteasome inhibitor lactacystin (10μM, 4hr) or vehicle (DMSO). The cells were then lysed by sonication in 25mM HEPES pH 7.4, 1mM ATP, 1mM DTT, 5mM MgCl 2 and 10% glycerol. Lysates were subsequently centrifuged for one hour at 100,000 x g. Protein concentrations of the lysates were determined using the DC Protein Assay (Biorad) and equal amounts of protein were separated by SDS-PAGE and analyzed by western blotting. α-tubulin served as a loading control. One representative experiment out of three independent experiments with similar outcomes is shown. The induction of LMP7 by IFNγ stimulation was confirmed by immunoblot analysis; GAPDH served as a loading control. The ubiquitin levels were determined by densitometric analyses (ImageJ) of five different western blots; shown are the mean values ± SD obtained after normalization to the loading control and relative to the value for unstimulated cells which was set to unity. (C and D) HeLa cells were treated with 100U/ml IFNγ as described (C) or with 10 μM lactacystin for 4 hr (D). The cells were lysed with nonionic detergent according to the methods described by Seifert et al. (20 mM TRIS-HCl, pH 7.5, 10 mM EDTA, 100 mM NaCl, 1% NP40, 10 μM MG-132, 5 mM NEM and Complete Protease Inhibitor Cocktail (Roche)) and immunblotted for ubiquitin (C, left). The ubiquitin levels, (relative to the β-actin control) were determined by densitometric analyses (ImageJ) as described (C, right). The mean values ± SEM for three replicates are shown (C).

Techniques Used: Sonication, DC Protein Assay, SDS Page, Western Blot, Protease Inhibitor

3) Product Images from "The histone chaperone sNASP binds a conserved peptide motif within the globular core of histone H3 through its TPR repeats"

Article Title: The histone chaperone sNASP binds a conserved peptide motif within the globular core of histone H3 through its TPR repeats

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkv1372

Mutations within the TPR domain of sNASP abolish H3 peptide binding. ( A ) Alignment of the TPR motifs from NASP homologues that retained the H3 116–135 interaction, displayed alongside secondary structural elements and the TPR repeat motif consensus. The positions predicted to face the concave binding channel of the TPR domain fold are indicated by arrows. Asterisks indicate two conserved clusters of residues that are outside of the TPR consensus. Residue numbers for the human homologue are given. The junction of the reconstituted TPR2 is shown as a vertical black line. ( B ) Pulldown experiments of two triple mutants corresponding to the conserved residues identified in (A) under conditions of low stringency (in 20 mM Hepes-KOH pH 7.5, 200 mM sodium chloride) and high stringency (50 mM potassium hydrogen phosphate pH 7.5, 300 mM sodium chloride, 0.1% NP40 and 20% imidazole). ( C ) F2H analysis of GFP-sNASP mutants E211A, E215A, E217A (EEE > AAA) and E246A, Y249S, L253S (EYL > ASS) with mCherry-LacI-H3 or H3 116–135 as indicated. ( D ) Quantification of the enrichment ratio for F2H pairs shown in (C). As comparisons, values for GFP-sNASP recruitment to mCherry-LacI, mCherry-LacI-H3 and mCherry-LacI-H3 116–135 from Figure 1C are shown alongside. Boxes represent the lower quartile, median and upper quartile. Whiskers represent 1.5× the interquartile range. Outliers are indicated by circles. Asterisks indicate a significant difference in enrichment compared to the empty vector control ( P -value
Figure Legend Snippet: Mutations within the TPR domain of sNASP abolish H3 peptide binding. ( A ) Alignment of the TPR motifs from NASP homologues that retained the H3 116–135 interaction, displayed alongside secondary structural elements and the TPR repeat motif consensus. The positions predicted to face the concave binding channel of the TPR domain fold are indicated by arrows. Asterisks indicate two conserved clusters of residues that are outside of the TPR consensus. Residue numbers for the human homologue are given. The junction of the reconstituted TPR2 is shown as a vertical black line. ( B ) Pulldown experiments of two triple mutants corresponding to the conserved residues identified in (A) under conditions of low stringency (in 20 mM Hepes-KOH pH 7.5, 200 mM sodium chloride) and high stringency (50 mM potassium hydrogen phosphate pH 7.5, 300 mM sodium chloride, 0.1% NP40 and 20% imidazole). ( C ) F2H analysis of GFP-sNASP mutants E211A, E215A, E217A (EEE > AAA) and E246A, Y249S, L253S (EYL > ASS) with mCherry-LacI-H3 or H3 116–135 as indicated. ( D ) Quantification of the enrichment ratio for F2H pairs shown in (C). As comparisons, values for GFP-sNASP recruitment to mCherry-LacI, mCherry-LacI-H3 and mCherry-LacI-H3 116–135 from Figure 1C are shown alongside. Boxes represent the lower quartile, median and upper quartile. Whiskers represent 1.5× the interquartile range. Outliers are indicated by circles. Asterisks indicate a significant difference in enrichment compared to the empty vector control ( P -value

Techniques Used: Binding Assay, Plasmid Preparation

4) Product Images from "Leoligin, the Major Lignan from Edelweiss (Leontopodium nivale subsp. alpinum), Promotes Cholesterol Efflux from THP-1 Macrophages"

Article Title: Leoligin, the Major Lignan from Edelweiss (Leontopodium nivale subsp. alpinum), Promotes Cholesterol Efflux from THP-1 Macrophages

Journal: Journal of Natural Products

doi: 10.1021/acs.jnatprod.6b00227

Leoligin increases ABCA1 and ABCG1, but not SR-B1 protein expression. THP-1 macrophages were treated with leoligin (LEO, 10 μM), pioglitazone (PIO, 10 μM), or solvent control (DMSO) for 24 h. Then, cells were lysed with NP40 buffer. A 20 μg amount of total protein was analyzed by Western blot using anti-ABCA1 (A), -ABCG1 (B), or -SR-B1 (C) antibodies. Band intensities of four independent experiments were quantified. The bar graphs represent mean ± SD, and the statistical evaluation was performed by one-way ANOVA with the Bonferroni post-test. * p
Figure Legend Snippet: Leoligin increases ABCA1 and ABCG1, but not SR-B1 protein expression. THP-1 macrophages were treated with leoligin (LEO, 10 μM), pioglitazone (PIO, 10 μM), or solvent control (DMSO) for 24 h. Then, cells were lysed with NP40 buffer. A 20 μg amount of total protein was analyzed by Western blot using anti-ABCA1 (A), -ABCG1 (B), or -SR-B1 (C) antibodies. Band intensities of four independent experiments were quantified. The bar graphs represent mean ± SD, and the statistical evaluation was performed by one-way ANOVA with the Bonferroni post-test. * p

Techniques Used: Expressing, Western Blot

5) Product Images from "PTENα regulates mitophagy and maintains mitochondrial quality control"

Article Title: PTENα regulates mitophagy and maintains mitochondrial quality control

Journal: Autophagy

doi: 10.1080/15548627.2018.1489477

PRKN/PARK2 is identified as a PTENα-associated protein upon mitochondrial depolarization. (a) Silver staining and MS analysis of PTENα-associated proteins. The empty vector (PSA), S•Tag PTENα and -PTEN were purified from HEK293T cells and incubated with mouse cardiac homogenate; associated proteins were identified by mass spectrometry. (b) Co-IP with HEK293T cells co-transfected with plasmids encoding PTENα-GFP and PRKN-FLAG. Cells were treated with 10 μM CCCP or DMSO for 1 h before harvest. Lysates immunoprecipitated with anti-FLAG were immunoblotted with anti-GFP or anti-FLAG. The asterisk indicates degradation of PTENα. (c) Co-IP in HEK293T cells co-transfected with plasmids encoding PRKN-GFP and FLAG-PTENα. Cells were treated with 10 μM CCCP or DMSO for 1 h before harvest. Lysates immunoprecipitated with anti-FLAG were immunoblotted with anti-GFP or anti-FLAG. (d) In vitro binding assay of PTENα-His with GST-PRKN. PTENα-His (1 µg) and 3 μg GST-PRKN were mixed in PBS containing 0.1% NP40, and PRKN was immunoprecipitated with a PRKN monoclonal antibody or mouse lgG and immunoblotted with anti-PTEN or anti-PRKN. PTENα-His was expressed and purified from insect cells, and GST-PRKN was expressed and purified from E. coli . The asterisk indicates degradation of PTENα. (e) Interaction of FLAG-PTENα and its truncations with PRKN-GFP in HEK293T cells with CCCP treatment. Cells were treated with 10 μM CCCP for 1 h before harvest. Lysates immunoprecipitated with anti-FLAG were immunoblotted with anti-GFP or anti-FLAG. IgH, heavy chain of IgG. (F) Interaction of PRKN-GFP and its truncations with HA-PTENα in HEK293T cells with CCCP treatment. Cells were treated with 10 μM CCCP for 1 h before harvest. Lysates immunoprecipitated with anti-GFP were immunoblotted with anti-HA or anti-GFP.
Figure Legend Snippet: PRKN/PARK2 is identified as a PTENα-associated protein upon mitochondrial depolarization. (a) Silver staining and MS analysis of PTENα-associated proteins. The empty vector (PSA), S•Tag PTENα and -PTEN were purified from HEK293T cells and incubated with mouse cardiac homogenate; associated proteins were identified by mass spectrometry. (b) Co-IP with HEK293T cells co-transfected with plasmids encoding PTENα-GFP and PRKN-FLAG. Cells were treated with 10 μM CCCP or DMSO for 1 h before harvest. Lysates immunoprecipitated with anti-FLAG were immunoblotted with anti-GFP or anti-FLAG. The asterisk indicates degradation of PTENα. (c) Co-IP in HEK293T cells co-transfected with plasmids encoding PRKN-GFP and FLAG-PTENα. Cells were treated with 10 μM CCCP or DMSO for 1 h before harvest. Lysates immunoprecipitated with anti-FLAG were immunoblotted with anti-GFP or anti-FLAG. (d) In vitro binding assay of PTENα-His with GST-PRKN. PTENα-His (1 µg) and 3 μg GST-PRKN were mixed in PBS containing 0.1% NP40, and PRKN was immunoprecipitated with a PRKN monoclonal antibody or mouse lgG and immunoblotted with anti-PTEN or anti-PRKN. PTENα-His was expressed and purified from insect cells, and GST-PRKN was expressed and purified from E. coli . The asterisk indicates degradation of PTENα. (e) Interaction of FLAG-PTENα and its truncations with PRKN-GFP in HEK293T cells with CCCP treatment. Cells were treated with 10 μM CCCP for 1 h before harvest. Lysates immunoprecipitated with anti-FLAG were immunoblotted with anti-GFP or anti-FLAG. IgH, heavy chain of IgG. (F) Interaction of PRKN-GFP and its truncations with HA-PTENα in HEK293T cells with CCCP treatment. Cells were treated with 10 μM CCCP for 1 h before harvest. Lysates immunoprecipitated with anti-GFP were immunoblotted with anti-HA or anti-GFP.

Techniques Used: Silver Staining, Mass Spectrometry, Plasmid Preparation, Purification, Incubation, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, In Vitro, Binding Assay

6) Product Images from "Stoichiometry of HLA Class II-Invariant Chain Oligomers"

Article Title: Stoichiometry of HLA Class II-Invariant Chain Oligomers

Journal: PLoS ONE

doi: 10.1371/journal.pone.0017257

Inspection for co-isolation of DP and DQ isotypes with DR. A) MelJuSo cells were transiently transfected with cDNAs encoding V5-tagged DPα or DPβ encoding cDNAs. Cells lysed in 1% NP40 were immunoprecipitated for DRαβ (monoclonal antibody ISCR3) (lanes 1,2,3,5,6 and 7). Lanes 4 and 8 contain cell lysates. The SDS-separated polypeptides were immunoblotted for DRα (lanes 1 and 5), for Ii (lanes 2 and 6), and for DPαV5 (lanes 3 and 4), or for DPβV5 chains (lanes 7 and 8). The position of DRα, Ii and Ab-epitope tagged DPαV5 or DPβV5 bands is indicated. H and L chain bands derived from the Ab used for immunoprecipitation are labeled on the left. B) Immunoprecipitates of the V5-tagged DPβ (lanes 1 and 4) and of DRαβ (lanes 2 and 5) chains were separated by SDS-PAGE and either blotted for DPβV5 (lanes 1 to 3) or for DRα (lanes 4 and 5). To monitor expression of DPβV5, lane 3 contains cell lysate. The position of DPβV5, of DRα and of H and L Ig chains are indicated. C) MelJuSo cells were transfected with V5-tagged DQα cDNA. Cell lysate was immunoprecipitated for the V5-tagged DQα chains. Immunoprecipitates (lanes 4 to 6) and cell lysates (lanes 1 to 3) were digested with EndoH (E H ), or with PNGase F (P F ) or left untreated (Ø), separated by SDS-PAGE and western blotted as indicated. In lanes 7 to 9 MelJuSo cell lysates untreated and glycosidase treated were western blotted for DRα.
Figure Legend Snippet: Inspection for co-isolation of DP and DQ isotypes with DR. A) MelJuSo cells were transiently transfected with cDNAs encoding V5-tagged DPα or DPβ encoding cDNAs. Cells lysed in 1% NP40 were immunoprecipitated for DRαβ (monoclonal antibody ISCR3) (lanes 1,2,3,5,6 and 7). Lanes 4 and 8 contain cell lysates. The SDS-separated polypeptides were immunoblotted for DRα (lanes 1 and 5), for Ii (lanes 2 and 6), and for DPαV5 (lanes 3 and 4), or for DPβV5 chains (lanes 7 and 8). The position of DRα, Ii and Ab-epitope tagged DPαV5 or DPβV5 bands is indicated. H and L chain bands derived from the Ab used for immunoprecipitation are labeled on the left. B) Immunoprecipitates of the V5-tagged DPβ (lanes 1 and 4) and of DRαβ (lanes 2 and 5) chains were separated by SDS-PAGE and either blotted for DPβV5 (lanes 1 to 3) or for DRα (lanes 4 and 5). To monitor expression of DPβV5, lane 3 contains cell lysate. The position of DPβV5, of DRα and of H and L Ig chains are indicated. C) MelJuSo cells were transfected with V5-tagged DQα cDNA. Cell lysate was immunoprecipitated for the V5-tagged DQα chains. Immunoprecipitates (lanes 4 to 6) and cell lysates (lanes 1 to 3) were digested with EndoH (E H ), or with PNGase F (P F ) or left untreated (Ø), separated by SDS-PAGE and western blotted as indicated. In lanes 7 to 9 MelJuSo cell lysates untreated and glycosidase treated were western blotted for DRα.

Techniques Used: Isolation, Transfection, Immunoprecipitation, Derivative Assay, Labeling, SDS Page, Expressing, Western Blot

7) Product Images from "Substrate ectodomain is critical for substrate preference and inhibition of ?-secretase"

Article Title: Substrate ectodomain is critical for substrate preference and inhibition of ?-secretase

Journal: Nature Communications

doi: 10.1038/ncomms3529

Schematic diagrams of generating γ-secretase substrates. γ-Secretase substrates were expressed as fusion proteins with the APP signal peptide and the Profinity eXact tag (Bio-Rad) in sf9 cells ( a ). The Profinity eXact tag protein purification system offers purification of recombinant proteins with a native amino terminus. Once Profinity eXact-tagged γ-secretase substrates were captured using S189 subtilisin-immobilized resin, addition of sodium fluoride triggered precise cleavage at the carboxyl terminus of the cleavage-recognition sequence (shown in green) and the release of substrates with a bona fide N terminus. Purified substrates were recaptured using anti-FLAG M2 agarose beads (Sigma) and eluted by addition of 0.2 M glycine, pH 2.7 and 0.3% NP40. 1/10th volume of 3 M Tris-HCl, pH 8.0 was added to the eluted substrates for neutralization. The flanking region of the S189 subtilisin cleavage site in each substrate is shown in b .
Figure Legend Snippet: Schematic diagrams of generating γ-secretase substrates. γ-Secretase substrates were expressed as fusion proteins with the APP signal peptide and the Profinity eXact tag (Bio-Rad) in sf9 cells ( a ). The Profinity eXact tag protein purification system offers purification of recombinant proteins with a native amino terminus. Once Profinity eXact-tagged γ-secretase substrates were captured using S189 subtilisin-immobilized resin, addition of sodium fluoride triggered precise cleavage at the carboxyl terminus of the cleavage-recognition sequence (shown in green) and the release of substrates with a bona fide N terminus. Purified substrates were recaptured using anti-FLAG M2 agarose beads (Sigma) and eluted by addition of 0.2 M glycine, pH 2.7 and 0.3% NP40. 1/10th volume of 3 M Tris-HCl, pH 8.0 was added to the eluted substrates for neutralization. The flanking region of the S189 subtilisin cleavage site in each substrate is shown in b .

Techniques Used: Protein Purification, Purification, Recombinant, Sequencing, Neutralization

8) Product Images from "Voluntary resistance wheel exercise from mid-life prevents sarcopenia and increases markers of mitochondrial function and autophagy in muscles of old male and female C57BL/6J mice"

Article Title: Voluntary resistance wheel exercise from mid-life prevents sarcopenia and increases markers of mitochondrial function and autophagy in muscles of old male and female C57BL/6J mice

Journal: Skeletal Muscle

doi: 10.1186/s13395-016-0117-3

Markers of autophagy in the quadriceps muscles of 15-month SED, 23-month SED, and 23-month RWE mice, of both sexes. P-ULK1(Ser757) was quantified relative to t-ULK1 ( a , b ), and t-ULK1 to the loading control GAPDH ( a , c ). Ratios of LC3II/LC3I were detected in the 1% NP40 soluble protein fraction, with GAPDH displayed to demonstrate equal loading ( a , d ). Protein amounts of p62 were quantified in both the 1% NP40 soluble and insoluble fractions, and standardized relative to GAPDH and Ponceau S (stained band between 50 and 37 kDa), respectively ( a , e , f ). Data were analyzed by ANOVA, using age and sex and sex and activity as variables. Data are mean ± SEM. Asterisk denotes significance at * P
Figure Legend Snippet: Markers of autophagy in the quadriceps muscles of 15-month SED, 23-month SED, and 23-month RWE mice, of both sexes. P-ULK1(Ser757) was quantified relative to t-ULK1 ( a , b ), and t-ULK1 to the loading control GAPDH ( a , c ). Ratios of LC3II/LC3I were detected in the 1% NP40 soluble protein fraction, with GAPDH displayed to demonstrate equal loading ( a , d ). Protein amounts of p62 were quantified in both the 1% NP40 soluble and insoluble fractions, and standardized relative to GAPDH and Ponceau S (stained band between 50 and 37 kDa), respectively ( a , e , f ). Data were analyzed by ANOVA, using age and sex and sex and activity as variables. Data are mean ± SEM. Asterisk denotes significance at * P

Techniques Used: Mouse Assay, Staining, Activity Assay

9) Product Images from "Optimization of non-denaturing protein extraction conditions for plant PPR proteins"

Article Title: Optimization of non-denaturing protein extraction conditions for plant PPR proteins

Journal: PLoS ONE

doi: 10.1371/journal.pone.0187753

Optimization of the extraction buffer components for MEF57. Total protein extracts from N . benthamiana leaves infiltrated with SLO2-HA construct together with MEF57-GFP. The extraction buffer was complemented with 50 μM MG132 proteasome inhibitor, 1x phosphatase inhibitor, 5 mM ATP and 1 mM PMSF. The pH, the NP40 detergent and the NaCl content were modified to pH 8, 1% Triton X-100 and 0 mM NaCl (a) . 0.1% SDS, 14 mM β-mercaptoethanol and 10% glycerol were tested (a) . Several concentrations of DTT and β-mercaptoethanol were also tested (b) . Samples were analyzed by western blot, stained with Ponceau and probed with anti-HA and anti-GFP antibodies (HA-Ab and GFP-Ab, respectively). The respective molecular weights are: SLO2-HA, 84.03 kDa; DYW2-GFP, 92.56 kDa; MEF57-GFP, 100.62 kDa; and HSP60.3B-GFP, 87.42 kDa. The Ponceau membrane staining of the most intense band at 55 kDa (presumably Rubisco) was used as a loading control. Full-length blots are shown in S1 Fig . Histograms of GFP/HA-tagged protein, relative to Ponceau, are shown in S2 Fig .
Figure Legend Snippet: Optimization of the extraction buffer components for MEF57. Total protein extracts from N . benthamiana leaves infiltrated with SLO2-HA construct together with MEF57-GFP. The extraction buffer was complemented with 50 μM MG132 proteasome inhibitor, 1x phosphatase inhibitor, 5 mM ATP and 1 mM PMSF. The pH, the NP40 detergent and the NaCl content were modified to pH 8, 1% Triton X-100 and 0 mM NaCl (a) . 0.1% SDS, 14 mM β-mercaptoethanol and 10% glycerol were tested (a) . Several concentrations of DTT and β-mercaptoethanol were also tested (b) . Samples were analyzed by western blot, stained with Ponceau and probed with anti-HA and anti-GFP antibodies (HA-Ab and GFP-Ab, respectively). The respective molecular weights are: SLO2-HA, 84.03 kDa; DYW2-GFP, 92.56 kDa; MEF57-GFP, 100.62 kDa; and HSP60.3B-GFP, 87.42 kDa. The Ponceau membrane staining of the most intense band at 55 kDa (presumably Rubisco) was used as a loading control. Full-length blots are shown in S1 Fig . Histograms of GFP/HA-tagged protein, relative to Ponceau, are shown in S2 Fig .

Techniques Used: Construct, Modification, Western Blot, Staining

10) Product Images from "Intracellular Regulation of Cross-Presentation during Dendritic Cell Maturation"

Article Title: Intracellular Regulation of Cross-Presentation during Dendritic Cell Maturation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0076801

OVA degradation in phagosomes of matured and immature DCs. Immature or mature DCs were pulsed with OVA/BSA coated beads for 5min, washed and separated into two samples. One sample was chased for 1.5h and then stained for BSA while the other sample (t=0) was stained directly with m anti-BSA followed by anti-m A647 to mark non-internalized beads. After BSA staining, cells were lysed in 0.5% NP40. Beads recovered from lysates were stained for intact and degraded OVA with rb anti-OVA followed by anti-rb A488. For samples indicated with “+”, CpG or LPS was added in combination with OVA/BSA beads during pulse. (A) Intensities of OVA fluorescence were detected by flow cytometry, gating on single beads (left panel). Extracellular beads were excluded based on a-m A647 staining (middle panel). At t=0 (after pulse), all samples showed a uniform, non-degraded OVA peak (right panel). (B) After 1.5h chase, control samples chased in the presence of Concanamycin B had reduced OVA degradation (both panels). OVA degradation patterns for all samples are shown in the right panel, the respective MFI is graphed in (C). One representative of three is shown.
Figure Legend Snippet: OVA degradation in phagosomes of matured and immature DCs. Immature or mature DCs were pulsed with OVA/BSA coated beads for 5min, washed and separated into two samples. One sample was chased for 1.5h and then stained for BSA while the other sample (t=0) was stained directly with m anti-BSA followed by anti-m A647 to mark non-internalized beads. After BSA staining, cells were lysed in 0.5% NP40. Beads recovered from lysates were stained for intact and degraded OVA with rb anti-OVA followed by anti-rb A488. For samples indicated with “+”, CpG or LPS was added in combination with OVA/BSA beads during pulse. (A) Intensities of OVA fluorescence were detected by flow cytometry, gating on single beads (left panel). Extracellular beads were excluded based on a-m A647 staining (middle panel). At t=0 (after pulse), all samples showed a uniform, non-degraded OVA peak (right panel). (B) After 1.5h chase, control samples chased in the presence of Concanamycin B had reduced OVA degradation (both panels). OVA degradation patterns for all samples are shown in the right panel, the respective MFI is graphed in (C). One representative of three is shown.

Techniques Used: Staining, Fluorescence, Flow Cytometry, Cytometry

11) Product Images from "The temporal program of peripheral blood gene expression in the response of nonhuman primates to Ebola hemorrhagic fever"

Article Title: The temporal program of peripheral blood gene expression in the response of nonhuman primates to Ebola hemorrhagic fever

Journal: Genome Biology

doi: 10.1186/gb-2007-8-8-r174

Expression levels of the metalloprotease responsible for cleavage of Ebola glycoprotein. Shown are (a) expression levels of tumor necrosis factor (TNF)-α converting enzyme/α-disintegrin and metalloproteinase (ADAM)-17 from the overview cluster and (b) in graph form. (c) Glycoprotein (GP) in the serum from infected rhesus macaques over the course of infection. Serum was diluted 1:3 in NP40 lysis buffer. Samples were run on a 10% Bis-Tris gel under reducing conditions, as shown. Mock cell lysate from 293T cells transfected with vector only (pDisplay) is shown as a negative control (lane 1); Zaire Ebola virus (ZEBOV; lane 2) is supernatant from in vitro Ebola infected Vero E6 cells at day 8 after infection. Lanes 3 and 4 are transfection controls expressing glycoprotein (GP) 1,2 (cell lysate) and GP 1,2Δ supernatant) [31]. Serum from infected rhesus macaques, before infection (lane 5), and on day 4 and 6 after infection (lanes 6 to 9) were diluted 1:3 in NP40 lysis buffer and 22.5 μl was loaded per lane. Samples included two animals per day (after infection) analyzed. Note the lack of GP in the prebleed control sample (lane 5). GP 2Δ is seen in the transfection control (lane 4) and NHP sera samples from days 4 (lane 6, albeit weakly) and 6 days after infection (lanes 8 and 9).
Figure Legend Snippet: Expression levels of the metalloprotease responsible for cleavage of Ebola glycoprotein. Shown are (a) expression levels of tumor necrosis factor (TNF)-α converting enzyme/α-disintegrin and metalloproteinase (ADAM)-17 from the overview cluster and (b) in graph form. (c) Glycoprotein (GP) in the serum from infected rhesus macaques over the course of infection. Serum was diluted 1:3 in NP40 lysis buffer. Samples were run on a 10% Bis-Tris gel under reducing conditions, as shown. Mock cell lysate from 293T cells transfected with vector only (pDisplay) is shown as a negative control (lane 1); Zaire Ebola virus (ZEBOV; lane 2) is supernatant from in vitro Ebola infected Vero E6 cells at day 8 after infection. Lanes 3 and 4 are transfection controls expressing glycoprotein (GP) 1,2 (cell lysate) and GP 1,2Δ supernatant) [31]. Serum from infected rhesus macaques, before infection (lane 5), and on day 4 and 6 after infection (lanes 6 to 9) were diluted 1:3 in NP40 lysis buffer and 22.5 μl was loaded per lane. Samples included two animals per day (after infection) analyzed. Note the lack of GP in the prebleed control sample (lane 5). GP 2Δ is seen in the transfection control (lane 4) and NHP sera samples from days 4 (lane 6, albeit weakly) and 6 days after infection (lanes 8 and 9).

Techniques Used: Expressing, Infection, Lysis, Transfection, Plasmid Preparation, Negative Control, In Vitro

12) Product Images from "CHOP-independent apoptosis and pathway-selective induction of the UPR in developing plasma cells"

Article Title: CHOP-independent apoptosis and pathway-selective induction of the UPR in developing plasma cells

Journal: Molecular immunology

doi: 10.1016/j.molimm.2009.12.003

CHOP promotes optimal assembly of IgM A) IgM polymerization was analyzed by non-reducing western blots. Total protein extracts from wt and chop -/- B cells after 4 days of LPS stimulation were subjected to SDS-PAGE in non-reducing conditions and blotted. The nitrocellulose filter was incubated with anti-μ antibodies and the main IgM assembly intermediates were identified. Immuno-decoration with anti-κ or anti-J antibodies (not shown) confirmed the identification of the assembly intermediates. B) Quantification of the relative amount of each IgM species in chop -/- ASC with respect to the wt. The percentage of IgM species (HMW complexes, polymers, μ 2 L 2 , μL, μ) relative to the sum of all the species was calculated after densitometric analyses of non-reducing western blots as the one shown in A. The histogram reports the ratios between each IgM species in chop -/- and wt samples as determined in four independent experiments (n=4; average ± SEM). C) Wt and chop -/- B splenocytes, stimulated in vitro with LPS (day 3), were pulsed for 10 min with 35 S-labeled aminoacids and chased for 0 or 4 hours. NP40 soluble (s) and insoluble (i) fractions were immunoprecipitated with anti-μ and resolved by SDS-PAGE under non-reducing conditions. More HMW complexes are found in the insoluble fraction of chop -/- cells (compare lanes 7 and 3). D) Lysates from wt and chop -/- B splenocytes, stimulated in vitro with LPS for 4 days, were centrifuged on continuous sucrose gradients. 39 fractions were collected and aliquots analyzed by dot-blot assays with anti-μ antibodies. E) Individual or pooled fractions of the gradient shown in D, were resolved by SDS-PAGE under non-reducing conditions and blots immuno-decorated with anti-μ to highlight the main assembly intermediates. An enhancement of the two lanes corresponding to fractions 27-39 is shown on the right (Enhanced Pixel Intensity, EPI) to better appreciate the presence of HMW complex. Note that in chop -/- cells HMW complexes (and also some polymers and μ 2 L 2 ) are more abundant in fractions 27-39 than in control splenocytes.
Figure Legend Snippet: CHOP promotes optimal assembly of IgM A) IgM polymerization was analyzed by non-reducing western blots. Total protein extracts from wt and chop -/- B cells after 4 days of LPS stimulation were subjected to SDS-PAGE in non-reducing conditions and blotted. The nitrocellulose filter was incubated with anti-μ antibodies and the main IgM assembly intermediates were identified. Immuno-decoration with anti-κ or anti-J antibodies (not shown) confirmed the identification of the assembly intermediates. B) Quantification of the relative amount of each IgM species in chop -/- ASC with respect to the wt. The percentage of IgM species (HMW complexes, polymers, μ 2 L 2 , μL, μ) relative to the sum of all the species was calculated after densitometric analyses of non-reducing western blots as the one shown in A. The histogram reports the ratios between each IgM species in chop -/- and wt samples as determined in four independent experiments (n=4; average ± SEM). C) Wt and chop -/- B splenocytes, stimulated in vitro with LPS (day 3), were pulsed for 10 min with 35 S-labeled aminoacids and chased for 0 or 4 hours. NP40 soluble (s) and insoluble (i) fractions were immunoprecipitated with anti-μ and resolved by SDS-PAGE under non-reducing conditions. More HMW complexes are found in the insoluble fraction of chop -/- cells (compare lanes 7 and 3). D) Lysates from wt and chop -/- B splenocytes, stimulated in vitro with LPS for 4 days, were centrifuged on continuous sucrose gradients. 39 fractions were collected and aliquots analyzed by dot-blot assays with anti-μ antibodies. E) Individual or pooled fractions of the gradient shown in D, were resolved by SDS-PAGE under non-reducing conditions and blots immuno-decorated with anti-μ to highlight the main assembly intermediates. An enhancement of the two lanes corresponding to fractions 27-39 is shown on the right (Enhanced Pixel Intensity, EPI) to better appreciate the presence of HMW complex. Note that in chop -/- cells HMW complexes (and also some polymers and μ 2 L 2 ) are more abundant in fractions 27-39 than in control splenocytes.

Techniques Used: Western Blot, SDS Page, Incubation, In Vitro, Labeling, Immunoprecipitation, Dot Blot

13) Product Images from "E1^E4-mediated keratin phosphorylation and ubiquitylation: a mechanism for keratin depletion in HPV16-infected epithelium"

Article Title: E1^E4-mediated keratin phosphorylation and ubiquitylation: a mechanism for keratin depletion in HPV16-infected epithelium

Journal: Journal of Cell Science

doi: 10.1242/jcs.061978

Ubiquitylated keratin associates with 16E1^E4 aggregates and its accumulation results in proteasomal inhibition ( A ) SiHa cells infected to express 16E1^E4 were immunostained for 16E1^E4 (green) and ubiquitin (red); DAPI stain is blue. The merged images clearly show that 16E1^E4-keratin aggregate structures co-localise with ubiquitin (arrows). Scale bars: 5 μm. ( B ) Fractionation of 16E1^E4 and keratin from SiHa cells expressing 16E1^E4 using 1% NP40 (N), 1% empigen (E) and 9M urea (U). The high molecular weight keratin species, which accumulate in the presence of E1^E4, fractionate exclusively in the insoluble fraction. ( C ) SiHa cells were infected to express 16E1^E4 or the keratin-binding mutant ΔLLKLL and harvested at the times shown. Cell lysates were probed to detect proteins as indicated. Accumulation of 16E1^E4 coincides with accumulation of K18 ladders and ubiquitylated protein. Accumulation of ubiquitylated protein is not observed in cells expressing the keratin binding mutant, indicating that 16E1^E4-keratin association results in an impairment of proteasomal function.
Figure Legend Snippet: Ubiquitylated keratin associates with 16E1^E4 aggregates and its accumulation results in proteasomal inhibition ( A ) SiHa cells infected to express 16E1^E4 were immunostained for 16E1^E4 (green) and ubiquitin (red); DAPI stain is blue. The merged images clearly show that 16E1^E4-keratin aggregate structures co-localise with ubiquitin (arrows). Scale bars: 5 μm. ( B ) Fractionation of 16E1^E4 and keratin from SiHa cells expressing 16E1^E4 using 1% NP40 (N), 1% empigen (E) and 9M urea (U). The high molecular weight keratin species, which accumulate in the presence of E1^E4, fractionate exclusively in the insoluble fraction. ( C ) SiHa cells were infected to express 16E1^E4 or the keratin-binding mutant ΔLLKLL and harvested at the times shown. Cell lysates were probed to detect proteins as indicated. Accumulation of 16E1^E4 coincides with accumulation of K18 ladders and ubiquitylated protein. Accumulation of ubiquitylated protein is not observed in cells expressing the keratin binding mutant, indicating that 16E1^E4-keratin association results in an impairment of proteasomal function.

Techniques Used: Inhibition, Infection, Staining, Fractionation, Expressing, Molecular Weight, Binding Assay, Mutagenesis

14) Product Images from "ERG-associated protein with SET domain (ESET)-Oct4 interaction regulates pluripotency and represses the trophectoderm lineage"

Article Title: ERG-associated protein with SET domain (ESET)-Oct4 interaction regulates pluripotency and represses the trophectoderm lineage

Journal: Epigenetics & Chromatin

doi: 10.1186/1756-8935-2-12

Delocalisation of ERG-associated protein with SET domain (ESET) from promyelocytic leukaemia (PML) nuclear bodies promotes the interaction of small ubiquitin-related modifier (SUMO)ylated ESET and Oct4 . (a) In PML (green)-depleted embryonic stem (ES) cells (white border), ESET (red) punctate foci were delocalised from PML nuclear bodies. Nuclei are labelled in blue. Scale bar, 3 μm. Cultures also contain cells which were not depleted of PML, where ESET exhibit punctate foci (arrow heads) associated with PML bodies. (b) ES cells transfected with Pml short hairpin RNA (shRNA) for 4 days were immunoprecipitated (IP) with anti-ESET antibody (kind gift of HH Ng; see text) under mild conditions in buffer containing digitonin and ethidium bromide, and were subjected to western blotting (WB) with the antibodies indicated. Note that more Oct4 is precipitated by ESET in Pml knockdown ES cells (*lane 4, top panel). Asterisk (lane 3, middle panel) indicates PML being precipitated by ESET in control cells transfected with an empty vector but not in Pml knockdown ES cells (compare with lane 4, middle panel). (c) ES cells transfected with Pml shRNA for 4 days were immunoprecipitated (IP) with anti-Oct4 antibody in buffer containing NP40, N -ethylmaleimide, and ethidium bromide and subjected to WB using 4% to 15% Tris-HCl gradient gel. More SUMOylated ESET was precipitated by Oct4 in two independent experiments (Exp 1 and Exp 2) in Pml knockdown cells as indicated (*; lane 4 and 6, top panel).
Figure Legend Snippet: Delocalisation of ERG-associated protein with SET domain (ESET) from promyelocytic leukaemia (PML) nuclear bodies promotes the interaction of small ubiquitin-related modifier (SUMO)ylated ESET and Oct4 . (a) In PML (green)-depleted embryonic stem (ES) cells (white border), ESET (red) punctate foci were delocalised from PML nuclear bodies. Nuclei are labelled in blue. Scale bar, 3 μm. Cultures also contain cells which were not depleted of PML, where ESET exhibit punctate foci (arrow heads) associated with PML bodies. (b) ES cells transfected with Pml short hairpin RNA (shRNA) for 4 days were immunoprecipitated (IP) with anti-ESET antibody (kind gift of HH Ng; see text) under mild conditions in buffer containing digitonin and ethidium bromide, and were subjected to western blotting (WB) with the antibodies indicated. Note that more Oct4 is precipitated by ESET in Pml knockdown ES cells (*lane 4, top panel). Asterisk (lane 3, middle panel) indicates PML being precipitated by ESET in control cells transfected with an empty vector but not in Pml knockdown ES cells (compare with lane 4, middle panel). (c) ES cells transfected with Pml shRNA for 4 days were immunoprecipitated (IP) with anti-Oct4 antibody in buffer containing NP40, N -ethylmaleimide, and ethidium bromide and subjected to WB using 4% to 15% Tris-HCl gradient gel. More SUMOylated ESET was precipitated by Oct4 in two independent experiments (Exp 1 and Exp 2) in Pml knockdown cells as indicated (*; lane 4 and 6, top panel).

Techniques Used: Transfection, shRNA, Immunoprecipitation, Western Blot, Plasmid Preparation

ERG-associated protein with SET domain (ESET) localises to promyelocytic leukaemia (PML) nuclear bodies and is post-translationally modified by small ubiquitin-related modifier (SUMO) . (a) ESET (red) localises to distinct, punctate foci in euchromatin regions, as indicated by the absence of DAPI (blue) staining in embryonic stem (ES) cells. Scale bar, 3 μm. (b) ESET (red) localises to distinct, punctate foci that overlaps with PML nuclear bodies (green) in ES cells. Nuclei are labelled in blue. Scale bar, 5 μm. (c) ES cell lysates were immunoprecipitated (IP) with anti-ESET antibody (kind gift of HH Ng; see text) under mild conditions in digitonin-containing buffer and subjected to western blotting (WB) with the antibodies indicated. Rabbit IgG was used as a negative control. (d) Western blot analysis of coimmunoprecipitation experiment in 293T cells transfected with haemagglutinin (HA)-ESET and/or Flag-SUMO-1. S-ESET represents SUMOylated ESET. (e) ES cell lysates were immunoprecipitated (IP) with the indicated antibodies in NP40-containing buffer either in the presence or absence of N -ethylmaleimide (NEM) and subjected to western blotting (WB) using 4% to 15% Tris-HCl gradient gel. A rabbit anti-HA antibody was used as negative control. (f) Quantitative polymerase chain reaction (Q-PCR) analysis of the enrichment of SUMO-1 on different positions along Cdx2 promoter relative to the least enriched region (C5) after normalising against their respective input and IgG controls. Error bars, standard deviation (SD) of three technical replicates.
Figure Legend Snippet: ERG-associated protein with SET domain (ESET) localises to promyelocytic leukaemia (PML) nuclear bodies and is post-translationally modified by small ubiquitin-related modifier (SUMO) . (a) ESET (red) localises to distinct, punctate foci in euchromatin regions, as indicated by the absence of DAPI (blue) staining in embryonic stem (ES) cells. Scale bar, 3 μm. (b) ESET (red) localises to distinct, punctate foci that overlaps with PML nuclear bodies (green) in ES cells. Nuclei are labelled in blue. Scale bar, 5 μm. (c) ES cell lysates were immunoprecipitated (IP) with anti-ESET antibody (kind gift of HH Ng; see text) under mild conditions in digitonin-containing buffer and subjected to western blotting (WB) with the antibodies indicated. Rabbit IgG was used as a negative control. (d) Western blot analysis of coimmunoprecipitation experiment in 293T cells transfected with haemagglutinin (HA)-ESET and/or Flag-SUMO-1. S-ESET represents SUMOylated ESET. (e) ES cell lysates were immunoprecipitated (IP) with the indicated antibodies in NP40-containing buffer either in the presence or absence of N -ethylmaleimide (NEM) and subjected to western blotting (WB) using 4% to 15% Tris-HCl gradient gel. A rabbit anti-HA antibody was used as negative control. (f) Quantitative polymerase chain reaction (Q-PCR) analysis of the enrichment of SUMO-1 on different positions along Cdx2 promoter relative to the least enriched region (C5) after normalising against their respective input and IgG controls. Error bars, standard deviation (SD) of three technical replicates.

Techniques Used: Modification, Staining, Immunoprecipitation, Western Blot, Negative Control, Transfection, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Standard Deviation

15) Product Images from "A novel Leishmania infantum nuclear phosphoprotein Lepp12 which stimulates IL1-beta synthesis in THP-1 transfectants"

Article Title: A novel Leishmania infantum nuclear phosphoprotein Lepp12 which stimulates IL1-beta synthesis in THP-1 transfectants

Journal: BMC Microbiology

doi: 10.1186/1471-2180-3-7

Natural Lepp12 protein is non-secreted protein located in promastigote nucleus. Western blotting was performed using immunopurified anti-Lepp12 antibodies (at 2 microg/ml) on total promastigote 3 % SDS lysate (positive control, lane 1), 20 % NP40 lysate (lane 2), cytosol (s1, lane 3), cell membranes (c1, lane 4), nuclear extract prepared as described in Methods section before (lane 5) or after (lane 6) 3% SDS lysis, and PEG concentrated culture supernatant (lane 7). Proteins were prepared from 1.6 × 10 8 cells. In each lane, 30 microg of proteins were separated on a 14% polyacrylamide SDS-gel, corresponding to an equivalent of 4 millions (lane 1), 8.6 millions (lane 2), 5.3 millions (lane 3), 4.8 millions (lane 4), 18.5 millions (lane 5), 12 millions (lane 6) and 2.2 millions (lane 7) promastigotes. Molecular mass markers are indicated in kDa.
Figure Legend Snippet: Natural Lepp12 protein is non-secreted protein located in promastigote nucleus. Western blotting was performed using immunopurified anti-Lepp12 antibodies (at 2 microg/ml) on total promastigote 3 % SDS lysate (positive control, lane 1), 20 % NP40 lysate (lane 2), cytosol (s1, lane 3), cell membranes (c1, lane 4), nuclear extract prepared as described in Methods section before (lane 5) or after (lane 6) 3% SDS lysis, and PEG concentrated culture supernatant (lane 7). Proteins were prepared from 1.6 × 10 8 cells. In each lane, 30 microg of proteins were separated on a 14% polyacrylamide SDS-gel, corresponding to an equivalent of 4 millions (lane 1), 8.6 millions (lane 2), 5.3 millions (lane 3), 4.8 millions (lane 4), 18.5 millions (lane 5), 12 millions (lane 6) and 2.2 millions (lane 7) promastigotes. Molecular mass markers are indicated in kDa.

Techniques Used: Western Blot, Positive Control, Lysis, SDS-Gel

16) Product Images from "Angiopoietin-like 4 promotes intracellular degradation of lipoprotein lipase in adipocytes"

Article Title: Angiopoietin-like 4 promotes intracellular degradation of lipoprotein lipase in adipocytes

Journal: Journal of Lipid Research

doi: 10.1194/jlr.M067363

ANGPTL4-mediated loss of LPL protein occurs in a post-ER compartment. A: Western blot of EndoH-treated cell lysates of adipocytes that had been differentiated from stromal vascular fractions of WAT from Angptl4 −/− and wild-type mice. Cells were treated with 5 μg/ml brefeldin A for 2 h. Western blots were probed with antibodies against LPL and HSP90 (as a loading control). B: Western blot of cell lysates of adipocytes that had been differentiated from stromal vascular fractions from WAT of Angptl4 −/− and wild-type mice. Cells were lysed in NP-40 lysis buffer. Western blots were probed with antibodies against LPL, HSP90 (as a loading control), and H2A (as a loading control). NP40S, NP40-soluble LPL; NP40P, NP40-precipitated LPL; s.e., short exposure; l.e., long exposure. C: Western blot of EndoH-treated cell lysates of adipocytes that had been differentiated from the stromal vascular fractions of WAT from Angptl4 −/− and wild-type mice. Cells were treated with 10 μM monensin (Mon.) for 3 h. Western blots were probed with antibodies against LPL and HSP90 (as a loading control). D: Western blot of EndoH-treated cell lysates from adipocytes that had been differentiated from stromal vascular fractions of WAT from Angptl4 −/− and wild-type mice. The cells were treated with 20 μM E64D for 24 h. Western blots were probed with antibodies against LPL and HSP90 (as a loading control). E: Western blot of EndoH-treated cell lysates of adipocytes that had been differentiated from stromal vascular fractions of WAT from Angptl4 −/− and wild-type mice. Cells were treated with 5 mM 3MA for 10 h. Western blots were probed with antibodies against LPL and HSP90 (as a loading control). F: Western blot of EndoH-treated cell lysates of adipocytes that had been differentiated from stromal vascular fractions from WAT of Angptl4 −/− and wild-type mice. The cells had been treated with 10 mM leupeptin (Leup.) for 10 h. Western blots were probed with antibodies against LPL and HSP90 (as a loading control). EndoH-resistant LPL (high-mannose oligosaccharides, ER LPL) is indicated with R; EndoH-sensitive LPL (complex oligosaccharides; Golgi and cell surface LPL) is indicated with S.
Figure Legend Snippet: ANGPTL4-mediated loss of LPL protein occurs in a post-ER compartment. A: Western blot of EndoH-treated cell lysates of adipocytes that had been differentiated from stromal vascular fractions of WAT from Angptl4 −/− and wild-type mice. Cells were treated with 5 μg/ml brefeldin A for 2 h. Western blots were probed with antibodies against LPL and HSP90 (as a loading control). B: Western blot of cell lysates of adipocytes that had been differentiated from stromal vascular fractions from WAT of Angptl4 −/− and wild-type mice. Cells were lysed in NP-40 lysis buffer. Western blots were probed with antibodies against LPL, HSP90 (as a loading control), and H2A (as a loading control). NP40S, NP40-soluble LPL; NP40P, NP40-precipitated LPL; s.e., short exposure; l.e., long exposure. C: Western blot of EndoH-treated cell lysates of adipocytes that had been differentiated from the stromal vascular fractions of WAT from Angptl4 −/− and wild-type mice. Cells were treated with 10 μM monensin (Mon.) for 3 h. Western blots were probed with antibodies against LPL and HSP90 (as a loading control). D: Western blot of EndoH-treated cell lysates from adipocytes that had been differentiated from stromal vascular fractions of WAT from Angptl4 −/− and wild-type mice. The cells were treated with 20 μM E64D for 24 h. Western blots were probed with antibodies against LPL and HSP90 (as a loading control). E: Western blot of EndoH-treated cell lysates of adipocytes that had been differentiated from stromal vascular fractions of WAT from Angptl4 −/− and wild-type mice. Cells were treated with 5 mM 3MA for 10 h. Western blots were probed with antibodies against LPL and HSP90 (as a loading control). F: Western blot of EndoH-treated cell lysates of adipocytes that had been differentiated from stromal vascular fractions from WAT of Angptl4 −/− and wild-type mice. The cells had been treated with 10 mM leupeptin (Leup.) for 10 h. Western blots were probed with antibodies against LPL and HSP90 (as a loading control). EndoH-resistant LPL (high-mannose oligosaccharides, ER LPL) is indicated with R; EndoH-sensitive LPL (complex oligosaccharides; Golgi and cell surface LPL) is indicated with S.

Techniques Used: Western Blot, Mouse Assay, Lysis

17) Product Images from "ERG-associated protein with SET domain (ESET)-Oct4 interaction regulates pluripotency and represses the trophectoderm lineage"

Article Title: ERG-associated protein with SET domain (ESET)-Oct4 interaction regulates pluripotency and represses the trophectoderm lineage

Journal: Epigenetics & Chromatin

doi: 10.1186/1756-8935-2-12

Delocalisation of ERG-associated protein with SET domain (ESET) from promyelocytic leukaemia (PML) nuclear bodies promotes the interaction of small ubiquitin-related modifier (SUMO)ylated ESET and Oct4 . (a) In PML (green)-depleted embryonic stem (ES) cells (white border), ESET (red) punctate foci were delocalised from PML nuclear bodies. Nuclei are labelled in blue. Scale bar, 3 μm. Cultures also contain cells which were not depleted of PML, where ESET exhibit punctate foci (arrow heads) associated with PML bodies. (b) ES cells transfected with Pml short hairpin RNA (shRNA) for 4 days were immunoprecipitated (IP) with anti-ESET antibody (kind gift of HH Ng; see text) under mild conditions in buffer containing digitonin and ethidium bromide, and were subjected to western blotting (WB) with the antibodies indicated. Note that more Oct4 is precipitated by ESET in Pml knockdown ES cells (*lane 4, top panel). Asterisk (lane 3, middle panel) indicates PML being precipitated by ESET in control cells transfected with an empty vector but not in Pml knockdown ES cells (compare with lane 4, middle panel). (c) ES cells transfected with Pml shRNA for 4 days were immunoprecipitated (IP) with anti-Oct4 antibody in buffer containing NP40, N -ethylmaleimide, and ethidium bromide and subjected to WB using 4% to 15% Tris-HCl gradient gel. More SUMOylated ESET was precipitated by Oct4 in two independent experiments (Exp 1 and Exp 2) in Pml knockdown cells as indicated (*; lane 4 and 6, top panel).
Figure Legend Snippet: Delocalisation of ERG-associated protein with SET domain (ESET) from promyelocytic leukaemia (PML) nuclear bodies promotes the interaction of small ubiquitin-related modifier (SUMO)ylated ESET and Oct4 . (a) In PML (green)-depleted embryonic stem (ES) cells (white border), ESET (red) punctate foci were delocalised from PML nuclear bodies. Nuclei are labelled in blue. Scale bar, 3 μm. Cultures also contain cells which were not depleted of PML, where ESET exhibit punctate foci (arrow heads) associated with PML bodies. (b) ES cells transfected with Pml short hairpin RNA (shRNA) for 4 days were immunoprecipitated (IP) with anti-ESET antibody (kind gift of HH Ng; see text) under mild conditions in buffer containing digitonin and ethidium bromide, and were subjected to western blotting (WB) with the antibodies indicated. Note that more Oct4 is precipitated by ESET in Pml knockdown ES cells (*lane 4, top panel). Asterisk (lane 3, middle panel) indicates PML being precipitated by ESET in control cells transfected with an empty vector but not in Pml knockdown ES cells (compare with lane 4, middle panel). (c) ES cells transfected with Pml shRNA for 4 days were immunoprecipitated (IP) with anti-Oct4 antibody in buffer containing NP40, N -ethylmaleimide, and ethidium bromide and subjected to WB using 4% to 15% Tris-HCl gradient gel. More SUMOylated ESET was precipitated by Oct4 in two independent experiments (Exp 1 and Exp 2) in Pml knockdown cells as indicated (*; lane 4 and 6, top panel).

Techniques Used: Transfection, shRNA, Immunoprecipitation, Western Blot, Plasmid Preparation

ERG-associated protein with SET domain (ESET) localises to promyelocytic leukaemia (PML) nuclear bodies and is post-translationally modified by small ubiquitin-related modifier (SUMO) . (a) ESET (red) localises to distinct, punctate foci in euchromatin regions, as indicated by the absence of DAPI (blue) staining in embryonic stem (ES) cells. Scale bar, 3 μm. (b) ESET (red) localises to distinct, punctate foci that overlaps with PML nuclear bodies (green) in ES cells. Nuclei are labelled in blue. Scale bar, 5 μm. (c) ES cell lysates were immunoprecipitated (IP) with anti-ESET antibody (kind gift of HH Ng; see text) under mild conditions in digitonin-containing buffer and subjected to western blotting (WB) with the antibodies indicated. Rabbit IgG was used as a negative control. (d) Western blot analysis of coimmunoprecipitation experiment in 293T cells transfected with haemagglutinin (HA)-ESET and/or Flag-SUMO-1. S-ESET represents SUMOylated ESET. (e) ES cell lysates were immunoprecipitated (IP) with the indicated antibodies in NP40-containing buffer either in the presence or absence of N -ethylmaleimide (NEM) and subjected to western blotting (WB) using 4% to 15% Tris-HCl gradient gel. A rabbit anti-HA antibody was used as negative control. (f) Quantitative polymerase chain reaction (Q-PCR) analysis of the enrichment of SUMO-1 on different positions along Cdx2 promoter relative to the least enriched region (C5) after normalising against their respective input and IgG controls. Error bars, standard deviation (SD) of three technical replicates.
Figure Legend Snippet: ERG-associated protein with SET domain (ESET) localises to promyelocytic leukaemia (PML) nuclear bodies and is post-translationally modified by small ubiquitin-related modifier (SUMO) . (a) ESET (red) localises to distinct, punctate foci in euchromatin regions, as indicated by the absence of DAPI (blue) staining in embryonic stem (ES) cells. Scale bar, 3 μm. (b) ESET (red) localises to distinct, punctate foci that overlaps with PML nuclear bodies (green) in ES cells. Nuclei are labelled in blue. Scale bar, 5 μm. (c) ES cell lysates were immunoprecipitated (IP) with anti-ESET antibody (kind gift of HH Ng; see text) under mild conditions in digitonin-containing buffer and subjected to western blotting (WB) with the antibodies indicated. Rabbit IgG was used as a negative control. (d) Western blot analysis of coimmunoprecipitation experiment in 293T cells transfected with haemagglutinin (HA)-ESET and/or Flag-SUMO-1. S-ESET represents SUMOylated ESET. (e) ES cell lysates were immunoprecipitated (IP) with the indicated antibodies in NP40-containing buffer either in the presence or absence of N -ethylmaleimide (NEM) and subjected to western blotting (WB) using 4% to 15% Tris-HCl gradient gel. A rabbit anti-HA antibody was used as negative control. (f) Quantitative polymerase chain reaction (Q-PCR) analysis of the enrichment of SUMO-1 on different positions along Cdx2 promoter relative to the least enriched region (C5) after normalising against their respective input and IgG controls. Error bars, standard deviation (SD) of three technical replicates.

Techniques Used: Modification, Staining, Immunoprecipitation, Western Blot, Negative Control, Transfection, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Standard Deviation

18) Product Images from "ERG-associated protein with SET domain (ESET)-Oct4 interaction regulates pluripotency and represses the trophectoderm lineage"

Article Title: ERG-associated protein with SET domain (ESET)-Oct4 interaction regulates pluripotency and represses the trophectoderm lineage

Journal: Epigenetics & Chromatin

doi: 10.1186/1756-8935-2-12

Delocalisation of ERG-associated protein with SET domain (ESET) from promyelocytic leukaemia (PML) nuclear bodies promotes the interaction of small ubiquitin-related modifier (SUMO)ylated ESET and Oct4 . (a) In PML (green)-depleted embryonic stem (ES) cells (white border), ESET (red) punctate foci were delocalised from PML nuclear bodies. Nuclei are labelled in blue. Scale bar, 3 μm. Cultures also contain cells which were not depleted of PML, where ESET exhibit punctate foci (arrow heads) associated with PML bodies. (b) ES cells transfected with Pml short hairpin RNA (shRNA) for 4 days were immunoprecipitated (IP) with anti-ESET antibody (kind gift of HH Ng; see text) under mild conditions in buffer containing digitonin and ethidium bromide, and were subjected to western blotting (WB) with the antibodies indicated. Note that more Oct4 is precipitated by ESET in Pml knockdown ES cells (*lane 4, top panel). Asterisk (lane 3, middle panel) indicates PML being precipitated by ESET in control cells transfected with an empty vector but not in Pml knockdown ES cells (compare with lane 4, middle panel). (c) ES cells transfected with Pml shRNA for 4 days were immunoprecipitated (IP) with anti-Oct4 antibody in buffer containing NP40, N -ethylmaleimide, and ethidium bromide and subjected to WB using 4% to 15% Tris-HCl gradient gel. More SUMOylated ESET was precipitated by Oct4 in two independent experiments (Exp 1 and Exp 2) in Pml knockdown cells as indicated (*; lane 4 and 6, top panel).
Figure Legend Snippet: Delocalisation of ERG-associated protein with SET domain (ESET) from promyelocytic leukaemia (PML) nuclear bodies promotes the interaction of small ubiquitin-related modifier (SUMO)ylated ESET and Oct4 . (a) In PML (green)-depleted embryonic stem (ES) cells (white border), ESET (red) punctate foci were delocalised from PML nuclear bodies. Nuclei are labelled in blue. Scale bar, 3 μm. Cultures also contain cells which were not depleted of PML, where ESET exhibit punctate foci (arrow heads) associated with PML bodies. (b) ES cells transfected with Pml short hairpin RNA (shRNA) for 4 days were immunoprecipitated (IP) with anti-ESET antibody (kind gift of HH Ng; see text) under mild conditions in buffer containing digitonin and ethidium bromide, and were subjected to western blotting (WB) with the antibodies indicated. Note that more Oct4 is precipitated by ESET in Pml knockdown ES cells (*lane 4, top panel). Asterisk (lane 3, middle panel) indicates PML being precipitated by ESET in control cells transfected with an empty vector but not in Pml knockdown ES cells (compare with lane 4, middle panel). (c) ES cells transfected with Pml shRNA for 4 days were immunoprecipitated (IP) with anti-Oct4 antibody in buffer containing NP40, N -ethylmaleimide, and ethidium bromide and subjected to WB using 4% to 15% Tris-HCl gradient gel. More SUMOylated ESET was precipitated by Oct4 in two independent experiments (Exp 1 and Exp 2) in Pml knockdown cells as indicated (*; lane 4 and 6, top panel).

Techniques Used: Transfection, shRNA, Immunoprecipitation, Western Blot, Plasmid Preparation

ERG-associated protein with SET domain (ESET) localises to promyelocytic leukaemia (PML) nuclear bodies and is post-translationally modified by small ubiquitin-related modifier (SUMO) . (a) ESET (red) localises to distinct, punctate foci in euchromatin regions, as indicated by the absence of DAPI (blue) staining in embryonic stem (ES) cells. Scale bar, 3 μm. (b) ESET (red) localises to distinct, punctate foci that overlaps with PML nuclear bodies (green) in ES cells. Nuclei are labelled in blue. Scale bar, 5 μm. (c) ES cell lysates were immunoprecipitated (IP) with anti-ESET antibody (kind gift of HH Ng; see text) under mild conditions in digitonin-containing buffer and subjected to western blotting (WB) with the antibodies indicated. Rabbit IgG was used as a negative control. (d) Western blot analysis of coimmunoprecipitation experiment in 293T cells transfected with haemagglutinin (HA)-ESET and/or Flag-SUMO-1. S-ESET represents SUMOylated ESET. (e) ES cell lysates were immunoprecipitated (IP) with the indicated antibodies in NP40-containing buffer either in the presence or absence of N -ethylmaleimide (NEM) and subjected to western blotting (WB) using 4% to 15% Tris-HCl gradient gel. A rabbit anti-HA antibody was used as negative control. (f) Quantitative polymerase chain reaction (Q-PCR) analysis of the enrichment of SUMO-1 on different positions along Cdx2 promoter relative to the least enriched region (C5) after normalising against their respective input and IgG controls. Error bars, standard deviation (SD) of three technical replicates.
Figure Legend Snippet: ERG-associated protein with SET domain (ESET) localises to promyelocytic leukaemia (PML) nuclear bodies and is post-translationally modified by small ubiquitin-related modifier (SUMO) . (a) ESET (red) localises to distinct, punctate foci in euchromatin regions, as indicated by the absence of DAPI (blue) staining in embryonic stem (ES) cells. Scale bar, 3 μm. (b) ESET (red) localises to distinct, punctate foci that overlaps with PML nuclear bodies (green) in ES cells. Nuclei are labelled in blue. Scale bar, 5 μm. (c) ES cell lysates were immunoprecipitated (IP) with anti-ESET antibody (kind gift of HH Ng; see text) under mild conditions in digitonin-containing buffer and subjected to western blotting (WB) with the antibodies indicated. Rabbit IgG was used as a negative control. (d) Western blot analysis of coimmunoprecipitation experiment in 293T cells transfected with haemagglutinin (HA)-ESET and/or Flag-SUMO-1. S-ESET represents SUMOylated ESET. (e) ES cell lysates were immunoprecipitated (IP) with the indicated antibodies in NP40-containing buffer either in the presence or absence of N -ethylmaleimide (NEM) and subjected to western blotting (WB) using 4% to 15% Tris-HCl gradient gel. A rabbit anti-HA antibody was used as negative control. (f) Quantitative polymerase chain reaction (Q-PCR) analysis of the enrichment of SUMO-1 on different positions along Cdx2 promoter relative to the least enriched region (C5) after normalising against their respective input and IgG controls. Error bars, standard deviation (SD) of three technical replicates.

Techniques Used: Modification, Staining, Immunoprecipitation, Western Blot, Negative Control, Transfection, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Standard Deviation

Related Articles

Centrifugation:

Article Title: C-Terminal Helical Domains of Dengue Virus Type 4 E Protein Affect the Expression/Stability of prM Protein and Conformation of prM and E Proteins
Article Snippet: .. At 48 h post-transfection, cells were washed with 1× PBS and treated with 1% NP40 lysis buffer (100 mM Tris [pH 7.5], 150 mM NaCl, 20 mM EDTA, 1% NP40, 0.5% Na deoxycholate and protease inhibitors [Roche Diagnostics]), followed by centrifugation at 20,000×g and 4°C for 30 min to obtain cell lysates . .. For Western blot analysis, cell lysates were added to non-reducing buffer (2% SDS, 0.5 M Tris [pH 6.8], 20% glycerol, 0.001% bromophenol blue [final concentration]) and subjected to 12% polyacrylamide gel electrophoresis (PAGE), followed by transfer to nitrocellulose membrane, blocking and incubation with primary antibody (human sera from confirmed dengue cases) and secondary antibody , .

Article Title: Voluntary resistance wheel exercise from mid-life prevents sarcopenia and increases markers of mitochondrial function and autophagy in muscles of old male and female C57BL/6J mice
Article Snippet: The muscles were ground in liquid nitrogen, and the powder homogenized in ice-cold PBS, 1% NP40, 1 mM EDTA buffer, supplemented with complete EDTA-free protease inhibitor and PhosSTOP phosphatase inhibitor tablets (Roche, Manheim, Germany), and centrifuged at 13,000g for 20 min at 4 °C. .. Resultant pellets were resuspended in a buffer containing 20 mM HEPES (pH 7.5) and 4% SDS, supplemented with protease and phosphatase inhibitor tablets (Roche, Manheim, Germany), and solubilized by sonication 4 × 5 s bursts at 40% amplitude (Vibra Cell, Sonics & Materials Inc. #VCX 130), followed by centrifugation at 19,600g for 10 min at 16 °C [ ].

Article Title: A novel Leishmania infantum nuclear phosphoprotein Lepp12 which stimulates IL1-beta synthesis in THP-1 transfectants
Article Snippet: .. Promastigote lysates used to stimulate phosphorylation of recombinant Lepp12 were obtained as supernatants of centrifugation at 20,600 g for 15 min at 4°C after lysis of PBS-washed cells for 30 min at 4°C in water containing 1% NP40, 1 mM NaVO4 , 25 mM beta-glycerophosphate, 50 microM NaF, 2.5 mM NaPPi and 1 tablet (per 1 ml) of complete protease-inhibitor-cocktail (Roche, Meylan France). .. Nuclear extracts were prepared as follows: washed promastigote pellet was incubated for 10 min on ice in 0.5 ml of lysis buffer 1 (Hepes 10 mM, pH 7.5, MgCl2 1.5 mM, KCl 10 mM, DTT 0.5 mM, NP40 0.5 %, supplemented with proteases and phosphatases inhibitors as above), centrifuged (1,460 g, 10 min, 4°C), and the resulting pellet was incubated for 20 min on ice with 0.1 ml of lysis buffer 2 (Hepes 200 mM, pH 7.5, MgCl2 1.5 mM, KCl 840 mM, DTT 0.5 mM, glycerol 25%, EDTA 0.2 mM, supplemented with proteases ans phosphatases inhibitors).

Article Title: Substrate ectodomain is critical for substrate preference and inhibition of ?-secretase
Article Snippet: .. The cell suspension was mixed with an equal amount of lysis buffer (50 mM PIPES (pH 7.0), 250 mM sucrose, 2% NP40 and 2 × protease inhibitor cocktail (Roche Diagnostics)) and incubated on ice for 1 h. After ultracentrifugation at 100,000 × g centrifugation for 1 h, the supernatant was agitated overnight with Profinity eXact (S189 subtilisin-immobilized) resin (Bio-Rad). .. The Profinity eXact tag protein purification system offers purification of recombinant proteins with a native N terminus.

Filtration:

Article Title: The histone chaperone sNASP binds a conserved peptide motif within the globular core of histone H3 through its TPR repeats
Article Snippet: Protein expression and purification sNASP was expressed in bacteria as an N-terminal (His)6 fusion construct and was purified over Nickel-NTA beads (GE Healthcare) in 300 mM sodium chloride, 50 mM Tris–HCl pH 8.0, 0.1% NP40, 20 mM imidazole and Complete protease inhibitor cocktail (Roche). .. Cleaved protein was further purified by ion-exchange using a ResourceQ column (GE Healthcare) before gel filtration chromatography in 200 mM sodium chloride, 20 mM Hepes-KOH pH 7.6 and 1 mM EDTA using a 26/60 Superdex 200 column (GE Healthcare).

Cytometry:

Article Title: Intracellular Regulation of Cross-Presentation during Dendritic Cell Maturation
Article Snippet: Cells were lysed in 50mM Tris pH 7.4, 0.5% NP40, Roche protease inhibitors, 200µg/ml DNAse for 1h on ice. .. Mean fluorescence intensities of OVA were detected by flow cytometry, gating on single beads.

Blocking Assay:

Article Title: C-Terminal Helical Domains of Dengue Virus Type 4 E Protein Affect the Expression/Stability of prM Protein and Conformation of prM and E Proteins
Article Snippet: At 48 h post-transfection, cells were washed with 1× PBS and treated with 1% NP40 lysis buffer (100 mM Tris [pH 7.5], 150 mM NaCl, 20 mM EDTA, 1% NP40, 0.5% Na deoxycholate and protease inhibitors [Roche Diagnostics]), followed by centrifugation at 20,000×g and 4°C for 30 min to obtain cell lysates . .. For Western blot analysis, cell lysates were added to non-reducing buffer (2% SDS, 0.5 M Tris [pH 6.8], 20% glycerol, 0.001% bromophenol blue [final concentration]) and subjected to 12% polyacrylamide gel electrophoresis (PAGE), followed by transfer to nitrocellulose membrane, blocking and incubation with primary antibody (human sera from confirmed dengue cases) and secondary antibody , .

Electrophoresis:

Article Title: A novel Leishmania infantum nuclear phosphoprotein Lepp12 which stimulates IL1-beta synthesis in THP-1 transfectants
Article Snippet: For western blot analyses promastigotes were lysed in an electrophoresis buffer previously heated at 100°C for 10 min (100 mM Tris pH 6.8, 3 % SDS, 12.5 % glycerol). .. Promastigote lysates used to stimulate phosphorylation of recombinant Lepp12 were obtained as supernatants of centrifugation at 20,600 g for 15 min at 4°C after lysis of PBS-washed cells for 30 min at 4°C in water containing 1% NP40, 1 mM NaVO4 , 25 mM beta-glycerophosphate, 50 microM NaF, 2.5 mM NaPPi and 1 tablet (per 1 ml) of complete protease-inhibitor-cocktail (Roche, Meylan France).

Modification:

Article Title: Optimization of non-denaturing protein extraction conditions for plant PPR proteins
Article Snippet: .. The initial extraction buffer used in this manuscript was slightly modified from the one used previously for chloroplastic PPR immunoprecipitation from stable transformed A . thaliana plants [ ], and the one for weak protein-protein interactions indicated in the μMACS Epitope Tag Protein Isolation Kit protocol (Miltenyi Biotec), plus EDTA-free protease inhibitor cocktail according to the manufacturer’s recommendations for very high proteolytic activity: 50 mM Tris-HCl pH7.5, 150 mM NaCl, 1% NP40 (Igepal CA630), 2x protease inhibitor mix cocktail without EDTA (EDTA-free Complete, Roche REF04 693 132 001). .. As a result of this manuscript, for an optimized composition of the extraction buffer, suitable for PPR proteins, the initial extraction buffer was complemented with: 1x phosphatase inhibitor (PhosSTOP, Roche), 50 μM MG132 proteasome inhibitor (C2211, SIGMA), 5 mM ATP and 1 mM PMSF.

Incubation:

Article Title: ERG-associated protein with SET domain (ESET)-Oct4 interaction regulates pluripotency and represses the trophectoderm lineage
Article Snippet: Endogenous immunoprecipitation To analyse SUMOylated ESET, one confluent 10 cm dish of ES cells was lysed in 100 μl buffer containing 50 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA, 15 mM MgCl2, 0.75% sodium deoxycholate, 1% NP40 supplemented with protease inhibitors (Roche, Basel, Switzerland), 1:100 diluted phosphatase inhibitors I and II (Sigma, St Louis, MO, USA) and 20 mM NEM (Sigma, St Louis, MO, USA). .. Cell lysate was incubated on ice for 30 min and centrifuged at 13,000 rpm for 30 min at 4°C.

Article Title: C-Terminal Helical Domains of Dengue Virus Type 4 E Protein Affect the Expression/Stability of prM Protein and Conformation of prM and E Proteins
Article Snippet: At 48 h post-transfection, cells were washed with 1× PBS and treated with 1% NP40 lysis buffer (100 mM Tris [pH 7.5], 150 mM NaCl, 20 mM EDTA, 1% NP40, 0.5% Na deoxycholate and protease inhibitors [Roche Diagnostics]), followed by centrifugation at 20,000×g and 4°C for 30 min to obtain cell lysates . .. For Western blot analysis, cell lysates were added to non-reducing buffer (2% SDS, 0.5 M Tris [pH 6.8], 20% glycerol, 0.001% bromophenol blue [final concentration]) and subjected to 12% polyacrylamide gel electrophoresis (PAGE), followed by transfer to nitrocellulose membrane, blocking and incubation with primary antibody (human sera from confirmed dengue cases) and secondary antibody , .

Article Title: CHOP-independent apoptosis and pathway-selective induction of the UPR in developing plasma cells
Article Snippet: Labelled cells were washed twice in HBSS (Sigma) 4°C and incubated for various time periods. .. At each time point cells were lysed in 1% NP40, protein inhibitors cocktail (Roche).

Article Title: PTENα regulates mitophagy and maintains mitochondrial quality control
Article Snippet: HEK293T cells were lysed in co-immunoprecipitation lysis buffer containing 150 mM NaCl, 0.1 mM EDTA, 10% glycerol (Aladdin, G116203), 0.5% NP40 (Fluka, ) and a cocktail of protease inhibitors (Roche, 04693132001). .. Cell lysate (500 µg) was incubated with antibody for 4 h, followed by incubation with protein A/G agarose (Santa Cruz Biotechnology, sc-2003) for 1 h. The protein-bead complex mixture was washed in washing buffer containing 0.1% NP40 and subjected to western blot to evaluate protein interaction.

Article Title: A novel Leishmania infantum nuclear phosphoprotein Lepp12 which stimulates IL1-beta synthesis in THP-1 transfectants
Article Snippet: Promastigote lysates used to stimulate phosphorylation of recombinant Lepp12 were obtained as supernatants of centrifugation at 20,600 g for 15 min at 4°C after lysis of PBS-washed cells for 30 min at 4°C in water containing 1% NP40, 1 mM NaVO4 , 25 mM beta-glycerophosphate, 50 microM NaF, 2.5 mM NaPPi and 1 tablet (per 1 ml) of complete protease-inhibitor-cocktail (Roche, Meylan France). .. Nuclear extracts were prepared as follows: washed promastigote pellet was incubated for 10 min on ice in 0.5 ml of lysis buffer 1 (Hepes 10 mM, pH 7.5, MgCl2 1.5 mM, KCl 10 mM, DTT 0.5 mM, NP40 0.5 %, supplemented with proteases and phosphatases inhibitors as above), centrifuged (1,460 g, 10 min, 4°C), and the resulting pellet was incubated for 20 min on ice with 0.1 ml of lysis buffer 2 (Hepes 200 mM, pH 7.5, MgCl2 1.5 mM, KCl 840 mM, DTT 0.5 mM, glycerol 25%, EDTA 0.2 mM, supplemented with proteases ans phosphatases inhibitors).

Article Title: Substrate ectodomain is critical for substrate preference and inhibition of ?-secretase
Article Snippet: .. The cell suspension was mixed with an equal amount of lysis buffer (50 mM PIPES (pH 7.0), 250 mM sucrose, 2% NP40 and 2 × protease inhibitor cocktail (Roche Diagnostics)) and incubated on ice for 1 h. After ultracentrifugation at 100,000 × g centrifugation for 1 h, the supernatant was agitated overnight with Profinity eXact (S189 subtilisin-immobilized) resin (Bio-Rad). .. The Profinity eXact tag protein purification system offers purification of recombinant proteins with a native N terminus.

Article Title: Intracellular Regulation of Cross-Presentation during Dendritic Cell Maturation
Article Snippet: One sample of immature DCs was pre-treated with 25nM concanamycin B (ConB) for 20min and bead incubation was performed in the presence of the drug. .. Cells were lysed in 50mM Tris pH 7.4, 0.5% NP40, Roche protease inhibitors, 200µg/ml DNAse for 1h on ice.

Article Title: E1^E4-mediated keratin phosphorylation and ubiquitylation: a mechanism for keratin depletion in HPV16-infected epithelium
Article Snippet: The soluble fraction was incubated (3 hours, 4°C) with protein-G-Sepharose beads covalently conjugated (1 hours, 4°C) to either the PanKeratin (Sigma, C2562) or the K8-K18 [L2A1 ( )] antibody. .. For analysis of active kinases, cell extracts were prepared in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% NP40, 1 mM Vanadate and 1 tablet of miniprotease inhibitor cocktail (Roche) per 10 ml.

Activity Assay:

Article Title: Optimization of non-denaturing protein extraction conditions for plant PPR proteins
Article Snippet: .. The initial extraction buffer used in this manuscript was slightly modified from the one used previously for chloroplastic PPR immunoprecipitation from stable transformed A . thaliana plants [ ], and the one for weak protein-protein interactions indicated in the μMACS Epitope Tag Protein Isolation Kit protocol (Miltenyi Biotec), plus EDTA-free protease inhibitor cocktail according to the manufacturer’s recommendations for very high proteolytic activity: 50 mM Tris-HCl pH7.5, 150 mM NaCl, 1% NP40 (Igepal CA630), 2x protease inhibitor mix cocktail without EDTA (EDTA-free Complete, Roche REF04 693 132 001). .. As a result of this manuscript, for an optimized composition of the extraction buffer, suitable for PPR proteins, the initial extraction buffer was complemented with: 1x phosphatase inhibitor (PhosSTOP, Roche), 50 μM MG132 proteasome inhibitor (C2211, SIGMA), 5 mM ATP and 1 mM PMSF.

Cell Culture:

Article Title: A novel Leishmania infantum nuclear phosphoprotein Lepp12 which stimulates IL1-beta synthesis in THP-1 transfectants
Article Snippet: Cell cultures and cell preparations The promastigote form of L. infantum MON-1 (MHOM/FR/94/LPN101) was cultured in a complete RPMI medium at 25°C under usual conditions [ ], except in some experiments indicated in the text where Fetal Calf Serum (FCS) was substituted by 0.1 % BSA. .. Promastigote lysates used to stimulate phosphorylation of recombinant Lepp12 were obtained as supernatants of centrifugation at 20,600 g for 15 min at 4°C after lysis of PBS-washed cells for 30 min at 4°C in water containing 1% NP40, 1 mM NaVO4 , 25 mM beta-glycerophosphate, 50 microM NaF, 2.5 mM NaPPi and 1 tablet (per 1 ml) of complete protease-inhibitor-cocktail (Roche, Meylan France).

Expressing:

Article Title: The histone chaperone sNASP binds a conserved peptide motif within the globular core of histone H3 through its TPR repeats
Article Snippet: .. Protein expression and purification sNASP was expressed in bacteria as an N-terminal (His)6 fusion construct and was purified over Nickel-NTA beads (GE Healthcare) in 300 mM sodium chloride, 50 mM Tris–HCl pH 8.0, 0.1% NP40, 20 mM imidazole and Complete protease inhibitor cocktail (Roche). ..

Article Title: Optimization of non-denaturing protein extraction conditions for plant PPR proteins
Article Snippet: Paragraph title: Protein expression and extraction ... The initial extraction buffer used in this manuscript was slightly modified from the one used previously for chloroplastic PPR immunoprecipitation from stable transformed A . thaliana plants [ ], and the one for weak protein-protein interactions indicated in the μMACS Epitope Tag Protein Isolation Kit protocol (Miltenyi Biotec), plus EDTA-free protease inhibitor cocktail according to the manufacturer’s recommendations for very high proteolytic activity: 50 mM Tris-HCl pH7.5, 150 mM NaCl, 1% NP40 (Igepal CA630), 2x protease inhibitor mix cocktail without EDTA (EDTA-free Complete, Roche REF04 693 132 001).

BIA-KA:

Article Title: Immuno- and Constitutive Proteasomes Do Not Differ in Their Abilities to Degrade Ubiquitinated Proteins
Article Snippet: .. The murine B8 cells and MEFs were stimulated with 200 U/ml murine recombinant IFNγ (Peprotech) for the indicated time points and lysed either in 20 mM TRIS-HCl, pH 7.5, 10 mM EDTA, 100 mM NaCl, 1% NP40, 10 μM MG-132, 5 mM NEM, and Complete Protease Inhibitor Cocktail (Roche) ( B), or in 10 mM Tris, 150 mM NaCl, 1% Triton X-100 ( C) and the amount of protein was determined with a Pierce BCA protein assay kit (Thermo scientific). ..

Acrylamide Gel Assay:

Article Title: The temporal program of peripheral blood gene expression in the response of nonhuman primates to Ebola hemorrhagic fever
Article Snippet: Western blotting for truncated glycoprotein 2 (GP2Δ ) in ZEBOV infected animals Monkey sera were diluted 1:3 in NP-40 lysis buffer (10 mmol/l Tris [pH 7.5], 3% 5 mol/l NaCl, 1% NP40 and complete protease inhibitor tablet [Roche Applied Science, Indianapolis, IN, USA]). .. Samples were diluted in NuPage LDS sample buffer and NuPage sample reducing agent (Invitrogen Corporation), boiled and then loaded on a 10% Bis-Tris acrylamide gel, and run using NuPage MES buffer (Invitrogen Corporation).

Western Blot:

Article Title: Stoichiometry of HLA Class II-Invariant Chain Oligomers
Article Snippet: .. Immunoprecipitation, western blotting, and immunofluorescence microscopy Cells were lysed with 1% NP40 or with 1% Triton X-100 in Tris-buffered saline pH 7.4 containing protease inhibitors (Complete, Roche Diagnostics, Mannheim Germany). ..

Article Title: C-Terminal Helical Domains of Dengue Virus Type 4 E Protein Affect the Expression/Stability of prM Protein and Conformation of prM and E Proteins
Article Snippet: Paragraph title: Transfection, cell lysates, Western blot analysis and dot blot assay ... At 48 h post-transfection, cells were washed with 1× PBS and treated with 1% NP40 lysis buffer (100 mM Tris [pH 7.5], 150 mM NaCl, 20 mM EDTA, 1% NP40, 0.5% Na deoxycholate and protease inhibitors [Roche Diagnostics]), followed by centrifugation at 20,000×g and 4°C for 30 min to obtain cell lysates .

Article Title: Immuno- and Constitutive Proteasomes Do Not Differ in Their Abilities to Degrade Ubiquitinated Proteins
Article Snippet: Paragraph title: Western Blotting and IFNγ Stimulation ... The murine B8 cells and MEFs were stimulated with 200 U/ml murine recombinant IFNγ (Peprotech) for the indicated time points and lysed either in 20 mM TRIS-HCl, pH 7.5, 10 mM EDTA, 100 mM NaCl, 1% NP40, 10 μM MG-132, 5 mM NEM, and Complete Protease Inhibitor Cocktail (Roche) ( B), or in 10 mM Tris, 150 mM NaCl, 1% Triton X-100 ( C) and the amount of protein was determined with a Pierce BCA protein assay kit (Thermo scientific).

Article Title: The temporal program of peripheral blood gene expression in the response of nonhuman primates to Ebola hemorrhagic fever
Article Snippet: .. Western blotting for truncated glycoprotein 2 (GP2Δ ) in ZEBOV infected animals Monkey sera were diluted 1:3 in NP-40 lysis buffer (10 mmol/l Tris [pH 7.5], 3% 5 mol/l NaCl, 1% NP40 and complete protease inhibitor tablet [Roche Applied Science, Indianapolis, IN, USA]). .. Controls included ZEBOV seed stock diluted 1:3 with NP40 lysis buffer, and glycoprotein controls were generated by transfecting 293T cells with GP1,2 or GP1,2Δ plasmids, as described previously [ ].

Article Title: PTENα regulates mitophagy and maintains mitochondrial quality control
Article Snippet: HEK293T cells were lysed in co-immunoprecipitation lysis buffer containing 150 mM NaCl, 0.1 mM EDTA, 10% glycerol (Aladdin, G116203), 0.5% NP40 (Fluka, ) and a cocktail of protease inhibitors (Roche, 04693132001). .. Cell lysate (500 µg) was incubated with antibody for 4 h, followed by incubation with protein A/G agarose (Santa Cruz Biotechnology, sc-2003) for 1 h. The protein-bead complex mixture was washed in washing buffer containing 0.1% NP40 and subjected to western blot to evaluate protein interaction.

Article Title: A novel Leishmania infantum nuclear phosphoprotein Lepp12 which stimulates IL1-beta synthesis in THP-1 transfectants
Article Snippet: For western blot analyses promastigotes were lysed in an electrophoresis buffer previously heated at 100°C for 10 min (100 mM Tris pH 6.8, 3 % SDS, 12.5 % glycerol). .. Promastigote lysates used to stimulate phosphorylation of recombinant Lepp12 were obtained as supernatants of centrifugation at 20,600 g for 15 min at 4°C after lysis of PBS-washed cells for 30 min at 4°C in water containing 1% NP40, 1 mM NaVO4 , 25 mM beta-glycerophosphate, 50 microM NaF, 2.5 mM NaPPi and 1 tablet (per 1 ml) of complete protease-inhibitor-cocktail (Roche, Meylan France).

Article Title: Leoligin, the Major Lignan from Edelweiss (Leontopodium nivale subsp. alpinum), Promotes Cholesterol Efflux from THP-1 Macrophages
Article Snippet: .. Western Blot Analysis THP-1 macrophages treated as indicated in the figure legends were washed with cold PBS (4 °C) and lysed with NP40 buffer (150 mM NaCl; 50 mM HEPES (pH 7.4); 1% NP40) containing a protease inhibitor mixture (1% Complete (Roche); 1% phenylmethylsulfonyl fluoride; 0.5% Na3 VO4 ; 0.5% NaF). ..

Transformation Assay:

Article Title: Optimization of non-denaturing protein extraction conditions for plant PPR proteins
Article Snippet: .. The initial extraction buffer used in this manuscript was slightly modified from the one used previously for chloroplastic PPR immunoprecipitation from stable transformed A . thaliana plants [ ], and the one for weak protein-protein interactions indicated in the μMACS Epitope Tag Protein Isolation Kit protocol (Miltenyi Biotec), plus EDTA-free protease inhibitor cocktail according to the manufacturer’s recommendations for very high proteolytic activity: 50 mM Tris-HCl pH7.5, 150 mM NaCl, 1% NP40 (Igepal CA630), 2x protease inhibitor mix cocktail without EDTA (EDTA-free Complete, Roche REF04 693 132 001). .. As a result of this manuscript, for an optimized composition of the extraction buffer, suitable for PPR proteins, the initial extraction buffer was complemented with: 1x phosphatase inhibitor (PhosSTOP, Roche), 50 μM MG132 proteasome inhibitor (C2211, SIGMA), 5 mM ATP and 1 mM PMSF.

Transfection:

Article Title: C-Terminal Helical Domains of Dengue Virus Type 4 E Protein Affect the Expression/Stability of prM Protein and Conformation of prM and E Proteins
Article Snippet: Paragraph title: Transfection, cell lysates, Western blot analysis and dot blot assay ... At 48 h post-transfection, cells were washed with 1× PBS and treated with 1% NP40 lysis buffer (100 mM Tris [pH 7.5], 150 mM NaCl, 20 mM EDTA, 1% NP40, 0.5% Na deoxycholate and protease inhibitors [Roche Diagnostics]), followed by centrifugation at 20,000×g and 4°C for 30 min to obtain cell lysates .

Pulse Chase:

Article Title: CHOP-independent apoptosis and pathway-selective induction of the UPR in developing plasma cells
Article Snippet: Paragraph title: 2.6 Pulse chase assays ... At each time point cells were lysed in 1% NP40, protein inhibitors cocktail (Roche).

Concentration Assay:

Article Title: C-Terminal Helical Domains of Dengue Virus Type 4 E Protein Affect the Expression/Stability of prM Protein and Conformation of prM and E Proteins
Article Snippet: At 48 h post-transfection, cells were washed with 1× PBS and treated with 1% NP40 lysis buffer (100 mM Tris [pH 7.5], 150 mM NaCl, 20 mM EDTA, 1% NP40, 0.5% Na deoxycholate and protease inhibitors [Roche Diagnostics]), followed by centrifugation at 20,000×g and 4°C for 30 min to obtain cell lysates . .. For Western blot analysis, cell lysates were added to non-reducing buffer (2% SDS, 0.5 M Tris [pH 6.8], 20% glycerol, 0.001% bromophenol blue [final concentration]) and subjected to 12% polyacrylamide gel electrophoresis (PAGE), followed by transfer to nitrocellulose membrane, blocking and incubation with primary antibody (human sera from confirmed dengue cases) and secondary antibody , .

Protease Inhibitor:

Article Title: Voluntary resistance wheel exercise from mid-life prevents sarcopenia and increases markers of mitochondrial function and autophagy in muscles of old male and female C57BL/6J mice
Article Snippet: .. The muscles were ground in liquid nitrogen, and the powder homogenized in ice-cold PBS, 1% NP40, 1 mM EDTA buffer, supplemented with complete EDTA-free protease inhibitor and PhosSTOP phosphatase inhibitor tablets (Roche, Manheim, Germany), and centrifuged at 13,000g for 20 min at 4 °C. ..

Article Title: Immuno- and Constitutive Proteasomes Do Not Differ in Their Abilities to Degrade Ubiquitinated Proteins
Article Snippet: .. The murine B8 cells and MEFs were stimulated with 200 U/ml murine recombinant IFNγ (Peprotech) for the indicated time points and lysed either in 20 mM TRIS-HCl, pH 7.5, 10 mM EDTA, 100 mM NaCl, 1% NP40, 10 μM MG-132, 5 mM NEM, and Complete Protease Inhibitor Cocktail (Roche) ( B), or in 10 mM Tris, 150 mM NaCl, 1% Triton X-100 ( C) and the amount of protein was determined with a Pierce BCA protein assay kit (Thermo scientific). ..

Article Title: The temporal program of peripheral blood gene expression in the response of nonhuman primates to Ebola hemorrhagic fever
Article Snippet: .. Western blotting for truncated glycoprotein 2 (GP2Δ ) in ZEBOV infected animals Monkey sera were diluted 1:3 in NP-40 lysis buffer (10 mmol/l Tris [pH 7.5], 3% 5 mol/l NaCl, 1% NP40 and complete protease inhibitor tablet [Roche Applied Science, Indianapolis, IN, USA]). .. Controls included ZEBOV seed stock diluted 1:3 with NP40 lysis buffer, and glycoprotein controls were generated by transfecting 293T cells with GP1,2 or GP1,2Δ plasmids, as described previously [ ].

Article Title: The histone chaperone sNASP binds a conserved peptide motif within the globular core of histone H3 through its TPR repeats
Article Snippet: .. Protein expression and purification sNASP was expressed in bacteria as an N-terminal (His)6 fusion construct and was purified over Nickel-NTA beads (GE Healthcare) in 300 mM sodium chloride, 50 mM Tris–HCl pH 8.0, 0.1% NP40, 20 mM imidazole and Complete protease inhibitor cocktail (Roche). ..

Article Title: Substrate ectodomain is critical for substrate preference and inhibition of ?-secretase
Article Snippet: .. The cell suspension was mixed with an equal amount of lysis buffer (50 mM PIPES (pH 7.0), 250 mM sucrose, 2% NP40 and 2 × protease inhibitor cocktail (Roche Diagnostics)) and incubated on ice for 1 h. After ultracentrifugation at 100,000 × g centrifugation for 1 h, the supernatant was agitated overnight with Profinity eXact (S189 subtilisin-immobilized) resin (Bio-Rad). .. The Profinity eXact tag protein purification system offers purification of recombinant proteins with a native N terminus.

Article Title: Optimization of non-denaturing protein extraction conditions for plant PPR proteins
Article Snippet: .. The initial extraction buffer used in this manuscript was slightly modified from the one used previously for chloroplastic PPR immunoprecipitation from stable transformed A . thaliana plants [ ], and the one for weak protein-protein interactions indicated in the μMACS Epitope Tag Protein Isolation Kit protocol (Miltenyi Biotec), plus EDTA-free protease inhibitor cocktail according to the manufacturer’s recommendations for very high proteolytic activity: 50 mM Tris-HCl pH7.5, 150 mM NaCl, 1% NP40 (Igepal CA630), 2x protease inhibitor mix cocktail without EDTA (EDTA-free Complete, Roche REF04 693 132 001). .. As a result of this manuscript, for an optimized composition of the extraction buffer, suitable for PPR proteins, the initial extraction buffer was complemented with: 1x phosphatase inhibitor (PhosSTOP, Roche), 50 μM MG132 proteasome inhibitor (C2211, SIGMA), 5 mM ATP and 1 mM PMSF.

Article Title: Leoligin, the Major Lignan from Edelweiss (Leontopodium nivale subsp. alpinum), Promotes Cholesterol Efflux from THP-1 Macrophages
Article Snippet: .. Western Blot Analysis THP-1 macrophages treated as indicated in the figure legends were washed with cold PBS (4 °C) and lysed with NP40 buffer (150 mM NaCl; 50 mM HEPES (pH 7.4); 1% NP40) containing a protease inhibitor mixture (1% Complete (Roche); 1% phenylmethylsulfonyl fluoride; 0.5% Na3 VO4 ; 0.5% NaF). ..

Infection:

Article Title: The temporal program of peripheral blood gene expression in the response of nonhuman primates to Ebola hemorrhagic fever
Article Snippet: .. Western blotting for truncated glycoprotein 2 (GP2Δ ) in ZEBOV infected animals Monkey sera were diluted 1:3 in NP-40 lysis buffer (10 mmol/l Tris [pH 7.5], 3% 5 mol/l NaCl, 1% NP40 and complete protease inhibitor tablet [Roche Applied Science, Indianapolis, IN, USA]). .. Controls included ZEBOV seed stock diluted 1:3 with NP40 lysis buffer, and glycoprotein controls were generated by transfecting 293T cells with GP1,2 or GP1,2Δ plasmids, as described previously [ ].

Article Title: Substrate ectodomain is critical for substrate preference and inhibition of ?-secretase
Article Snippet: Infected cells were harvested after 36 h and resuspended in 50 mM PIPES (pH 7.0), 250 mM sucrose. .. The cell suspension was mixed with an equal amount of lysis buffer (50 mM PIPES (pH 7.0), 250 mM sucrose, 2% NP40 and 2 × protease inhibitor cocktail (Roche Diagnostics)) and incubated on ice for 1 h. After ultracentrifugation at 100,000 × g centrifugation for 1 h, the supernatant was agitated overnight with Profinity eXact (S189 subtilisin-immobilized) resin (Bio-Rad).

Sedimentation:

Article Title: A novel Leishmania infantum nuclear phosphoprotein Lepp12 which stimulates IL1-beta synthesis in THP-1 transfectants
Article Snippet: Early stationary phase promastigotes (5-day-old cultures) were used to carry out various cell preparations, unless indicated otherwise in figure legends, and were washed 3 times by sedimentation at 206 g for 5 min at 4°C in PBS containing 1 mM NaVO4 . .. Promastigote lysates used to stimulate phosphorylation of recombinant Lepp12 were obtained as supernatants of centrifugation at 20,600 g for 15 min at 4°C after lysis of PBS-washed cells for 30 min at 4°C in water containing 1% NP40, 1 mM NaVO4 , 25 mM beta-glycerophosphate, 50 microM NaF, 2.5 mM NaPPi and 1 tablet (per 1 ml) of complete protease-inhibitor-cocktail (Roche, Meylan France).

Generated:

Article Title: The temporal program of peripheral blood gene expression in the response of nonhuman primates to Ebola hemorrhagic fever
Article Snippet: Western blotting for truncated glycoprotein 2 (GP2Δ ) in ZEBOV infected animals Monkey sera were diluted 1:3 in NP-40 lysis buffer (10 mmol/l Tris [pH 7.5], 3% 5 mol/l NaCl, 1% NP40 and complete protease inhibitor tablet [Roche Applied Science, Indianapolis, IN, USA]). .. Controls included ZEBOV seed stock diluted 1:3 with NP40 lysis buffer, and glycoprotein controls were generated by transfecting 293T cells with GP1,2 or GP1,2Δ plasmids, as described previously [ ].

Protein Concentration:

Article Title: Immuno- and Constitutive Proteasomes Do Not Differ in Their Abilities to Degrade Ubiquitinated Proteins
Article Snippet: Protein concentration of the lysates were determined using the Bradford reagent (Pierce) and equal amounts of protein were separated by SDS PAGE and analyzed by western blotting. .. The murine B8 cells and MEFs were stimulated with 200 U/ml murine recombinant IFNγ (Peprotech) for the indicated time points and lysed either in 20 mM TRIS-HCl, pH 7.5, 10 mM EDTA, 100 mM NaCl, 1% NP40, 10 μM MG-132, 5 mM NEM, and Complete Protease Inhibitor Cocktail (Roche) ( B), or in 10 mM Tris, 150 mM NaCl, 1% Triton X-100 ( C) and the amount of protein was determined with a Pierce BCA protein assay kit (Thermo scientific).

Sequencing:

Article Title: Substrate ectodomain is critical for substrate preference and inhibition of ?-secretase
Article Snippet: The cell suspension was mixed with an equal amount of lysis buffer (50 mM PIPES (pH 7.0), 250 mM sucrose, 2% NP40 and 2 × protease inhibitor cocktail (Roche Diagnostics)) and incubated on ice for 1 h. After ultracentrifugation at 100,000 × g centrifugation for 1 h, the supernatant was agitated overnight with Profinity eXact (S189 subtilisin-immobilized) resin (Bio-Rad). .. Once Profinity eXact-tagged γ-secretase substrates were captured using S189 subtilisin-immobilized resin, addition of sodium fluoride triggered precise cleavage at the C terminus of the cleavage-recognition sequence and the release of substrates with a bona fide N terminus.

Sonication:

Article Title: Voluntary resistance wheel exercise from mid-life prevents sarcopenia and increases markers of mitochondrial function and autophagy in muscles of old male and female C57BL/6J mice
Article Snippet: The muscles were ground in liquid nitrogen, and the powder homogenized in ice-cold PBS, 1% NP40, 1 mM EDTA buffer, supplemented with complete EDTA-free protease inhibitor and PhosSTOP phosphatase inhibitor tablets (Roche, Manheim, Germany), and centrifuged at 13,000g for 20 min at 4 °C. .. Resultant pellets were resuspended in a buffer containing 20 mM HEPES (pH 7.5) and 4% SDS, supplemented with protease and phosphatase inhibitor tablets (Roche, Manheim, Germany), and solubilized by sonication 4 × 5 s bursts at 40% amplitude (Vibra Cell, Sonics & Materials Inc. #VCX 130), followed by centrifugation at 19,600g for 10 min at 16 °C [ ].

Article Title: Immuno- and Constitutive Proteasomes Do Not Differ in Their Abilities to Degrade Ubiquitinated Proteins
Article Snippet: The cells were then lysed by sonication in 25 mM HEPES pH 7.4, 1 mM ATP, 1 mM DTT, 5 mM MgCl2 , and 10% glycerol. .. The murine B8 cells and MEFs were stimulated with 200 U/ml murine recombinant IFNγ (Peprotech) for the indicated time points and lysed either in 20 mM TRIS-HCl, pH 7.5, 10 mM EDTA, 100 mM NaCl, 1% NP40, 10 μM MG-132, 5 mM NEM, and Complete Protease Inhibitor Cocktail (Roche) ( B), or in 10 mM Tris, 150 mM NaCl, 1% Triton X-100 ( C) and the amount of protein was determined with a Pierce BCA protein assay kit (Thermo scientific).

Recombinant:

Article Title: Immuno- and Constitutive Proteasomes Do Not Differ in Their Abilities to Degrade Ubiquitinated Proteins
Article Snippet: .. The murine B8 cells and MEFs were stimulated with 200 U/ml murine recombinant IFNγ (Peprotech) for the indicated time points and lysed either in 20 mM TRIS-HCl, pH 7.5, 10 mM EDTA, 100 mM NaCl, 1% NP40, 10 μM MG-132, 5 mM NEM, and Complete Protease Inhibitor Cocktail (Roche) ( B), or in 10 mM Tris, 150 mM NaCl, 1% Triton X-100 ( C) and the amount of protein was determined with a Pierce BCA protein assay kit (Thermo scientific). ..

Article Title: A novel Leishmania infantum nuclear phosphoprotein Lepp12 which stimulates IL1-beta synthesis in THP-1 transfectants
Article Snippet: .. Promastigote lysates used to stimulate phosphorylation of recombinant Lepp12 were obtained as supernatants of centrifugation at 20,600 g for 15 min at 4°C after lysis of PBS-washed cells for 30 min at 4°C in water containing 1% NP40, 1 mM NaVO4 , 25 mM beta-glycerophosphate, 50 microM NaF, 2.5 mM NaPPi and 1 tablet (per 1 ml) of complete protease-inhibitor-cocktail (Roche, Meylan France). .. Nuclear extracts were prepared as follows: washed promastigote pellet was incubated for 10 min on ice in 0.5 ml of lysis buffer 1 (Hepes 10 mM, pH 7.5, MgCl2 1.5 mM, KCl 10 mM, DTT 0.5 mM, NP40 0.5 %, supplemented with proteases and phosphatases inhibitors as above), centrifuged (1,460 g, 10 min, 4°C), and the resulting pellet was incubated for 20 min on ice with 0.1 ml of lysis buffer 2 (Hepes 200 mM, pH 7.5, MgCl2 1.5 mM, KCl 840 mM, DTT 0.5 mM, glycerol 25%, EDTA 0.2 mM, supplemented with proteases ans phosphatases inhibitors).

Article Title: Substrate ectodomain is critical for substrate preference and inhibition of ?-secretase
Article Snippet: Sf9 cells were infected with recombinant baculovirus according to the manufacturer’s instructions. .. The cell suspension was mixed with an equal amount of lysis buffer (50 mM PIPES (pH 7.0), 250 mM sucrose, 2% NP40 and 2 × protease inhibitor cocktail (Roche Diagnostics)) and incubated on ice for 1 h. After ultracentrifugation at 100,000 × g centrifugation for 1 h, the supernatant was agitated overnight with Profinity eXact (S189 subtilisin-immobilized) resin (Bio-Rad).

Immunofluorescence:

Article Title: Stoichiometry of HLA Class II-Invariant Chain Oligomers
Article Snippet: .. Immunoprecipitation, western blotting, and immunofluorescence microscopy Cells were lysed with 1% NP40 or with 1% Triton X-100 in Tris-buffered saline pH 7.4 containing protease inhibitors (Complete, Roche Diagnostics, Mannheim Germany). ..

Nucleic Acid Electrophoresis:

Article Title: Leoligin, the Major Lignan from Edelweiss (Leontopodium nivale subsp. alpinum), Promotes Cholesterol Efflux from THP-1 Macrophages
Article Snippet: Western Blot Analysis THP-1 macrophages treated as indicated in the figure legends were washed with cold PBS (4 °C) and lysed with NP40 buffer (150 mM NaCl; 50 mM HEPES (pH 7.4); 1% NP40) containing a protease inhibitor mixture (1% Complete (Roche); 1% phenylmethylsulfonyl fluoride; 0.5% Na3 VO4 ; 0.5% NaF). .. An equal amount of protein samples (20 μg) was resolved via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a PVDF membrane (Bio-Rad).

Fluorescence:

Article Title: Intracellular Regulation of Cross-Presentation during Dendritic Cell Maturation
Article Snippet: Cells were lysed in 50mM Tris pH 7.4, 0.5% NP40, Roche protease inhibitors, 200µg/ml DNAse for 1h on ice. .. Mean fluorescence intensities of OVA were detected by flow cytometry, gating on single beads.

Isolation:

Article Title: Stoichiometry of HLA Class II-Invariant Chain Oligomers
Article Snippet: Immunoprecipitation, western blotting, and immunofluorescence microscopy Cells were lysed with 1% NP40 or with 1% Triton X-100 in Tris-buffered saline pH 7.4 containing protease inhibitors (Complete, Roche Diagnostics, Mannheim Germany). .. Immunoprecipitates were isolated with protein A Sepharose, separated by SDS-PAGE, and blotted onto Immobilon P membrane (Millipore, Schwalbach, Germany).

Article Title: Optimization of non-denaturing protein extraction conditions for plant PPR proteins
Article Snippet: .. The initial extraction buffer used in this manuscript was slightly modified from the one used previously for chloroplastic PPR immunoprecipitation from stable transformed A . thaliana plants [ ], and the one for weak protein-protein interactions indicated in the μMACS Epitope Tag Protein Isolation Kit protocol (Miltenyi Biotec), plus EDTA-free protease inhibitor cocktail according to the manufacturer’s recommendations for very high proteolytic activity: 50 mM Tris-HCl pH7.5, 150 mM NaCl, 1% NP40 (Igepal CA630), 2x protease inhibitor mix cocktail without EDTA (EDTA-free Complete, Roche REF04 693 132 001). .. As a result of this manuscript, for an optimized composition of the extraction buffer, suitable for PPR proteins, the initial extraction buffer was complemented with: 1x phosphatase inhibitor (PhosSTOP, Roche), 50 μM MG132 proteasome inhibitor (C2211, SIGMA), 5 mM ATP and 1 mM PMSF.

Flow Cytometry:

Article Title: Intracellular Regulation of Cross-Presentation during Dendritic Cell Maturation
Article Snippet: Cells were lysed in 50mM Tris pH 7.4, 0.5% NP40, Roche protease inhibitors, 200µg/ml DNAse for 1h on ice. .. Mean fluorescence intensities of OVA were detected by flow cytometry, gating on single beads.

Microscopy:

Article Title: Stoichiometry of HLA Class II-Invariant Chain Oligomers
Article Snippet: .. Immunoprecipitation, western blotting, and immunofluorescence microscopy Cells were lysed with 1% NP40 or with 1% Triton X-100 in Tris-buffered saline pH 7.4 containing protease inhibitors (Complete, Roche Diagnostics, Mannheim Germany). ..

Purification:

Article Title: The histone chaperone sNASP binds a conserved peptide motif within the globular core of histone H3 through its TPR repeats
Article Snippet: .. Protein expression and purification sNASP was expressed in bacteria as an N-terminal (His)6 fusion construct and was purified over Nickel-NTA beads (GE Healthcare) in 300 mM sodium chloride, 50 mM Tris–HCl pH 8.0, 0.1% NP40, 20 mM imidazole and Complete protease inhibitor cocktail (Roche). ..

Article Title: Substrate ectodomain is critical for substrate preference and inhibition of ?-secretase
Article Snippet: The cell suspension was mixed with an equal amount of lysis buffer (50 mM PIPES (pH 7.0), 250 mM sucrose, 2% NP40 and 2 × protease inhibitor cocktail (Roche Diagnostics)) and incubated on ice for 1 h. After ultracentrifugation at 100,000 × g centrifugation for 1 h, the supernatant was agitated overnight with Profinity eXact (S189 subtilisin-immobilized) resin (Bio-Rad). .. The Profinity eXact tag protein purification system offers purification of recombinant proteins with a native N terminus.

Dot Blot:

Article Title: C-Terminal Helical Domains of Dengue Virus Type 4 E Protein Affect the Expression/Stability of prM Protein and Conformation of prM and E Proteins
Article Snippet: Paragraph title: Transfection, cell lysates, Western blot analysis and dot blot assay ... At 48 h post-transfection, cells were washed with 1× PBS and treated with 1% NP40 lysis buffer (100 mM Tris [pH 7.5], 150 mM NaCl, 20 mM EDTA, 1% NP40, 0.5% Na deoxycholate and protease inhibitors [Roche Diagnostics]), followed by centrifugation at 20,000×g and 4°C for 30 min to obtain cell lysates .

Protein Extraction:

Article Title: Voluntary resistance wheel exercise from mid-life prevents sarcopenia and increases markers of mitochondrial function and autophagy in muscles of old male and female C57BL/6J mice
Article Snippet: Paragraph title: Protein extraction and immunoblotting ... The muscles were ground in liquid nitrogen, and the powder homogenized in ice-cold PBS, 1% NP40, 1 mM EDTA buffer, supplemented with complete EDTA-free protease inhibitor and PhosSTOP phosphatase inhibitor tablets (Roche, Manheim, Germany), and centrifuged at 13,000g for 20 min at 4 °C.

Construct:

Article Title: The histone chaperone sNASP binds a conserved peptide motif within the globular core of histone H3 through its TPR repeats
Article Snippet: .. Protein expression and purification sNASP was expressed in bacteria as an N-terminal (His)6 fusion construct and was purified over Nickel-NTA beads (GE Healthcare) in 300 mM sodium chloride, 50 mM Tris–HCl pH 8.0, 0.1% NP40, 20 mM imidazole and Complete protease inhibitor cocktail (Roche). ..

Polyacrylamide Gel Electrophoresis:

Article Title: C-Terminal Helical Domains of Dengue Virus Type 4 E Protein Affect the Expression/Stability of prM Protein and Conformation of prM and E Proteins
Article Snippet: At 48 h post-transfection, cells were washed with 1× PBS and treated with 1% NP40 lysis buffer (100 mM Tris [pH 7.5], 150 mM NaCl, 20 mM EDTA, 1% NP40, 0.5% Na deoxycholate and protease inhibitors [Roche Diagnostics]), followed by centrifugation at 20,000×g and 4°C for 30 min to obtain cell lysates . .. For Western blot analysis, cell lysates were added to non-reducing buffer (2% SDS, 0.5 M Tris [pH 6.8], 20% glycerol, 0.001% bromophenol blue [final concentration]) and subjected to 12% polyacrylamide gel electrophoresis (PAGE), followed by transfer to nitrocellulose membrane, blocking and incubation with primary antibody (human sera from confirmed dengue cases) and secondary antibody , .

Lysis:

Article Title: ERG-associated protein with SET domain (ESET)-Oct4 interaction regulates pluripotency and represses the trophectoderm lineage
Article Snippet: Endogenous immunoprecipitation To analyse SUMOylated ESET, one confluent 10 cm dish of ES cells was lysed in 100 μl buffer containing 50 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA, 15 mM MgCl2, 0.75% sodium deoxycholate, 1% NP40 supplemented with protease inhibitors (Roche, Basel, Switzerland), 1:100 diluted phosphatase inhibitors I and II (Sigma, St Louis, MO, USA) and 20 mM NEM (Sigma, St Louis, MO, USA). .. Where indicated, lysis buffer was also added with 50 μg/ml of ethidium bromide.

Article Title: C-Terminal Helical Domains of Dengue Virus Type 4 E Protein Affect the Expression/Stability of prM Protein and Conformation of prM and E Proteins
Article Snippet: .. At 48 h post-transfection, cells were washed with 1× PBS and treated with 1% NP40 lysis buffer (100 mM Tris [pH 7.5], 150 mM NaCl, 20 mM EDTA, 1% NP40, 0.5% Na deoxycholate and protease inhibitors [Roche Diagnostics]), followed by centrifugation at 20,000×g and 4°C for 30 min to obtain cell lysates . .. For Western blot analysis, cell lysates were added to non-reducing buffer (2% SDS, 0.5 M Tris [pH 6.8], 20% glycerol, 0.001% bromophenol blue [final concentration]) and subjected to 12% polyacrylamide gel electrophoresis (PAGE), followed by transfer to nitrocellulose membrane, blocking and incubation with primary antibody (human sera from confirmed dengue cases) and secondary antibody , .

Article Title: The temporal program of peripheral blood gene expression in the response of nonhuman primates to Ebola hemorrhagic fever
Article Snippet: .. Western blotting for truncated glycoprotein 2 (GP2Δ ) in ZEBOV infected animals Monkey sera were diluted 1:3 in NP-40 lysis buffer (10 mmol/l Tris [pH 7.5], 3% 5 mol/l NaCl, 1% NP40 and complete protease inhibitor tablet [Roche Applied Science, Indianapolis, IN, USA]). .. Controls included ZEBOV seed stock diluted 1:3 with NP40 lysis buffer, and glycoprotein controls were generated by transfecting 293T cells with GP1,2 or GP1,2Δ plasmids, as described previously [ ].

Article Title: PTENα regulates mitophagy and maintains mitochondrial quality control
Article Snippet: .. HEK293T cells were lysed in co-immunoprecipitation lysis buffer containing 150 mM NaCl, 0.1 mM EDTA, 10% glycerol (Aladdin, G116203), 0.5% NP40 (Fluka, ) and a cocktail of protease inhibitors (Roche, 04693132001). .. Cell lysate (500 µg) was incubated with antibody for 4 h, followed by incubation with protein A/G agarose (Santa Cruz Biotechnology, sc-2003) for 1 h. The protein-bead complex mixture was washed in washing buffer containing 0.1% NP40 and subjected to western blot to evaluate protein interaction.

Article Title: A novel Leishmania infantum nuclear phosphoprotein Lepp12 which stimulates IL1-beta synthesis in THP-1 transfectants
Article Snippet: .. Promastigote lysates used to stimulate phosphorylation of recombinant Lepp12 were obtained as supernatants of centrifugation at 20,600 g for 15 min at 4°C after lysis of PBS-washed cells for 30 min at 4°C in water containing 1% NP40, 1 mM NaVO4 , 25 mM beta-glycerophosphate, 50 microM NaF, 2.5 mM NaPPi and 1 tablet (per 1 ml) of complete protease-inhibitor-cocktail (Roche, Meylan France). .. Nuclear extracts were prepared as follows: washed promastigote pellet was incubated for 10 min on ice in 0.5 ml of lysis buffer 1 (Hepes 10 mM, pH 7.5, MgCl2 1.5 mM, KCl 10 mM, DTT 0.5 mM, NP40 0.5 %, supplemented with proteases and phosphatases inhibitors as above), centrifuged (1,460 g, 10 min, 4°C), and the resulting pellet was incubated for 20 min on ice with 0.1 ml of lysis buffer 2 (Hepes 200 mM, pH 7.5, MgCl2 1.5 mM, KCl 840 mM, DTT 0.5 mM, glycerol 25%, EDTA 0.2 mM, supplemented with proteases ans phosphatases inhibitors).

Article Title: Substrate ectodomain is critical for substrate preference and inhibition of ?-secretase
Article Snippet: .. The cell suspension was mixed with an equal amount of lysis buffer (50 mM PIPES (pH 7.0), 250 mM sucrose, 2% NP40 and 2 × protease inhibitor cocktail (Roche Diagnostics)) and incubated on ice for 1 h. After ultracentrifugation at 100,000 × g centrifugation for 1 h, the supernatant was agitated overnight with Profinity eXact (S189 subtilisin-immobilized) resin (Bio-Rad). .. The Profinity eXact tag protein purification system offers purification of recombinant proteins with a native N terminus.

Article Title: Intracellular Regulation of Cross-Presentation during Dendritic Cell Maturation
Article Snippet: For all samples (t=0 and t=1.5h), extracellular beads were marked with mouse anti-BSA antibody (7G10, Abcam) followed by A647-conjugated F(ab’)2 of goat anti-mouse IgG before cell lysis. .. Cells were lysed in 50mM Tris pH 7.4, 0.5% NP40, Roche protease inhibitors, 200µg/ml DNAse for 1h on ice.

SDS Page:

Article Title: Stoichiometry of HLA Class II-Invariant Chain Oligomers
Article Snippet: Immunoprecipitation, western blotting, and immunofluorescence microscopy Cells were lysed with 1% NP40 or with 1% Triton X-100 in Tris-buffered saline pH 7.4 containing protease inhibitors (Complete, Roche Diagnostics, Mannheim Germany). .. Immunoprecipitates were isolated with protein A Sepharose, separated by SDS-PAGE, and blotted onto Immobilon P membrane (Millipore, Schwalbach, Germany).

Article Title: Immuno- and Constitutive Proteasomes Do Not Differ in Their Abilities to Degrade Ubiquitinated Proteins
Article Snippet: Protein concentration of the lysates were determined using the Bradford reagent (Pierce) and equal amounts of protein were separated by SDS PAGE and analyzed by western blotting. .. The murine B8 cells and MEFs were stimulated with 200 U/ml murine recombinant IFNγ (Peprotech) for the indicated time points and lysed either in 20 mM TRIS-HCl, pH 7.5, 10 mM EDTA, 100 mM NaCl, 1% NP40, 10 μM MG-132, 5 mM NEM, and Complete Protease Inhibitor Cocktail (Roche) ( B), or in 10 mM Tris, 150 mM NaCl, 1% Triton X-100 ( C) and the amount of protein was determined with a Pierce BCA protein assay kit (Thermo scientific).

Article Title: Leoligin, the Major Lignan from Edelweiss (Leontopodium nivale subsp. alpinum), Promotes Cholesterol Efflux from THP-1 Macrophages
Article Snippet: Western Blot Analysis THP-1 macrophages treated as indicated in the figure legends were washed with cold PBS (4 °C) and lysed with NP40 buffer (150 mM NaCl; 50 mM HEPES (pH 7.4); 1% NP40) containing a protease inhibitor mixture (1% Complete (Roche); 1% phenylmethylsulfonyl fluoride; 0.5% Na3 VO4 ; 0.5% NaF). .. An equal amount of protein samples (20 μg) was resolved via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a PVDF membrane (Bio-Rad).

Plasmid Preparation:

Article Title: C-Terminal Helical Domains of Dengue Virus Type 4 E Protein Affect the Expression/Stability of prM Protein and Conformation of prM and E Proteins
Article Snippet: Transfection, cell lysates, Western blot analysis and dot blot assay 293T cells (from ATCC) prepared in a 10 cm-culture dish at 5×105 cells per dish one day earlier were transfected with 10 µg of plasmid DNA by calcium phosphate method . .. At 48 h post-transfection, cells were washed with 1× PBS and treated with 1% NP40 lysis buffer (100 mM Tris [pH 7.5], 150 mM NaCl, 20 mM EDTA, 1% NP40, 0.5% Na deoxycholate and protease inhibitors [Roche Diagnostics]), followed by centrifugation at 20,000×g and 4°C for 30 min to obtain cell lysates .

Article Title: Optimization of non-denaturing protein extraction conditions for plant PPR proteins
Article Snippet: 2-day cultures of Agrobacterium tumefaciens harbouring the desired constructs and the silencing-suppressor p19 plasmid [ ] were pelleted, diluted to OD600 0.1–0.5 in infiltration buffer [0.5% (w/v) D-glucose; 10 mM MES; 10 mM MgCl2 ; 0.1 mM acetosyringone], and co-infiltrated in young N . benthamiana leaves. .. The initial extraction buffer used in this manuscript was slightly modified from the one used previously for chloroplastic PPR immunoprecipitation from stable transformed A . thaliana plants [ ], and the one for weak protein-protein interactions indicated in the μMACS Epitope Tag Protein Isolation Kit protocol (Miltenyi Biotec), plus EDTA-free protease inhibitor cocktail according to the manufacturer’s recommendations for very high proteolytic activity: 50 mM Tris-HCl pH7.5, 150 mM NaCl, 1% NP40 (Igepal CA630), 2x protease inhibitor mix cocktail without EDTA (EDTA-free Complete, Roche REF04 693 132 001).

Solubility:

Article Title: E1^E4-mediated keratin phosphorylation and ubiquitylation: a mechanism for keratin depletion in HPV16-infected epithelium
Article Snippet: To assess protein solubility, cell extracts were sequentially fractionated in 1% NP40, 1% empigen and 9 M urea. .. For analysis of active kinases, cell extracts were prepared in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% NP40, 1 mM Vanadate and 1 tablet of miniprotease inhibitor cocktail (Roche) per 10 ml.

Protein Purification:

Article Title: Substrate ectodomain is critical for substrate preference and inhibition of ?-secretase
Article Snippet: The cell suspension was mixed with an equal amount of lysis buffer (50 mM PIPES (pH 7.0), 250 mM sucrose, 2% NP40 and 2 × protease inhibitor cocktail (Roche Diagnostics)) and incubated on ice for 1 h. After ultracentrifugation at 100,000 × g centrifugation for 1 h, the supernatant was agitated overnight with Profinity eXact (S189 subtilisin-immobilized) resin (Bio-Rad). .. The Profinity eXact tag protein purification system offers purification of recombinant proteins with a native N terminus.

Chromatography:

Article Title: The histone chaperone sNASP binds a conserved peptide motif within the globular core of histone H3 through its TPR repeats
Article Snippet: Protein expression and purification sNASP was expressed in bacteria as an N-terminal (His)6 fusion construct and was purified over Nickel-NTA beads (GE Healthcare) in 300 mM sodium chloride, 50 mM Tris–HCl pH 8.0, 0.1% NP40, 20 mM imidazole and Complete protease inhibitor cocktail (Roche). .. Cleaved protein was further purified by ion-exchange using a ResourceQ column (GE Healthcare) before gel filtration chromatography in 200 mM sodium chloride, 20 mM Hepes-KOH pH 7.6 and 1 mM EDTA using a 26/60 Superdex 200 column (GE Healthcare).

Immunoprecipitation:

Article Title: ERG-associated protein with SET domain (ESET)-Oct4 interaction regulates pluripotency and represses the trophectoderm lineage
Article Snippet: .. Endogenous immunoprecipitation To analyse SUMOylated ESET, one confluent 10 cm dish of ES cells was lysed in 100 μl buffer containing 50 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA, 15 mM MgCl2, 0.75% sodium deoxycholate, 1% NP40 supplemented with protease inhibitors (Roche, Basel, Switzerland), 1:100 diluted phosphatase inhibitors I and II (Sigma, St Louis, MO, USA) and 20 mM NEM (Sigma, St Louis, MO, USA). .. Where indicated, lysis buffer was also added with 50 μg/ml of ethidium bromide.

Article Title: Stoichiometry of HLA Class II-Invariant Chain Oligomers
Article Snippet: .. Immunoprecipitation, western blotting, and immunofluorescence microscopy Cells were lysed with 1% NP40 or with 1% Triton X-100 in Tris-buffered saline pH 7.4 containing protease inhibitors (Complete, Roche Diagnostics, Mannheim Germany). ..

Article Title: A novel Leishmania infantum nuclear phosphoprotein Lepp12 which stimulates IL1-beta synthesis in THP-1 transfectants
Article Snippet: Promastigote lysates used to stimulate phosphorylation of recombinant Lepp12 were obtained as supernatants of centrifugation at 20,600 g for 15 min at 4°C after lysis of PBS-washed cells for 30 min at 4°C in water containing 1% NP40, 1 mM NaVO4 , 25 mM beta-glycerophosphate, 50 microM NaF, 2.5 mM NaPPi and 1 tablet (per 1 ml) of complete protease-inhibitor-cocktail (Roche, Meylan France). .. For immunoprecipitation experiments 0.1 ml of nuclear extract was first incubated with 23 microL of immunopurified anti-Lepp12 antibodies (or control, irrelevant human antibodies) for 5 h at 4°C under gentle agitation, then after adding 20 microL settled volume of mixed (1:4, v/v) protein A sepharose/sepharose 4B, incubation continued overnight as before.

Article Title: Optimization of non-denaturing protein extraction conditions for plant PPR proteins
Article Snippet: .. The initial extraction buffer used in this manuscript was slightly modified from the one used previously for chloroplastic PPR immunoprecipitation from stable transformed A . thaliana plants [ ], and the one for weak protein-protein interactions indicated in the μMACS Epitope Tag Protein Isolation Kit protocol (Miltenyi Biotec), plus EDTA-free protease inhibitor cocktail according to the manufacturer’s recommendations for very high proteolytic activity: 50 mM Tris-HCl pH7.5, 150 mM NaCl, 1% NP40 (Igepal CA630), 2x protease inhibitor mix cocktail without EDTA (EDTA-free Complete, Roche REF04 693 132 001). .. As a result of this manuscript, for an optimized composition of the extraction buffer, suitable for PPR proteins, the initial extraction buffer was complemented with: 1x phosphatase inhibitor (PhosSTOP, Roche), 50 μM MG132 proteasome inhibitor (C2211, SIGMA), 5 mM ATP and 1 mM PMSF.

Article Title: E1^E4-mediated keratin phosphorylation and ubiquitylation: a mechanism for keratin depletion in HPV16-infected epithelium
Article Snippet: Paragraph title: Fractionation, immunoprecipitation and immunoblotting ... For analysis of active kinases, cell extracts were prepared in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% NP40, 1 mM Vanadate and 1 tablet of miniprotease inhibitor cocktail (Roche) per 10 ml.

Fractionation:

Article Title: E1^E4-mediated keratin phosphorylation and ubiquitylation: a mechanism for keratin depletion in HPV16-infected epithelium
Article Snippet: Paragraph title: Fractionation, immunoprecipitation and immunoblotting ... For analysis of active kinases, cell extracts were prepared in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% NP40, 1 mM Vanadate and 1 tablet of miniprotease inhibitor cocktail (Roche) per 10 ml.

BAC Assay:

Article Title: Substrate ectodomain is critical for substrate preference and inhibition of ?-secretase
Article Snippet: γ-Secretase substrates γ-Secretase substrates were expressed as fusion proteins with an APP signal peptide and the Profinity eXact tag (Bio-Rad) in sf9 cells by using Bac-to-Bac Baculovirus Expression System (Invitrogen) ( ). .. The cell suspension was mixed with an equal amount of lysis buffer (50 mM PIPES (pH 7.0), 250 mM sucrose, 2% NP40 and 2 × protease inhibitor cocktail (Roche Diagnostics)) and incubated on ice for 1 h. After ultracentrifugation at 100,000 × g centrifugation for 1 h, the supernatant was agitated overnight with Profinity eXact (S189 subtilisin-immobilized) resin (Bio-Rad).

Staining:

Article Title: Intracellular Regulation of Cross-Presentation during Dendritic Cell Maturation
Article Snippet: Cells were lysed in 50mM Tris pH 7.4, 0.5% NP40, Roche protease inhibitors, 200µg/ml DNAse for 1h on ice. .. Free beads were stained for intact and degraded OVA with rabbit anti-OVA antiserum followed by A488-conjugated goat anti-rabbit IgG antibody.

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Roche np40 buffer
    Leoligin increases ABCA1 and ABCG1, but not SR-B1 protein expression. THP-1 macrophages were treated with leoligin (LEO, 10 μM), pioglitazone (PIO, 10 μM), or solvent control (DMSO) for 24 h. Then, cells were lysed with <t>NP40</t> buffer. A 20 μg amount of total protein was analyzed by Western blot using anti-ABCA1 (A), -ABCG1 (B), or -SR-B1 (C) antibodies. Band intensities of four independent experiments were quantified. The bar graphs represent mean ± SD, and the statistical evaluation was performed by one-way ANOVA with the Bonferroni post-test. * p
    Np40 Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 74 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/np40 buffer/product/Roche
    Average 99 stars, based on 74 article reviews
    Price from $9.99 to $1999.99
    np40 buffer - by Bioz Stars, 2020-03
    99/100 stars
      Buy from Supplier

    np40  (Roche)
    99
    Roche np40
    Effect of C-terminal E domains and prM protein on the recognition of E protein by different mouse anti-E mAbs. (A) Binding specificity of five mouse anti-E mAbs including GR (4G2 and DEN2-12), CR (DEN3-3), and DENV4 TS (1H10-6-7 and 1H10-5-7) mAbs. Western blot analysis was performed by using cell lysates derived from C6/36 cells infected with each of the four DENV serotypes, WNV or JEV. The size of molecular weight markers is shown in kDa. (B,C) Dot blot binding assay using these five mAbs to recognize WT E protein (expressed by prME), E protein alone and mutant E proteins containing C-terminal truncations (expressed by prME- or E-based constructs) in 1% <t>NP40</t> lysis buffer (NP40). Layout of the dot blot assay and the binding by mixed mAbs are shown in (B). Decreasing amount of native WT E protein in 1% NP40 lysis buffer (column B) as well as mixtures containing decreasing amount of native WT E protein in 1% NP40 lysis buffer and increasing amount of denatured WT E protein in reducing (R) buffer (column A) were also included to control for exposure and sensitivity of the assay signal. Relative intensities of each dot in columns A (black bars) and B (white bars) were shown below each membrane. Recognition indices of each mAb to mutant E protein = [intensity of mutant E dot/intensity of WT E dot] (recognized by a mAb) divided by [intensity of mutant E dot/intensity of WT E dot] (recognized by mixed mAbs) were shown in blue bars (column C, in the presence of prM protein) and yellow bars (column D, in the absence of prM protein) below each membrane [33] . Data are mean and standard errors from two experiments.
    Np40, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 201 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/np40/product/Roche
    Average 99 stars, based on 201 article reviews
    Price from $9.99 to $1999.99
    np40 - by Bioz Stars, 2020-03
    99/100 stars
      Buy from Supplier

    Image Search Results


    Leoligin increases ABCA1 and ABCG1, but not SR-B1 protein expression. THP-1 macrophages were treated with leoligin (LEO, 10 μM), pioglitazone (PIO, 10 μM), or solvent control (DMSO) for 24 h. Then, cells were lysed with NP40 buffer. A 20 μg amount of total protein was analyzed by Western blot using anti-ABCA1 (A), -ABCG1 (B), or -SR-B1 (C) antibodies. Band intensities of four independent experiments were quantified. The bar graphs represent mean ± SD, and the statistical evaluation was performed by one-way ANOVA with the Bonferroni post-test. * p

    Journal: Journal of Natural Products

    Article Title: Leoligin, the Major Lignan from Edelweiss (Leontopodium nivale subsp. alpinum), Promotes Cholesterol Efflux from THP-1 Macrophages

    doi: 10.1021/acs.jnatprod.6b00227

    Figure Lengend Snippet: Leoligin increases ABCA1 and ABCG1, but not SR-B1 protein expression. THP-1 macrophages were treated with leoligin (LEO, 10 μM), pioglitazone (PIO, 10 μM), or solvent control (DMSO) for 24 h. Then, cells were lysed with NP40 buffer. A 20 μg amount of total protein was analyzed by Western blot using anti-ABCA1 (A), -ABCG1 (B), or -SR-B1 (C) antibodies. Band intensities of four independent experiments were quantified. The bar graphs represent mean ± SD, and the statistical evaluation was performed by one-way ANOVA with the Bonferroni post-test. * p

    Article Snippet: Western Blot Analysis THP-1 macrophages treated as indicated in the figure legends were washed with cold PBS (4 °C) and lysed with NP40 buffer (150 mM NaCl; 50 mM HEPES (pH 7.4); 1% NP40) containing a protease inhibitor mixture (1% Complete (Roche); 1% phenylmethylsulfonyl fluoride; 0.5% Na3 VO4 ; 0.5% NaF).

    Techniques: Expressing, Western Blot

    Effect of C-terminal E domains and prM protein on the recognition of E protein by different mouse anti-E mAbs. (A) Binding specificity of five mouse anti-E mAbs including GR (4G2 and DEN2-12), CR (DEN3-3), and DENV4 TS (1H10-6-7 and 1H10-5-7) mAbs. Western blot analysis was performed by using cell lysates derived from C6/36 cells infected with each of the four DENV serotypes, WNV or JEV. The size of molecular weight markers is shown in kDa. (B,C) Dot blot binding assay using these five mAbs to recognize WT E protein (expressed by prME), E protein alone and mutant E proteins containing C-terminal truncations (expressed by prME- or E-based constructs) in 1% NP40 lysis buffer (NP40). Layout of the dot blot assay and the binding by mixed mAbs are shown in (B). Decreasing amount of native WT E protein in 1% NP40 lysis buffer (column B) as well as mixtures containing decreasing amount of native WT E protein in 1% NP40 lysis buffer and increasing amount of denatured WT E protein in reducing (R) buffer (column A) were also included to control for exposure and sensitivity of the assay signal. Relative intensities of each dot in columns A (black bars) and B (white bars) were shown below each membrane. Recognition indices of each mAb to mutant E protein = [intensity of mutant E dot/intensity of WT E dot] (recognized by a mAb) divided by [intensity of mutant E dot/intensity of WT E dot] (recognized by mixed mAbs) were shown in blue bars (column C, in the presence of prM protein) and yellow bars (column D, in the absence of prM protein) below each membrane [33] . Data are mean and standard errors from two experiments.

    Journal: PLoS ONE

    Article Title: C-Terminal Helical Domains of Dengue Virus Type 4 E Protein Affect the Expression/Stability of prM Protein and Conformation of prM and E Proteins

    doi: 10.1371/journal.pone.0052600

    Figure Lengend Snippet: Effect of C-terminal E domains and prM protein on the recognition of E protein by different mouse anti-E mAbs. (A) Binding specificity of five mouse anti-E mAbs including GR (4G2 and DEN2-12), CR (DEN3-3), and DENV4 TS (1H10-6-7 and 1H10-5-7) mAbs. Western blot analysis was performed by using cell lysates derived from C6/36 cells infected with each of the four DENV serotypes, WNV or JEV. The size of molecular weight markers is shown in kDa. (B,C) Dot blot binding assay using these five mAbs to recognize WT E protein (expressed by prME), E protein alone and mutant E proteins containing C-terminal truncations (expressed by prME- or E-based constructs) in 1% NP40 lysis buffer (NP40). Layout of the dot blot assay and the binding by mixed mAbs are shown in (B). Decreasing amount of native WT E protein in 1% NP40 lysis buffer (column B) as well as mixtures containing decreasing amount of native WT E protein in 1% NP40 lysis buffer and increasing amount of denatured WT E protein in reducing (R) buffer (column A) were also included to control for exposure and sensitivity of the assay signal. Relative intensities of each dot in columns A (black bars) and B (white bars) were shown below each membrane. Recognition indices of each mAb to mutant E protein = [intensity of mutant E dot/intensity of WT E dot] (recognized by a mAb) divided by [intensity of mutant E dot/intensity of WT E dot] (recognized by mixed mAbs) were shown in blue bars (column C, in the presence of prM protein) and yellow bars (column D, in the absence of prM protein) below each membrane [33] . Data are mean and standard errors from two experiments.

    Article Snippet: At 48 h post-transfection, cells were washed with 1× PBS and treated with 1% NP40 lysis buffer (100 mM Tris [pH 7.5], 150 mM NaCl, 20 mM EDTA, 1% NP40, 0.5% Na deoxycholate and protease inhibitors [Roche Diagnostics]), followed by centrifugation at 20,000×g and 4°C for 30 min to obtain cell lysates .

    Techniques: Binding Assay, Western Blot, Derivative Assay, Infection, Molecular Weight, Dot Blot, Mutagenesis, Construct, Lysis

    Effect of E protein on the recognition of prM protein by human anti-prM mAbs. (A) Binding specificity of 4 human anti-prM mAbs including 2 CR (DVB59.3 and DVB18.5) and 2 sCR (DVB65.5 and DVB32.4) mAbs was determined as in Figure 5 . (B) Dot blot binding assay using these 4 mAbs to recognize prM protein in the presence (expressed by prME) or absence (expressed by prM) of E protein in 1% NP40 lysis buffer (NP40). Decreasing amount of prM protein (expressed by prME) in 1% NP40 lysis buffer (column B, rows 3 to 7) and mixtures containing decreasing amount of prM protein in 1% NP40 lysis buffer and increasing amount of denatured prM protein in reducing (R) buffer (column A) were included to control for exposure and sensitivity of the assay signal, respectively. Twenty times more cell lysates derived from transfection of prM alone were loaded. Relative intensities of each dot in column A (black bars) and column B (blue bars) were shown below each membrane. Data are mean and standard errors from two experiments.

    Journal: PLoS ONE

    Article Title: C-Terminal Helical Domains of Dengue Virus Type 4 E Protein Affect the Expression/Stability of prM Protein and Conformation of prM and E Proteins

    doi: 10.1371/journal.pone.0052600

    Figure Lengend Snippet: Effect of E protein on the recognition of prM protein by human anti-prM mAbs. (A) Binding specificity of 4 human anti-prM mAbs including 2 CR (DVB59.3 and DVB18.5) and 2 sCR (DVB65.5 and DVB32.4) mAbs was determined as in Figure 5 . (B) Dot blot binding assay using these 4 mAbs to recognize prM protein in the presence (expressed by prME) or absence (expressed by prM) of E protein in 1% NP40 lysis buffer (NP40). Decreasing amount of prM protein (expressed by prME) in 1% NP40 lysis buffer (column B, rows 3 to 7) and mixtures containing decreasing amount of prM protein in 1% NP40 lysis buffer and increasing amount of denatured prM protein in reducing (R) buffer (column A) were included to control for exposure and sensitivity of the assay signal, respectively. Twenty times more cell lysates derived from transfection of prM alone were loaded. Relative intensities of each dot in column A (black bars) and column B (blue bars) were shown below each membrane. Data are mean and standard errors from two experiments.

    Article Snippet: At 48 h post-transfection, cells were washed with 1× PBS and treated with 1% NP40 lysis buffer (100 mM Tris [pH 7.5], 150 mM NaCl, 20 mM EDTA, 1% NP40, 0.5% Na deoxycholate and protease inhibitors [Roche Diagnostics]), followed by centrifugation at 20,000×g and 4°C for 30 min to obtain cell lysates .

    Techniques: Binding Assay, Dot Blot, Lysis, Derivative Assay, Transfection

    IFNγ Treatment Does Not Cause a Significant Accumulation of Polyubiquitin Conjugates during the Induction of Immunoproteasomes, Related to Figure 1 (A and B) Murine embryonic (A) and B8 (B) fibroblasts were stimulated with 100U/ml IFNγ for the indicated time periods. As a control for ubiquitin accumulation, cells were treated with the proteasome inhibitor lactacystin (10μM, 4hr) or vehicle (DMSO). The cells were then lysed by sonication in 25mM HEPES pH 7.4, 1mM ATP, 1mM DTT, 5mM MgCl 2 and 10% glycerol. Lysates were subsequently centrifuged for one hour at 100,000 x g. Protein concentrations of the lysates were determined using the DC Protein Assay (Biorad) and equal amounts of protein were separated by SDS-PAGE and analyzed by western blotting. α-tubulin served as a loading control. One representative experiment out of three independent experiments with similar outcomes is shown. The induction of LMP7 by IFNγ stimulation was confirmed by immunoblot analysis; GAPDH served as a loading control. The ubiquitin levels were determined by densitometric analyses (ImageJ) of five different western blots; shown are the mean values ± SD obtained after normalization to the loading control and relative to the value for unstimulated cells which was set to unity. (C and D) HeLa cells were treated with 100U/ml IFNγ as described (C) or with 10 μM lactacystin for 4 hr (D). The cells were lysed with nonionic detergent according to the methods described by Seifert et al. (20 mM TRIS-HCl, pH 7.5, 10 mM EDTA, 100 mM NaCl, 1% NP40, 10 μM MG-132, 5 mM NEM and Complete Protease Inhibitor Cocktail (Roche)) and immunblotted for ubiquitin (C, left). The ubiquitin levels, (relative to the β-actin control) were determined by densitometric analyses (ImageJ) as described (C, right). The mean values ± SEM for three replicates are shown (C).

    Journal: Cell

    Article Title: Immuno- and Constitutive Proteasomes Do Not Differ in Their Abilities to Degrade Ubiquitinated Proteins

    doi: 10.1016/j.cell.2013.01.037

    Figure Lengend Snippet: IFNγ Treatment Does Not Cause a Significant Accumulation of Polyubiquitin Conjugates during the Induction of Immunoproteasomes, Related to Figure 1 (A and B) Murine embryonic (A) and B8 (B) fibroblasts were stimulated with 100U/ml IFNγ for the indicated time periods. As a control for ubiquitin accumulation, cells were treated with the proteasome inhibitor lactacystin (10μM, 4hr) or vehicle (DMSO). The cells were then lysed by sonication in 25mM HEPES pH 7.4, 1mM ATP, 1mM DTT, 5mM MgCl 2 and 10% glycerol. Lysates were subsequently centrifuged for one hour at 100,000 x g. Protein concentrations of the lysates were determined using the DC Protein Assay (Biorad) and equal amounts of protein were separated by SDS-PAGE and analyzed by western blotting. α-tubulin served as a loading control. One representative experiment out of three independent experiments with similar outcomes is shown. The induction of LMP7 by IFNγ stimulation was confirmed by immunoblot analysis; GAPDH served as a loading control. The ubiquitin levels were determined by densitometric analyses (ImageJ) of five different western blots; shown are the mean values ± SD obtained after normalization to the loading control and relative to the value for unstimulated cells which was set to unity. (C and D) HeLa cells were treated with 100U/ml IFNγ as described (C) or with 10 μM lactacystin for 4 hr (D). The cells were lysed with nonionic detergent according to the methods described by Seifert et al. (20 mM TRIS-HCl, pH 7.5, 10 mM EDTA, 100 mM NaCl, 1% NP40, 10 μM MG-132, 5 mM NEM and Complete Protease Inhibitor Cocktail (Roche)) and immunblotted for ubiquitin (C, left). The ubiquitin levels, (relative to the β-actin control) were determined by densitometric analyses (ImageJ) as described (C, right). The mean values ± SEM for three replicates are shown (C).

    Article Snippet: The murine B8 cells and MEFs were stimulated with 200 U/ml murine recombinant IFNγ (Peprotech) for the indicated time points and lysed either in 20 mM TRIS-HCl, pH 7.5, 10 mM EDTA, 100 mM NaCl, 1% NP40, 10 μM MG-132, 5 mM NEM, and Complete Protease Inhibitor Cocktail (Roche) ( B), or in 10 mM Tris, 150 mM NaCl, 1% Triton X-100 ( C) and the amount of protein was determined with a Pierce BCA protein assay kit (Thermo scientific).

    Techniques: Sonication, DC Protein Assay, SDS Page, Western Blot, Protease Inhibitor

    Leoligin increases ABCA1 and ABCG1, but not SR-B1 protein expression. THP-1 macrophages were treated with leoligin (LEO, 10 μM), pioglitazone (PIO, 10 μM), or solvent control (DMSO) for 24 h. Then, cells were lysed with NP40 buffer. A 20 μg amount of total protein was analyzed by Western blot using anti-ABCA1 (A), -ABCG1 (B), or -SR-B1 (C) antibodies. Band intensities of four independent experiments were quantified. The bar graphs represent mean ± SD, and the statistical evaluation was performed by one-way ANOVA with the Bonferroni post-test. * p

    Journal: Journal of Natural Products

    Article Title: Leoligin, the Major Lignan from Edelweiss (Leontopodium nivale subsp. alpinum), Promotes Cholesterol Efflux from THP-1 Macrophages

    doi: 10.1021/acs.jnatprod.6b00227

    Figure Lengend Snippet: Leoligin increases ABCA1 and ABCG1, but not SR-B1 protein expression. THP-1 macrophages were treated with leoligin (LEO, 10 μM), pioglitazone (PIO, 10 μM), or solvent control (DMSO) for 24 h. Then, cells were lysed with NP40 buffer. A 20 μg amount of total protein was analyzed by Western blot using anti-ABCA1 (A), -ABCG1 (B), or -SR-B1 (C) antibodies. Band intensities of four independent experiments were quantified. The bar graphs represent mean ± SD, and the statistical evaluation was performed by one-way ANOVA with the Bonferroni post-test. * p

    Article Snippet: Western Blot Analysis THP-1 macrophages treated as indicated in the figure legends were washed with cold PBS (4 °C) and lysed with NP40 buffer (150 mM NaCl; 50 mM HEPES (pH 7.4); 1% NP40) containing a protease inhibitor mixture (1% Complete (Roche); 1% phenylmethylsulfonyl fluoride; 0.5% Na3 VO4 ; 0.5% NaF).

    Techniques: Expressing, Western Blot