sds lysis buffer  (Roche)


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    Structured Review

    Roche sds lysis buffer
    Functional characterization of modulation of LRRK2 at T1410. (A) A LanthaScreen assay was used to assess LRRK2 T1410A kinase activity. Titration of wildtype, G2019S, and T1410A (G2019S) LRRK2 proteins demonstrate that mutation T1410A does not affect phosphorylation of LRRKtide. (B) To investigate dimer formation HEK-293T cells were transfected with the indicated LRRK2 constructs. Cell pellets were split equally for lysis by freeze/thaw cycles directly in PBS or lysis with 1% <t>SDS</t> and PBS with sonication, and 10 ug of protein lysate was loaded onto a native gel (3–12% <t>Bis-Tris)</t> or an SDS gel (7% Tris acetate-SDS), respectively. LRRK2 complexes were visualized with the anti-HA antibody by Western blot with standard ECL. Dimer-sized structures are evident (signal from 480 to 550 kDa), monomeric LRRK2 (278 kDa) is only visible by overexposure (Supplemental Figure S1 , [25] ).Western blots representative of five independent experiments are shown. Normalization of the LRRK2 dimer to total LRRK2 protein (SDS-solubilized) was done using densitometry analysis. ** p
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    Images

    1) Product Images from "Identification and Characterization of a Leucine-Rich Repeat Kinase 2 (LRRK2) Consensus Phosphorylation Motif"

    Article Title: Identification and Characterization of a Leucine-Rich Repeat Kinase 2 (LRRK2) Consensus Phosphorylation Motif

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0013672

    Functional characterization of modulation of LRRK2 at T1410. (A) A LanthaScreen assay was used to assess LRRK2 T1410A kinase activity. Titration of wildtype, G2019S, and T1410A (G2019S) LRRK2 proteins demonstrate that mutation T1410A does not affect phosphorylation of LRRKtide. (B) To investigate dimer formation HEK-293T cells were transfected with the indicated LRRK2 constructs. Cell pellets were split equally for lysis by freeze/thaw cycles directly in PBS or lysis with 1% SDS and PBS with sonication, and 10 ug of protein lysate was loaded onto a native gel (3–12% Bis-Tris) or an SDS gel (7% Tris acetate-SDS), respectively. LRRK2 complexes were visualized with the anti-HA antibody by Western blot with standard ECL. Dimer-sized structures are evident (signal from 480 to 550 kDa), monomeric LRRK2 (278 kDa) is only visible by overexposure (Supplemental Figure S1 , [25] ).Western blots representative of five independent experiments are shown. Normalization of the LRRK2 dimer to total LRRK2 protein (SDS-solubilized) was done using densitometry analysis. ** p
    Figure Legend Snippet: Functional characterization of modulation of LRRK2 at T1410. (A) A LanthaScreen assay was used to assess LRRK2 T1410A kinase activity. Titration of wildtype, G2019S, and T1410A (G2019S) LRRK2 proteins demonstrate that mutation T1410A does not affect phosphorylation of LRRKtide. (B) To investigate dimer formation HEK-293T cells were transfected with the indicated LRRK2 constructs. Cell pellets were split equally for lysis by freeze/thaw cycles directly in PBS or lysis with 1% SDS and PBS with sonication, and 10 ug of protein lysate was loaded onto a native gel (3–12% Bis-Tris) or an SDS gel (7% Tris acetate-SDS), respectively. LRRK2 complexes were visualized with the anti-HA antibody by Western blot with standard ECL. Dimer-sized structures are evident (signal from 480 to 550 kDa), monomeric LRRK2 (278 kDa) is only visible by overexposure (Supplemental Figure S1 , [25] ).Western blots representative of five independent experiments are shown. Normalization of the LRRK2 dimer to total LRRK2 protein (SDS-solubilized) was done using densitometry analysis. ** p

    Techniques Used: Functional Assay, Activity Assay, Titration, Mutagenesis, Transfection, Construct, Lysis, Sonication, SDS-Gel, Western Blot

    2) Product Images from "Regulation of human papillomavirus type 31 gene expression during the differentiation-dependent life cycle through histone modifications and transcription factor binding"

    Article Title: Regulation of human papillomavirus type 31 gene expression during the differentiation-dependent life cycle through histone modifications and transcription factor binding

    Journal: Virology

    doi: 10.1016/j.virol.2007.12.011

    Western blot analysis of transcription factors present in whole cell lysates collected from primary human foreskin keratinocytes (HFKs) and HPV 31 cells grown in monolayer (0 hour) or suspended in methylcellulose for 7, 16, 24, and 40 hours. Cells pellets were resuspended in RIPA lysis buffer and analyzed on SDS-PAGE gels at examined by western analysis. Antibodies against Sp1, YY1, C/EBP-β, C/EBP-α, E2F2, Oct-1, and c-Jun were used to probe the membrane. GAPDH was used as a loading control.
    Figure Legend Snippet: Western blot analysis of transcription factors present in whole cell lysates collected from primary human foreskin keratinocytes (HFKs) and HPV 31 cells grown in monolayer (0 hour) or suspended in methylcellulose for 7, 16, 24, and 40 hours. Cells pellets were resuspended in RIPA lysis buffer and analyzed on SDS-PAGE gels at examined by western analysis. Antibodies against Sp1, YY1, C/EBP-β, C/EBP-α, E2F2, Oct-1, and c-Jun were used to probe the membrane. GAPDH was used as a loading control.

    Techniques Used: Western Blot, Lysis, SDS Page

    3) Product Images from "Dependence of Leucine-rich Repeat Kinase 2 (LRRK2) Kinase Activity on Dimerization *"

    Article Title: Dependence of Leucine-rich Repeat Kinase 2 (LRRK2) Kinase Activity on Dimerization *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.025437

    LRRK2 forms kinase-sensitive high molecular weight conformations. A , cell pellets derived from four lymphoblast cell lines (labeled Lymph1 to -4 ) were split equally for lysis by freeze/thaw cycles directly in PBS or lysis with 1% SDS and PBS with sonication,
    Figure Legend Snippet: LRRK2 forms kinase-sensitive high molecular weight conformations. A , cell pellets derived from four lymphoblast cell lines (labeled Lymph1 to -4 ) were split equally for lysis by freeze/thaw cycles directly in PBS or lysis with 1% SDS and PBS with sonication,

    Techniques Used: Molecular Weight, Derivative Assay, Labeling, Lysis, Sonication

    4) Product Images from "The LPS-inducible lncRNA Mirt2 is a negative regulator of inflammation"

    Article Title: The LPS-inducible lncRNA Mirt2 is a negative regulator of inflammation

    Journal: Nature Communications

    doi: 10.1038/s41467-017-02229-1

    Mirt2 inhibits TRAF6 oligomerization and auto-ubiquitination. a Silver-stained SDS-PAGE gel analysis of proteins in macrophages that are bound to biotinylated lncRNA-Mirt2. The highlighted regions were analyzed by mass spectrometry, identifying TRAF6 as a protein unique to Mirt2. b Immunoblotting analysis of proteins in macrophages bound to biotinylated Mirt2 using anti-TRAF6 antibody. c RNA immunoprecipitation (RIP) analysis to determine the recovery of Mirt2 in macrophages using anti-TRAF6 antibody. IgG served as control. Data represent the mean ± SEM of three independent experiments. * P
    Figure Legend Snippet: Mirt2 inhibits TRAF6 oligomerization and auto-ubiquitination. a Silver-stained SDS-PAGE gel analysis of proteins in macrophages that are bound to biotinylated lncRNA-Mirt2. The highlighted regions were analyzed by mass spectrometry, identifying TRAF6 as a protein unique to Mirt2. b Immunoblotting analysis of proteins in macrophages bound to biotinylated Mirt2 using anti-TRAF6 antibody. c RNA immunoprecipitation (RIP) analysis to determine the recovery of Mirt2 in macrophages using anti-TRAF6 antibody. IgG served as control. Data represent the mean ± SEM of three independent experiments. * P

    Techniques Used: Staining, SDS Page, Mass Spectrometry, Immunoprecipitation

    5) Product Images from "Motor axon navigation relies on Fidgetin-like 1–driven microtubule plus end dynamics"

    Article Title: Motor axon navigation relies on Fidgetin-like 1–driven microtubule plus end dynamics

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201604108

    The N-terminally truncated Fignl1 isoforms promote MT depolymerization at the cell cortex. (A) COS-7 cells transfected with Fignl1-HA or Fignl1Δ1–113-HA were treated with DMSO or latrunculin B 24 h posttransfection and labeled with phalloidin, HA, and tyrosinated tubulin (Tyr tubulin) antibodies. Right: Higher magnifications of boxed regions in corresponding panels. Dotted line outlines the plasma membrane. Arrow points out the colocalization between Fignl1Δ1–113 and cortical F-actin. Bracket indicates the reduced density of MTs beneath the cell cortex of Fignl1Δ1–113-HA–expressing cells. (B–D) Mean fluorescence intensity of F-actin and Fignl1-HA (B) or Fignl1Δ1–113-HA (C and D) in COS-7 cells treated with DMSO (B and C) or latrunculin B (D). Dist. from mb, distance from membrane. (E) MT fluorescence intensity beneath the cell cortex. NT, nontransfected. (F) Mean distance between MT plus ends and the plasma membrane (Dist. to mb). (G–M) Live TIRF recordings of MT behavior beneath the cell membrane of MEFs expressing α-tubulin–mCherry and Fignl1-GFP, Fignl1Δ1–113-GFP, or GFP. (G) Representative time series (left) and kymograms (right) of individual MT behavior at the cell membrane (dashed lines). White and yellow arrows, respectively, point at MT ends contacting the membrane or undergoing catastrophe. Black arrows are kymogram scale bars. (H–L and M) MT dynamic instability analyses. Catastrophe and rescue frequencies (I and L), shrinkage and growth rates (H and K), shrinking length (J), and the percentage of MT that never shrunk, grew, or paused (M) were calculated from kymograph analysis of at least 150 MTs per condition. **, P ≤ 0.01; ***, P ≤ 0.001; Kruskal–Wallis ANOVA test with Dunn’s post hoc test. Error bars are SEM. The total number of cells (E, F, and M) or MTs (M) analyzed per condition in three independent experiments is indicated under the histogram bar (E and F) or in the table (M). Nb., number. (N) Coomassie blue–stained SDS-PAGE gel showing purified His-Fignl1Δ1–113 (arrow). (O) In vitro MT-severing assay. Atto 565–labeled and biotinylated taxol-stabilized MTs were immobilized in perfusion chambers. GFP-spastin (top) or His-Fignl1Δ1–113 (middle and bottom) were diluted to 100 or 400 nM and perfused into the chambers. Panels are representative time frames of TIRF recordings acquired every 5 min over 30 min. Bars: (A) 20 µm; (G) 3 µm; (O) 5 µm.
    Figure Legend Snippet: The N-terminally truncated Fignl1 isoforms promote MT depolymerization at the cell cortex. (A) COS-7 cells transfected with Fignl1-HA or Fignl1Δ1–113-HA were treated with DMSO or latrunculin B 24 h posttransfection and labeled with phalloidin, HA, and tyrosinated tubulin (Tyr tubulin) antibodies. Right: Higher magnifications of boxed regions in corresponding panels. Dotted line outlines the plasma membrane. Arrow points out the colocalization between Fignl1Δ1–113 and cortical F-actin. Bracket indicates the reduced density of MTs beneath the cell cortex of Fignl1Δ1–113-HA–expressing cells. (B–D) Mean fluorescence intensity of F-actin and Fignl1-HA (B) or Fignl1Δ1–113-HA (C and D) in COS-7 cells treated with DMSO (B and C) or latrunculin B (D). Dist. from mb, distance from membrane. (E) MT fluorescence intensity beneath the cell cortex. NT, nontransfected. (F) Mean distance between MT plus ends and the plasma membrane (Dist. to mb). (G–M) Live TIRF recordings of MT behavior beneath the cell membrane of MEFs expressing α-tubulin–mCherry and Fignl1-GFP, Fignl1Δ1–113-GFP, or GFP. (G) Representative time series (left) and kymograms (right) of individual MT behavior at the cell membrane (dashed lines). White and yellow arrows, respectively, point at MT ends contacting the membrane or undergoing catastrophe. Black arrows are kymogram scale bars. (H–L and M) MT dynamic instability analyses. Catastrophe and rescue frequencies (I and L), shrinkage and growth rates (H and K), shrinking length (J), and the percentage of MT that never shrunk, grew, or paused (M) were calculated from kymograph analysis of at least 150 MTs per condition. **, P ≤ 0.01; ***, P ≤ 0.001; Kruskal–Wallis ANOVA test with Dunn’s post hoc test. Error bars are SEM. The total number of cells (E, F, and M) or MTs (M) analyzed per condition in three independent experiments is indicated under the histogram bar (E and F) or in the table (M). Nb., number. (N) Coomassie blue–stained SDS-PAGE gel showing purified His-Fignl1Δ1–113 (arrow). (O) In vitro MT-severing assay. Atto 565–labeled and biotinylated taxol-stabilized MTs were immobilized in perfusion chambers. GFP-spastin (top) or His-Fignl1Δ1–113 (middle and bottom) were diluted to 100 or 400 nM and perfused into the chambers. Panels are representative time frames of TIRF recordings acquired every 5 min over 30 min. Bars: (A) 20 µm; (G) 3 µm; (O) 5 µm.

    Techniques Used: Transfection, Labeling, Expressing, Fluorescence, Staining, SDS Page, Purification, In Vitro

    6) Product Images from "Nebulette is a powerful cytolinker organizing desmin and actin in mouse hearts"

    Article Title: Nebulette is a powerful cytolinker organizing desmin and actin in mouse hearts

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E16-04-0237

    Direct protein–protein interaction of nebulette and full-length desmin. A specific biochemical interaction of nebulette to desmin was detected under buffer conditions that restrict desmin filament maturation by sucrose gradient sedimentation. Soluble desmin oligomers and nebulette M1–5 recombinant proteins were precleared from small aggregates by ultracentrifugation. Desmin alone (A) or nebulette alone (B) or in a 1:1 M ratio mixture (C) were incubated for 1 h at 37°C and placed on top of 10–70% sucrose gradients made in a buffer (5 mM Tris-HCl, 1 mM EDTA, and 0.1 mM EGTA, pH 8.4) that favors desmin oligomer formation. After a 5 h sedimentation at 42,000 rpm, fractions (1–10 and pellet) were collected and analyzed by 12% SDS–PAGE and Western blot. Arrows point to bands for desmin or nebulette M1–5, migrating at their expected molecular weights of 52 and 25 kDa, respectively.
    Figure Legend Snippet: Direct protein–protein interaction of nebulette and full-length desmin. A specific biochemical interaction of nebulette to desmin was detected under buffer conditions that restrict desmin filament maturation by sucrose gradient sedimentation. Soluble desmin oligomers and nebulette M1–5 recombinant proteins were precleared from small aggregates by ultracentrifugation. Desmin alone (A) or nebulette alone (B) or in a 1:1 M ratio mixture (C) were incubated for 1 h at 37°C and placed on top of 10–70% sucrose gradients made in a buffer (5 mM Tris-HCl, 1 mM EDTA, and 0.1 mM EGTA, pH 8.4) that favors desmin oligomer formation. After a 5 h sedimentation at 42,000 rpm, fractions (1–10 and pellet) were collected and analyzed by 12% SDS–PAGE and Western blot. Arrows point to bands for desmin or nebulette M1–5, migrating at their expected molecular weights of 52 and 25 kDa, respectively.

    Techniques Used: Sedimentation, Recombinant, Incubation, SDS Page, Western Blot

    7) Product Images from "Berberine Chloride is an Alphavirus Inhibitor That Targets Nucleocapsid Assembly"

    Article Title: Berberine Chloride is an Alphavirus Inhibitor That Targets Nucleocapsid Assembly

    Journal: mBio

    doi: 10.1128/mBio.01382-20

    BBC treatment of infected cells decreases virus specific infectivity. BHK-21 cells were infected with SFV at an MOI of 10 and treated with 0.1% DMSO or 20 μM BBC at 4 hpi, and the cell media were collected at 8.5 hpi. (A) An aliquot of each sample was used to determine infectious particle number by plaque assay. The remaining sample was pelleted through a 20% sucrose cushion and resuspended in buffer. Parallel aliquots were analyzed for particle amounts by (B) SDS-PAGE and WB using a MAb to E2 or (C) used for RNA isolation and gRNA quantitation by RT-qPCR. For specific infectivity determination, the ratio of total infectious particle number to absolute E2 signal or to absolute gRNA copies (D and E, respectively) was expressed relative to DMSO treatment. The graphs represent the means with individual data points plotted for six independent experiments. Individual data points are paired for each treatment for each replicate, and paired points are displayed in the same color. The representative E2 WB and the quantitated signal displayed below in panel B is the sample corresponding to the orange point in panels A, C, D, and E. To assess the statistical significance between DMSO versus BBC treatment, two-tailed Wilcoxon matched-pairs signed rank tests were performed in panels A and C and two-tailed paired t tests were performed in panels D and E. ***, P
    Figure Legend Snippet: BBC treatment of infected cells decreases virus specific infectivity. BHK-21 cells were infected with SFV at an MOI of 10 and treated with 0.1% DMSO or 20 μM BBC at 4 hpi, and the cell media were collected at 8.5 hpi. (A) An aliquot of each sample was used to determine infectious particle number by plaque assay. The remaining sample was pelleted through a 20% sucrose cushion and resuspended in buffer. Parallel aliquots were analyzed for particle amounts by (B) SDS-PAGE and WB using a MAb to E2 or (C) used for RNA isolation and gRNA quantitation by RT-qPCR. For specific infectivity determination, the ratio of total infectious particle number to absolute E2 signal or to absolute gRNA copies (D and E, respectively) was expressed relative to DMSO treatment. The graphs represent the means with individual data points plotted for six independent experiments. Individual data points are paired for each treatment for each replicate, and paired points are displayed in the same color. The representative E2 WB and the quantitated signal displayed below in panel B is the sample corresponding to the orange point in panels A, C, D, and E. To assess the statistical significance between DMSO versus BBC treatment, two-tailed Wilcoxon matched-pairs signed rank tests were performed in panels A and C and two-tailed paired t tests were performed in panels D and E. ***, P

    Techniques Used: Infection, Plaque Assay, SDS Page, Western Blot, Isolation, Quantitation Assay, Quantitative RT-PCR, Two Tailed Test

    BBC does not affect structural protein synthesis or processing. (A) Steady-state structural protein production. BHK-21 cells were infected with ppSFV at an MOI of 10 and treated with dimethyl sulfoxide (DMSO) or BBC from 4 to 8.5 hpi. Cells were then solubilized in Triton X-100-containing lysis buffer and centrifuged to obtain detergent-insoluble pellets and lysates. Samples were analyzed by SDS-PAGE and Western blotting (WB) using polyclonal antibody (PAb) to E2 and E1 and monoclonal antibody (MAb) to Cp. Histone H3 and β-tubulin served as loading controls for the detergent-insoluble and cytoplasmic extracts, respectively. Treatment labels are as follows: D, 0.1% DMSO; 10, 10 μM BBC; 20, 20 μM BBC; 50, 50 μM BBC. (B) Pulse-chase analysis of protein synthesis and processing. BHK-21 cells were infected with ppSFV at an MOI of 10, treated with DMSO or BBC at 4 hpi, pulse labeled for 30 min with [ 35 S]methionine/cysteine at 5 hpi, and chased for the indicated times, all in the continued presence of DMSO or BBC. At each time point, the cells were lysed, and the envelope proteins were immunoprecipitated with a PAb to E2 and E1. Samples were analyzed by SDS-PAGE and fluorography. (C) WB analysis of E2 to Cp ratio of pelleted particles. BHK-21 cells were infected with ppSFV at an MOI of 10 and treated with 0.1% DMSO or 20 μM BBC at 4 hpi, and the cell media were collected at 8.5 hpi. Particles were pelleted through a 20% sucrose cushion and resuspended in buffer. Samples were analyzed by SDS-PAGE and WB using MAb to E2 and PAb to Cp. The results shown in panels A, B, and C are representative of three independent experiments.
    Figure Legend Snippet: BBC does not affect structural protein synthesis or processing. (A) Steady-state structural protein production. BHK-21 cells were infected with ppSFV at an MOI of 10 and treated with dimethyl sulfoxide (DMSO) or BBC from 4 to 8.5 hpi. Cells were then solubilized in Triton X-100-containing lysis buffer and centrifuged to obtain detergent-insoluble pellets and lysates. Samples were analyzed by SDS-PAGE and Western blotting (WB) using polyclonal antibody (PAb) to E2 and E1 and monoclonal antibody (MAb) to Cp. Histone H3 and β-tubulin served as loading controls for the detergent-insoluble and cytoplasmic extracts, respectively. Treatment labels are as follows: D, 0.1% DMSO; 10, 10 μM BBC; 20, 20 μM BBC; 50, 50 μM BBC. (B) Pulse-chase analysis of protein synthesis and processing. BHK-21 cells were infected with ppSFV at an MOI of 10, treated with DMSO or BBC at 4 hpi, pulse labeled for 30 min with [ 35 S]methionine/cysteine at 5 hpi, and chased for the indicated times, all in the continued presence of DMSO or BBC. At each time point, the cells were lysed, and the envelope proteins were immunoprecipitated with a PAb to E2 and E1. Samples were analyzed by SDS-PAGE and fluorography. (C) WB analysis of E2 to Cp ratio of pelleted particles. BHK-21 cells were infected with ppSFV at an MOI of 10 and treated with 0.1% DMSO or 20 μM BBC at 4 hpi, and the cell media were collected at 8.5 hpi. Particles were pelleted through a 20% sucrose cushion and resuspended in buffer. Samples were analyzed by SDS-PAGE and WB using MAb to E2 and PAb to Cp. The results shown in panels A, B, and C are representative of three independent experiments.

    Techniques Used: Infection, Lysis, SDS Page, Western Blot, Pulse Chase, Labeling, Immunoprecipitation

    8) Product Images from "Distinct functions of ATG16L1 isoforms in membrane binding and LC3B lipidation in autophagy-related processes"

    Article Title: Distinct functions of ATG16L1 isoforms in membrane binding and LC3B lipidation in autophagy-related processes

    Journal: Nature cell biology

    doi: 10.1038/s41556-019-0274-9

    ATG16L1 membrane binding is required for efficient in vitro LC3 and GABARAP lipidation a , In vitro lipidation reactions containing 0.5 μM ATG7, 1 μM ATG3, 0.5 μM ATG12–5 or 0.25 μM ATG12-5–16L1β/β FII/α VRV/α FII VRV, 10 μM LC3B, 3 mM lipid (liposomes composed of 10 mol% bl-PI, 50 mol% DOPE and 40 mol% POPC were sonicated or extruded to 400 nm), 1 mM dithiothreitol and 1 mM ATP were incubated at 30 °C for 90 min. Reactions were subjected to SDS–PAGE and Coomassie blue stain. b , Lipidation reactions were performed as in (a) but using sonicated or extruded liposomes composed of 10 mol% b1PI, 20 mol% DOPE and 70 mol% POPC. c , The extent of LC3B-II formation in (a) and (b) was determined and plotted as percentage of total LC3B based on n=3 independent experiments and presented as mean±SEM. Statistical analyses was performed by Two-way Anova followed by Bonferonis multiple comparison test. d , In vitro lipidation reactions containing 0.5 μM ATG7, 1 μM ATG3, 0.2 μM ATG12–5 or 0.1 μM ATG12-5–16L1β/β FII/α VRV/α FII VRV, 10 μM GABARAP, 3 mM lipid (liposomes composed of 10 mol% bl-PI, 50 mol% DOPE and 40 mol% POPC were sonicated or extruded to 400 nm), 1 mM dithiothreitol and 1 mM ATP were incubated at 30 °C for 90 min. Reactions were subjected to SDS–PAGE and Coomassie blue stain. e , Lipidation reactions were performed as in (d) but using sonicated or extruded liposomes composed of 10 mol% bl-PI, 20 mol% DOPE and 70 mol% POPC. Note the presence of accumulated GABARAP-AMP. f .
    Figure Legend Snippet: ATG16L1 membrane binding is required for efficient in vitro LC3 and GABARAP lipidation a , In vitro lipidation reactions containing 0.5 μM ATG7, 1 μM ATG3, 0.5 μM ATG12–5 or 0.25 μM ATG12-5–16L1β/β FII/α VRV/α FII VRV, 10 μM LC3B, 3 mM lipid (liposomes composed of 10 mol% bl-PI, 50 mol% DOPE and 40 mol% POPC were sonicated or extruded to 400 nm), 1 mM dithiothreitol and 1 mM ATP were incubated at 30 °C for 90 min. Reactions were subjected to SDS–PAGE and Coomassie blue stain. b , Lipidation reactions were performed as in (a) but using sonicated or extruded liposomes composed of 10 mol% b1PI, 20 mol% DOPE and 70 mol% POPC. c , The extent of LC3B-II formation in (a) and (b) was determined and plotted as percentage of total LC3B based on n=3 independent experiments and presented as mean±SEM. Statistical analyses was performed by Two-way Anova followed by Bonferonis multiple comparison test. d , In vitro lipidation reactions containing 0.5 μM ATG7, 1 μM ATG3, 0.2 μM ATG12–5 or 0.1 μM ATG12-5–16L1β/β FII/α VRV/α FII VRV, 10 μM GABARAP, 3 mM lipid (liposomes composed of 10 mol% bl-PI, 50 mol% DOPE and 40 mol% POPC were sonicated or extruded to 400 nm), 1 mM dithiothreitol and 1 mM ATP were incubated at 30 °C for 90 min. Reactions were subjected to SDS–PAGE and Coomassie blue stain. e , Lipidation reactions were performed as in (d) but using sonicated or extruded liposomes composed of 10 mol% bl-PI, 20 mol% DOPE and 70 mol% POPC. Note the presence of accumulated GABARAP-AMP. f .

    Techniques Used: Binding Assay, In Vitro, Sonication, Incubation, SDS Page, Staining

    The C-terminal end of ATG16L1 can compensate for WIPI2b function a , Size-exclusion chromatography fractions of purified WIPI2b visualized by Coomassie blue stain (molecular mass standard is indicated). (n=2 independent experiments). b, In vitro lipidation reactions containing 0.5 μM ATG7, 1 μM ATG3, 0.1 μM ATG12-5–16L1β or ATG12-5–16L1α VRV, 10 μM LC3B, 0.3 mM lipid (liposomes composed of 10 mol% bl-PI, 50 mol% DOPE, with/without 10 mol% PI(3)P and 40/30 mol% POPC extruded to 400nm), 1 mM dithiothreitol and 1 mM ATP were incubated at 30°C for 90 min in the absence or presence of 1 μM WIPI2b or WIPI2b R108E/R125E (EE). Reactions were subjected to SDS–PAGE and Coomassie blue stain. c, The extent of LC3B-II formation in (b) was determined and plotted as percentage of total LC3B from n=3 independent experiments and presented as mean±SEM. Statistical analyses were performed by Two-way Anova followed by Bonferonis multiple comparison test. d, LC3B/GABARAP lipidation in WT, WIPI2 KO, ATG16L1 KO or WIPI2/ATG16L1 double KO HEK293 cells rescued or not with EGFP-tagged ATG16L1 (aa 1–249), ATG16L1 (aa 1–249) F32A/I35A/I36A, ATG16L1α or ATG16L1β, treated as indicated for 2h. Cell lysates were immunoblotted against the indicated proteins. e, The levels of LC3B-II/Actin were quantified from immunoblots in (d) and normalized to WT cells in the starvation with Baf.A1 condition. Data is based on n=3 independent experiments and presented as mean±SEM. Statistical analyses were performed by Two-way Anova followed by Bonferonis multiple comparison test. f , LC3B/GABARAP lipidation in ATG16L1 KO or WIPI2/ATG16L1 double KO HEK293 cells rescued with EGFP-ATG16L1α or EGFP-ATG16L1β, treated as indicated for 2h. Cell lysates were immunoblotted against the indicated proteins (n=1 experiment). g .
    Figure Legend Snippet: The C-terminal end of ATG16L1 can compensate for WIPI2b function a , Size-exclusion chromatography fractions of purified WIPI2b visualized by Coomassie blue stain (molecular mass standard is indicated). (n=2 independent experiments). b, In vitro lipidation reactions containing 0.5 μM ATG7, 1 μM ATG3, 0.1 μM ATG12-5–16L1β or ATG12-5–16L1α VRV, 10 μM LC3B, 0.3 mM lipid (liposomes composed of 10 mol% bl-PI, 50 mol% DOPE, with/without 10 mol% PI(3)P and 40/30 mol% POPC extruded to 400nm), 1 mM dithiothreitol and 1 mM ATP were incubated at 30°C for 90 min in the absence or presence of 1 μM WIPI2b or WIPI2b R108E/R125E (EE). Reactions were subjected to SDS–PAGE and Coomassie blue stain. c, The extent of LC3B-II formation in (b) was determined and plotted as percentage of total LC3B from n=3 independent experiments and presented as mean±SEM. Statistical analyses were performed by Two-way Anova followed by Bonferonis multiple comparison test. d, LC3B/GABARAP lipidation in WT, WIPI2 KO, ATG16L1 KO or WIPI2/ATG16L1 double KO HEK293 cells rescued or not with EGFP-tagged ATG16L1 (aa 1–249), ATG16L1 (aa 1–249) F32A/I35A/I36A, ATG16L1α or ATG16L1β, treated as indicated for 2h. Cell lysates were immunoblotted against the indicated proteins. e, The levels of LC3B-II/Actin were quantified from immunoblots in (d) and normalized to WT cells in the starvation with Baf.A1 condition. Data is based on n=3 independent experiments and presented as mean±SEM. Statistical analyses were performed by Two-way Anova followed by Bonferonis multiple comparison test. f , LC3B/GABARAP lipidation in ATG16L1 KO or WIPI2/ATG16L1 double KO HEK293 cells rescued with EGFP-ATG16L1α or EGFP-ATG16L1β, treated as indicated for 2h. Cell lysates were immunoblotted against the indicated proteins (n=1 experiment). g .

    Techniques Used: Size-exclusion Chromatography, Purification, Staining, In Vitro, Incubation, SDS Page, Western Blot

    9) Product Images from "Mucin 1 (MUC1) is a novel partner for MAL2 in breast carcinoma cells"

    Article Title: Mucin 1 (MUC1) is a novel partner for MAL2 in breast carcinoma cells

    Journal: BMC Cell Biology

    doi: 10.1186/1471-2121-10-7

    Co-immunoprecipitation analyses to detect MAL2/MUC1 interactions in Triton X-100-soluble versus-insoluble fractions. MCF-10A/Myc-MAL2 cells were extracted with 1% Triton X-100 at 4°C, and subjected to sucrose gradient centrifugation . Antisera employed in Western blot analyses are shown at the left, and sizes of detected proteins are shown at the right. (a) Fractions (Fr, as shown above the top panel) of 1 ml were collected, and aliquots from each were subjected to SDS-PAGE and Western blot analysis with MUC1 and MAL2 antisera. Vertical lines between fractions 8 and 9 distinguish samples loaded on different gels. (b) Pooled fractions (fractions 1–4, which include lipid rafts, fractions 5–8 and fractions 9–12) were immunoprecipitated with MUC1 or MAL2 antisera either alone or with MAL2 peptides (+Pep), as shown at the top of the panel. Immunoprecipitates were separated by SDS-PAGE and subjected to Western blot analysis with MUC1 monoclonal antibody. Results shown represent those obtained from 3 independent experiments.
    Figure Legend Snippet: Co-immunoprecipitation analyses to detect MAL2/MUC1 interactions in Triton X-100-soluble versus-insoluble fractions. MCF-10A/Myc-MAL2 cells were extracted with 1% Triton X-100 at 4°C, and subjected to sucrose gradient centrifugation . Antisera employed in Western blot analyses are shown at the left, and sizes of detected proteins are shown at the right. (a) Fractions (Fr, as shown above the top panel) of 1 ml were collected, and aliquots from each were subjected to SDS-PAGE and Western blot analysis with MUC1 and MAL2 antisera. Vertical lines between fractions 8 and 9 distinguish samples loaded on different gels. (b) Pooled fractions (fractions 1–4, which include lipid rafts, fractions 5–8 and fractions 9–12) were immunoprecipitated with MUC1 or MAL2 antisera either alone or with MAL2 peptides (+Pep), as shown at the top of the panel. Immunoprecipitates were separated by SDS-PAGE and subjected to Western blot analysis with MUC1 monoclonal antibody. Results shown represent those obtained from 3 independent experiments.

    Techniques Used: Immunoprecipitation, Gradient Centrifugation, Western Blot, SDS Page

    Effect of MβCD treatment on MUC1 and MAL2 distribution in lipid raft fractions . SK-BR-3 and MDA-MB-435(M) cells were treated (+Mβ), or not (-Mβ), with 20 mM MβCD, extracted with 1% Triton X-100 at 4°C, and subjected to sucrose gradient centrifugation. Twelve fractions of 1 ml (Fr, as shown above each top panel) were collected and 15 μl aliquots were subjected to SDS-PAGE and Western blot analysis with antibodies to MUC1, MAL2 or CAV1, as indicated at the left, with sizes of detected proteins indicated at the right. In all panels, vertical lines between fractions 8 and 9 distinguish samples loaded on different gels. In fractions 2–4, MUC1, MAL2 and CAV1 were not detected in SK-BR-3 cells, or were significantly reduced in MDA-MB-435 [M] cells following MβCD treatment. Results shown represent those obtained from 3 independent experiments.
    Figure Legend Snippet: Effect of MβCD treatment on MUC1 and MAL2 distribution in lipid raft fractions . SK-BR-3 and MDA-MB-435(M) cells were treated (+Mβ), or not (-Mβ), with 20 mM MβCD, extracted with 1% Triton X-100 at 4°C, and subjected to sucrose gradient centrifugation. Twelve fractions of 1 ml (Fr, as shown above each top panel) were collected and 15 μl aliquots were subjected to SDS-PAGE and Western blot analysis with antibodies to MUC1, MAL2 or CAV1, as indicated at the left, with sizes of detected proteins indicated at the right. In all panels, vertical lines between fractions 8 and 9 distinguish samples loaded on different gels. In fractions 2–4, MUC1, MAL2 and CAV1 were not detected in SK-BR-3 cells, or were significantly reduced in MDA-MB-435 [M] cells following MβCD treatment. Results shown represent those obtained from 3 independent experiments.

    Techniques Used: Multiple Displacement Amplification, Gradient Centrifugation, SDS Page, Western Blot

    Effect of ectopic Myc-MAL2 expression on MCF-10A cell morphology and intracellular localisation of MUC1 . (a) MCF-10A and MCF-10A/Myc-MAL2 cells were grown in culture and their morphologies compared by light microscopy (X100 magnification). (b) MCF-10A and MCF-10A/Myc-MAL2 cells were co-stained for MUC1 (left panel) and MAL2 (middle panel). Overlap between the MUC1 and MAL2 proteins are shown in merged images (right panel). Images shown are single horizontal x-y sections. (c) MCF-10A and MCF-10A/Myc-MAL2 cells were extracted with 1% Triton X-100 at 4°C, and centrifuged to equilibrium in sucrose density gradients. Twelve fractions of 1 ml were collected (Fr, as shown above each top panel), and aliquots were subjected to SDS-PAGE and Western blot analysis with MUC1, MAL2 or CAV1 antisera, as indicated at the left, with sizes of detected proteins indicated at the right. Vertical lines between fractions 8 and 9 distinguish samples loaded on different gels. Results shown represent those obtained from 3 independent experiments.
    Figure Legend Snippet: Effect of ectopic Myc-MAL2 expression on MCF-10A cell morphology and intracellular localisation of MUC1 . (a) MCF-10A and MCF-10A/Myc-MAL2 cells were grown in culture and their morphologies compared by light microscopy (X100 magnification). (b) MCF-10A and MCF-10A/Myc-MAL2 cells were co-stained for MUC1 (left panel) and MAL2 (middle panel). Overlap between the MUC1 and MAL2 proteins are shown in merged images (right panel). Images shown are single horizontal x-y sections. (c) MCF-10A and MCF-10A/Myc-MAL2 cells were extracted with 1% Triton X-100 at 4°C, and centrifuged to equilibrium in sucrose density gradients. Twelve fractions of 1 ml were collected (Fr, as shown above each top panel), and aliquots were subjected to SDS-PAGE and Western blot analysis with MUC1, MAL2 or CAV1 antisera, as indicated at the left, with sizes of detected proteins indicated at the right. Vertical lines between fractions 8 and 9 distinguish samples loaded on different gels. Results shown represent those obtained from 3 independent experiments.

    Techniques Used: Expressing, Light Microscopy, Staining, SDS Page, Western Blot

    Co-immunoprecipitation analyses verifying interactions between MAL2 and MUC1 . Total cell lysate from MCF-10A/Myc-MAL2 cells was immunoprecipitated with MUC1, c-Myc, or rabbit MAL2 antisera, either alone or with MAL2 peptides, as indicated above the top panel. Total cell lysate and immunoprecipitates were separated by SDS-PAGE and subjected to Western blot analyses with antisera against MUC1, MAL2, c-Myc and D52, as indicated at the left. Sizes of detected proteins are indicated at the right. Results shown are representative of at least 3 independent experiments.
    Figure Legend Snippet: Co-immunoprecipitation analyses verifying interactions between MAL2 and MUC1 . Total cell lysate from MCF-10A/Myc-MAL2 cells was immunoprecipitated with MUC1, c-Myc, or rabbit MAL2 antisera, either alone or with MAL2 peptides, as indicated above the top panel. Total cell lysate and immunoprecipitates were separated by SDS-PAGE and subjected to Western blot analyses with antisera against MUC1, MAL2, c-Myc and D52, as indicated at the left. Sizes of detected proteins are indicated at the right. Results shown are representative of at least 3 independent experiments.

    Techniques Used: Immunoprecipitation, SDS Page, Western Blot

    Distribution of MUC1, MAL2 and D52 in membrane fractions from breast cancer cell lines . (a) SK-BR-3, (b) MDA-MB-435(V) (c) MDA-MB-435(M) (d) MDA-MB-453(V) and (e) MDA-MB-453(M) cells were extracted with 1% Triton X-100 at 4°C, and subjected to centrifugation to equilibrium in sucrose density gradients. Twelve 1 ml fractions (Fr, shown above each top panel) were collected from the top of the gradient and 15 μl aliquots from each were subjected to SDS-PAGE and Western blot analysis with antibodies against MUC1, MAL2 and D52, as indicated to the left of each panel. SK-BR-3 fractions (a) were also immunoblotted with CAV1 antibody. In all panels, vertical lines between fractions 8 and 9 distinguish samples loaded on different gels. Sizes of detected proteins are indicated at the right. Results shown represent those obtained from 2–3 independent experiments.
    Figure Legend Snippet: Distribution of MUC1, MAL2 and D52 in membrane fractions from breast cancer cell lines . (a) SK-BR-3, (b) MDA-MB-435(V) (c) MDA-MB-435(M) (d) MDA-MB-453(V) and (e) MDA-MB-453(M) cells were extracted with 1% Triton X-100 at 4°C, and subjected to centrifugation to equilibrium in sucrose density gradients. Twelve 1 ml fractions (Fr, shown above each top panel) were collected from the top of the gradient and 15 μl aliquots from each were subjected to SDS-PAGE and Western blot analysis with antibodies against MUC1, MAL2 and D52, as indicated to the left of each panel. SK-BR-3 fractions (a) were also immunoblotted with CAV1 antibody. In all panels, vertical lines between fractions 8 and 9 distinguish samples loaded on different gels. Sizes of detected proteins are indicated at the right. Results shown represent those obtained from 2–3 independent experiments.

    Techniques Used: Multiple Displacement Amplification, Centrifugation, SDS Page, Western Blot

    10) Product Images from "Motor axon navigation relies on Fidgetin-like 1–driven microtubule plus end dynamics"

    Article Title: Motor axon navigation relies on Fidgetin-like 1–driven microtubule plus end dynamics

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201604108

    The N-terminally truncated Fignl1 isoforms promote MT depolymerization at the cell cortex. (A) COS-7 cells transfected with Fignl1-HA or Fignl1Δ1–113-HA were treated with DMSO or latrunculin B 24 h posttransfection and labeled with phalloidin, HA, and tyrosinated tubulin (Tyr tubulin) antibodies. Right: Higher magnifications of boxed regions in corresponding panels. Dotted line outlines the plasma membrane. Arrow points out the colocalization between Fignl1Δ1–113 and cortical F-actin. Bracket indicates the reduced density of MTs beneath the cell cortex of Fignl1Δ1–113-HA–expressing cells. (B–D) Mean fluorescence intensity of F-actin and Fignl1-HA (B) or Fignl1Δ1–113-HA (C and D) in COS-7 cells treated with DMSO (B and C) or latrunculin B (D). Dist. from mb, distance from membrane. (E) MT fluorescence intensity beneath the cell cortex. NT, nontransfected. (F) Mean distance between MT plus ends and the plasma membrane (Dist. to mb). (G–M) Live TIRF recordings of MT behavior beneath the cell membrane of MEFs expressing α-tubulin–mCherry and Fignl1-GFP, Fignl1Δ1–113-GFP, or GFP. (G) Representative time series (left) and kymograms (right) of individual MT behavior at the cell membrane (dashed lines). White and yellow arrows, respectively, point at MT ends contacting the membrane or undergoing catastrophe. Black arrows are kymogram scale bars. (H–L and M) MT dynamic instability analyses. Catastrophe and rescue frequencies (I and L), shrinkage and growth rates (H and K), shrinking length (J), and the percentage of MT that never shrunk, grew, or paused (M) were calculated from kymograph analysis of at least 150 MTs per condition. **, P ≤ 0.01; ***, P ≤ 0.001; Kruskal–Wallis ANOVA test with Dunn’s post hoc test. Error bars are SEM. The total number of cells (E, F, and M) or MTs (M) analyzed per condition in three independent experiments is indicated under the histogram bar (E and F) or in the table (M). Nb., number. (N) Coomassie blue–stained SDS-PAGE gel showing purified His-Fignl1Δ1–113 (arrow). (O) In vitro MT-severing assay. Atto 565–labeled and biotinylated taxol-stabilized MTs were immobilized in perfusion chambers. GFP-spastin (top) or His-Fignl1Δ1–113 (middle and bottom) were diluted to 100 or 400 nM and perfused into the chambers. Panels are representative time frames of TIRF recordings acquired every 5 min over 30 min. Bars: (A) 20 µm; (G) 3 µm; (O) 5 µm.
    Figure Legend Snippet: The N-terminally truncated Fignl1 isoforms promote MT depolymerization at the cell cortex. (A) COS-7 cells transfected with Fignl1-HA or Fignl1Δ1–113-HA were treated with DMSO or latrunculin B 24 h posttransfection and labeled with phalloidin, HA, and tyrosinated tubulin (Tyr tubulin) antibodies. Right: Higher magnifications of boxed regions in corresponding panels. Dotted line outlines the plasma membrane. Arrow points out the colocalization between Fignl1Δ1–113 and cortical F-actin. Bracket indicates the reduced density of MTs beneath the cell cortex of Fignl1Δ1–113-HA–expressing cells. (B–D) Mean fluorescence intensity of F-actin and Fignl1-HA (B) or Fignl1Δ1–113-HA (C and D) in COS-7 cells treated with DMSO (B and C) or latrunculin B (D). Dist. from mb, distance from membrane. (E) MT fluorescence intensity beneath the cell cortex. NT, nontransfected. (F) Mean distance between MT plus ends and the plasma membrane (Dist. to mb). (G–M) Live TIRF recordings of MT behavior beneath the cell membrane of MEFs expressing α-tubulin–mCherry and Fignl1-GFP, Fignl1Δ1–113-GFP, or GFP. (G) Representative time series (left) and kymograms (right) of individual MT behavior at the cell membrane (dashed lines). White and yellow arrows, respectively, point at MT ends contacting the membrane or undergoing catastrophe. Black arrows are kymogram scale bars. (H–L and M) MT dynamic instability analyses. Catastrophe and rescue frequencies (I and L), shrinkage and growth rates (H and K), shrinking length (J), and the percentage of MT that never shrunk, grew, or paused (M) were calculated from kymograph analysis of at least 150 MTs per condition. **, P ≤ 0.01; ***, P ≤ 0.001; Kruskal–Wallis ANOVA test with Dunn’s post hoc test. Error bars are SEM. The total number of cells (E, F, and M) or MTs (M) analyzed per condition in three independent experiments is indicated under the histogram bar (E and F) or in the table (M). Nb., number. (N) Coomassie blue–stained SDS-PAGE gel showing purified His-Fignl1Δ1–113 (arrow). (O) In vitro MT-severing assay. Atto 565–labeled and biotinylated taxol-stabilized MTs were immobilized in perfusion chambers. GFP-spastin (top) or His-Fignl1Δ1–113 (middle and bottom) were diluted to 100 or 400 nM and perfused into the chambers. Panels are representative time frames of TIRF recordings acquired every 5 min over 30 min. Bars: (A) 20 µm; (G) 3 µm; (O) 5 µm.

    Techniques Used: Transfection, Labeling, Expressing, Fluorescence, Staining, SDS Page, Purification, In Vitro

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    Transfection:

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    Incubation:

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    Article Snippet: .. Infected or uninfected subconfluent monolayers of 17Cl-1 cells were lysed with 2× sample buffer containing SDS, protease and phosphatase inhibitors (Roche, Basal, Switzerland), β-mercaptoethanol, and Pierce Universal Nuclease (Thermo Fisher Scientific). .. Boiled cell lysates were separated by SDS-PAGE on polyacrylamide gels.

    Sonication:

    Article Title: Salmonella Type III Effector AvrA Stabilizes Cell Tight Junctions to Inhibit Inflammation in Intestinal Epithelial Cells
    Article Snippet: Equal volumes of total cell lysate were separated by SDS-PAGE, transferred to nitrocellulose, and processed for immunoblotting with Mouse anti-α-catenin, Rabbit anti-claudin-1, Mouse anti-occludin-1, Mouse anti-ZO-1 antibodies from Zymed Laboratories Inc.(South San Francisco, CA), or E-cadherin antibodies from BD Transduction Laboratories( Franklin Lakes, NJ). .. Immunoblotting for AvrA Bacteria were lysed in lysis buffer [in mM: 50 Tris, pH 8.0, 150 NaCl, 5 EDTA with a complete Mini protease inhibitor cocktail (1 tablet/10 ml, Roche), and 1% Triton X-100], and sonicated. .. Equal amounts of total proteins were loaded, separated by SDS-PAGE, and processed for immunoblotting with custom-made AvrA antibody.

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  • 86
    Roche sds lysis buffer
    Functional characterization of modulation of LRRK2 at T1410. (A) A LanthaScreen assay was used to assess LRRK2 T1410A kinase activity. Titration of wildtype, G2019S, and T1410A (G2019S) LRRK2 proteins demonstrate that mutation T1410A does not affect phosphorylation of LRRKtide. (B) To investigate dimer formation HEK-293T cells were transfected with the indicated LRRK2 constructs. Cell pellets were split equally for lysis by freeze/thaw cycles directly in PBS or lysis with 1% <t>SDS</t> and PBS with sonication, and 10 ug of protein lysate was loaded onto a native gel (3–12% <t>Bis-Tris)</t> or an SDS gel (7% Tris acetate-SDS), respectively. LRRK2 complexes were visualized with the anti-HA antibody by Western blot with standard ECL. Dimer-sized structures are evident (signal from 480 to 550 kDa), monomeric LRRK2 (278 kDa) is only visible by overexposure (Supplemental Figure S1 , [25] ).Western blots representative of five independent experiments are shown. Normalization of the LRRK2 dimer to total LRRK2 protein (SDS-solubilized) was done using densitometry analysis. ** p
    Sds Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Roche 2 de lysis buffer
    Silver-stained <t>2-DE</t> images of proteins differentially expressed in tuberculous and ulcerative colitis patients. Enlarged images of proteins found to be differentially expressed by silver-stained 2-DE from Fig. 1 . Arrows indicate spots downregulated or upregulated and numbers correspond to the spot numbers shown in Table 1 . Graphs show numerical data obtained from images in the left panel. Data were analyzed using the PDQuest software (n=3) * p
    2 De Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Roche np 40 lysis buffer
    4-BPB tags a large number of proteins by click chemistry. Parasites were treated with either DMSO, 4-PPB or 4-APB and then lysed. The supernatants were then treated with the alkyne- or azide-conjugated biotin-tags, and click chemistry was used to label the targets of the 4-BPB click derivatives. These targets were then purified using streptavidin-conjugated beads and resolved by SDS-PAGE and silver staining. The resulting bands in the silver-stained gel were cut out for identification by LC-MS/MS. Parasites were either treated with DMSO, 0.5 µM 4-PPB or 1 µM 4-APB for 10 minutes, followed by lysis in 1% <t>NP-40</t> buffer. Each of the lysates was treated with either the 4-APB-tag or the 4-PPB-tag during the click reaction. Lanes are labeled with the inhibitor (DMSO (lanes 1,2), 4-PPB (lanes 3,4), 4-APB (lanes 5,6)) and the click tag (4PPB-tag (lanes 1,3,5) or 4-APB-tag (lanes 2,4,6)) that the corresponding sample was treated with.
    Np 40 Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Functional characterization of modulation of LRRK2 at T1410. (A) A LanthaScreen assay was used to assess LRRK2 T1410A kinase activity. Titration of wildtype, G2019S, and T1410A (G2019S) LRRK2 proteins demonstrate that mutation T1410A does not affect phosphorylation of LRRKtide. (B) To investigate dimer formation HEK-293T cells were transfected with the indicated LRRK2 constructs. Cell pellets were split equally for lysis by freeze/thaw cycles directly in PBS or lysis with 1% SDS and PBS with sonication, and 10 ug of protein lysate was loaded onto a native gel (3–12% Bis-Tris) or an SDS gel (7% Tris acetate-SDS), respectively. LRRK2 complexes were visualized with the anti-HA antibody by Western blot with standard ECL. Dimer-sized structures are evident (signal from 480 to 550 kDa), monomeric LRRK2 (278 kDa) is only visible by overexposure (Supplemental Figure S1 , [25] ).Western blots representative of five independent experiments are shown. Normalization of the LRRK2 dimer to total LRRK2 protein (SDS-solubilized) was done using densitometry analysis. ** p

    Journal: PLoS ONE

    Article Title: Identification and Characterization of a Leucine-Rich Repeat Kinase 2 (LRRK2) Consensus Phosphorylation Motif

    doi: 10.1371/journal.pone.0013672

    Figure Lengend Snippet: Functional characterization of modulation of LRRK2 at T1410. (A) A LanthaScreen assay was used to assess LRRK2 T1410A kinase activity. Titration of wildtype, G2019S, and T1410A (G2019S) LRRK2 proteins demonstrate that mutation T1410A does not affect phosphorylation of LRRKtide. (B) To investigate dimer formation HEK-293T cells were transfected with the indicated LRRK2 constructs. Cell pellets were split equally for lysis by freeze/thaw cycles directly in PBS or lysis with 1% SDS and PBS with sonication, and 10 ug of protein lysate was loaded onto a native gel (3–12% Bis-Tris) or an SDS gel (7% Tris acetate-SDS), respectively. LRRK2 complexes were visualized with the anti-HA antibody by Western blot with standard ECL. Dimer-sized structures are evident (signal from 480 to 550 kDa), monomeric LRRK2 (278 kDa) is only visible by overexposure (Supplemental Figure S1 , [25] ).Western blots representative of five independent experiments are shown. Normalization of the LRRK2 dimer to total LRRK2 protein (SDS-solubilized) was done using densitometry analysis. ** p

    Article Snippet: Cell pellets were resuspended with either native lysis buffer (phosphate-buffered saline (PBS), pH 7.4, 1x protease and phosphatase inhibitors (Roche Applied Science), lysed by four cycles of freezing and thawing, and analyzed on a 3–12% bis-tris Blue native polyacrylamide gel (Invitrogen) or lysates combined with SDS-lysis buffer (PBS, pH 7.4, 1% SDS, 1xprotease and phosphatase inhibitors (Roche Applied Science) and sonicated with 10% power for 10 s on a Branson dismembranator and analyzed on a 7% Tris acetate SDS gel (Invitrogen).

    Techniques: Functional Assay, Activity Assay, Titration, Mutagenesis, Transfection, Construct, Lysis, Sonication, SDS-Gel, Western Blot

    Western blot analysis of transcription factors present in whole cell lysates collected from primary human foreskin keratinocytes (HFKs) and HPV 31 cells grown in monolayer (0 hour) or suspended in methylcellulose for 7, 16, 24, and 40 hours. Cells pellets were resuspended in RIPA lysis buffer and analyzed on SDS-PAGE gels at examined by western analysis. Antibodies against Sp1, YY1, C/EBP-β, C/EBP-α, E2F2, Oct-1, and c-Jun were used to probe the membrane. GAPDH was used as a loading control.

    Journal: Virology

    Article Title: Regulation of human papillomavirus type 31 gene expression during the differentiation-dependent life cycle through histone modifications and transcription factor binding

    doi: 10.1016/j.virol.2007.12.011

    Figure Lengend Snippet: Western blot analysis of transcription factors present in whole cell lysates collected from primary human foreskin keratinocytes (HFKs) and HPV 31 cells grown in monolayer (0 hour) or suspended in methylcellulose for 7, 16, 24, and 40 hours. Cells pellets were resuspended in RIPA lysis buffer and analyzed on SDS-PAGE gels at examined by western analysis. Antibodies against Sp1, YY1, C/EBP-β, C/EBP-α, E2F2, Oct-1, and c-Jun were used to probe the membrane. GAPDH was used as a loading control.

    Article Snippet: Cells were then resuspended in SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris, pH 8.1) in the presence of Complete Mini protease inhibitors (Roche Diagnostics, NJ, USA) and incubated on ice for ten minutes.

    Techniques: Western Blot, Lysis, SDS Page

    Silver-stained 2-DE images of proteins differentially expressed in tuberculous and ulcerative colitis patients. Enlarged images of proteins found to be differentially expressed by silver-stained 2-DE from Fig. 1 . Arrows indicate spots downregulated or upregulated and numbers correspond to the spot numbers shown in Table 1 . Graphs show numerical data obtained from images in the left panel. Data were analyzed using the PDQuest software (n=3) * p

    Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

    Article Title: Proteomic Analysis of Colonic Mucosal Tissue from Tuberculous and Ulcerative Colitis Patients

    doi: 10.4196/kjpp.2012.16.3.193

    Figure Lengend Snippet: Silver-stained 2-DE images of proteins differentially expressed in tuberculous and ulcerative colitis patients. Enlarged images of proteins found to be differentially expressed by silver-stained 2-DE from Fig. 1 . Arrows indicate spots downregulated or upregulated and numbers correspond to the spot numbers shown in Table 1 . Graphs show numerical data obtained from images in the left panel. Data were analyzed using the PDQuest software (n=3) * p

    Article Snippet: Two-dimensional electrophoresis Isolated colon tissue was homogenized in 2-DE lysis buffer (8 M urea, 2 M thiourea, 65 mM dithiothreitol, 2% CHAPS, and 1× complete protease inhibitor cocktail) (Roche Applied Science, Germany) and centrifuged at 12,000× g for 10 min at 10℃.

    Techniques: Staining, Software

    Silver-stained 2-DE gels of the colonic proteome of normal, tuberculous and ulcerative colitis patients. Proteins from the colonic mucosa of patients were loaded on IPG pH 3~10 nonlinear (NL) strips and separated by 12% (w/v) polyacrylamide SDS-PAGE. Protein spots were visualized by silver staining. Arrows and numbers correspond to Table 1 . 2-DE, 2-dimensional electrophoresis; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TC, tuberculous colitis; UC, ulcerative colitis.

    Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

    Article Title: Proteomic Analysis of Colonic Mucosal Tissue from Tuberculous and Ulcerative Colitis Patients

    doi: 10.4196/kjpp.2012.16.3.193

    Figure Lengend Snippet: Silver-stained 2-DE gels of the colonic proteome of normal, tuberculous and ulcerative colitis patients. Proteins from the colonic mucosa of patients were loaded on IPG pH 3~10 nonlinear (NL) strips and separated by 12% (w/v) polyacrylamide SDS-PAGE. Protein spots were visualized by silver staining. Arrows and numbers correspond to Table 1 . 2-DE, 2-dimensional electrophoresis; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TC, tuberculous colitis; UC, ulcerative colitis.

    Article Snippet: Two-dimensional electrophoresis Isolated colon tissue was homogenized in 2-DE lysis buffer (8 M urea, 2 M thiourea, 65 mM dithiothreitol, 2% CHAPS, and 1× complete protease inhibitor cocktail) (Roche Applied Science, Germany) and centrifuged at 12,000× g for 10 min at 10℃.

    Techniques: Staining, SDS Page, Silver Staining, Electrophoresis, Polyacrylamide Gel Electrophoresis

    4-BPB tags a large number of proteins by click chemistry. Parasites were treated with either DMSO, 4-PPB or 4-APB and then lysed. The supernatants were then treated with the alkyne- or azide-conjugated biotin-tags, and click chemistry was used to label the targets of the 4-BPB click derivatives. These targets were then purified using streptavidin-conjugated beads and resolved by SDS-PAGE and silver staining. The resulting bands in the silver-stained gel were cut out for identification by LC-MS/MS. Parasites were either treated with DMSO, 0.5 µM 4-PPB or 1 µM 4-APB for 10 minutes, followed by lysis in 1% NP-40 buffer. Each of the lysates was treated with either the 4-APB-tag or the 4-PPB-tag during the click reaction. Lanes are labeled with the inhibitor (DMSO (lanes 1,2), 4-PPB (lanes 3,4), 4-APB (lanes 5,6)) and the click tag (4PPB-tag (lanes 1,3,5) or 4-APB-tag (lanes 2,4,6)) that the corresponding sample was treated with.

    Journal: PLoS ONE

    Article Title: 4-Bromophenacyl Bromide Specifically Inhibits Rhoptry Secretion during Toxoplasma Invasion

    doi: 10.1371/journal.pone.0008143

    Figure Lengend Snippet: 4-BPB tags a large number of proteins by click chemistry. Parasites were treated with either DMSO, 4-PPB or 4-APB and then lysed. The supernatants were then treated with the alkyne- or azide-conjugated biotin-tags, and click chemistry was used to label the targets of the 4-BPB click derivatives. These targets were then purified using streptavidin-conjugated beads and resolved by SDS-PAGE and silver staining. The resulting bands in the silver-stained gel were cut out for identification by LC-MS/MS. Parasites were either treated with DMSO, 0.5 µM 4-PPB or 1 µM 4-APB for 10 minutes, followed by lysis in 1% NP-40 buffer. Each of the lysates was treated with either the 4-APB-tag or the 4-PPB-tag during the click reaction. Lanes are labeled with the inhibitor (DMSO (lanes 1,2), 4-PPB (lanes 3,4), 4-APB (lanes 5,6)) and the click tag (4PPB-tag (lanes 1,3,5) or 4-APB-tag (lanes 2,4,6)) that the corresponding sample was treated with.

    Article Snippet: Parasites were spun down and washed three times in PBS, and resuspended in 1% NP-40 lysis buffer containing Complete (Roche) protease inhibitors.

    Techniques: Purification, SDS Page, Silver Staining, Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Lysis, Labeling