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Roche nonidet p‑40
Nonidet P‑40, supplied by Roche, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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nonidet p‑40 - by Bioz Stars, 2020-03
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Centrifugation:

Article Title: HIGH CHLOROPHYLL FLUORESCENCE145 Binds to and Stabilizes the psaA 5′ UTR via a Newly Defined Repeat Motif in Embryophyta
Article Snippet: The chloroplast pellet was lysed by incubation for 15 min on ice in coimmunoprecipitation buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% [v/v] Nonidet P‑40, and Complete Protease Inhibitor Cocktail Tablets [Roche]). .. Membranes were then pelleted by centrifugation at 36,000 g for 30 min. One milligram of protein was incubated with 25 μL coimmunoprecipitation buffer-washed GFP-Trap-M beads (Chromotek) for 1 h at 4°C with rotation.

Protein Concentration:

Article Title: Defining the functional binding sites of interleukin 12 receptor β1 and interleukin 23 receptor to Janus kinases
Article Snippet: Subsequently cells were harvested and lysed in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1 mM NaF, 1 mM Na3 VO4 , 1% Nonidet P‑40, and 1% Triton X-100 supplemented with complete protease inhibitor cocktail tablets (Roche Diagnostics, Mannheim, Germany). .. Protein concentration of cell lysates was determined by bicinchoninic acid protein assay (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Protease Inhibitor:

Article Title: HIGH CHLOROPHYLL FLUORESCENCE145 Binds to and Stabilizes the psaA 5′ UTR via a Newly Defined Repeat Motif in Embryophyta
Article Snippet: .. The chloroplast pellet was lysed by incubation for 15 min on ice in coimmunoprecipitation buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% [v/v] Nonidet P‑40, and Complete Protease Inhibitor Cocktail Tablets [Roche]). .. Membranes were then pelleted by centrifugation at 36,000 g for 30 min. One milligram of protein was incubated with 25 μL coimmunoprecipitation buffer-washed GFP-Trap-M beads (Chromotek) for 1 h at 4°C with rotation.

Article Title: Defining the functional binding sites of interleukin 12 receptor β1 and interleukin 23 receptor to Janus kinases
Article Snippet: .. Subsequently cells were harvested and lysed in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1 mM NaF, 1 mM Na3 VO4 , 1% Nonidet P‑40, and 1% Triton X-100 supplemented with complete protease inhibitor cocktail tablets (Roche Diagnostics, Mannheim, Germany). .. Ba/F3-gp130 cell lines were washed three times with sterile PBS and incubated in serum-free DMEM for at least 4 h. Cells were stimulated with HIL‑23, HIL‑12, or HIL-6 as indicated, harvested, frozen in liquid nitrogen, and lysed as described.

Bicinchoninic Acid Protein Assay:

Article Title: Defining the functional binding sites of interleukin 12 receptor β1 and interleukin 23 receptor to Janus kinases
Article Snippet: Subsequently cells were harvested and lysed in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1 mM NaF, 1 mM Na3 VO4 , 1% Nonidet P‑40, and 1% Triton X-100 supplemented with complete protease inhibitor cocktail tablets (Roche Diagnostics, Mannheim, Germany). .. Protein concentration of cell lysates was determined by bicinchoninic acid protein assay (Thermo Fisher Scientific) according to the manufacturer’s instructions.

RNA Extraction:

Article Title: HIGH CHLOROPHYLL FLUORESCENCE145 Binds to and Stabilizes the psaA 5′ UTR via a Newly Defined Repeat Motif in Embryophyta
Article Snippet: The chloroplast pellet was lysed by incubation for 15 min on ice in coimmunoprecipitation buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% [v/v] Nonidet P‑40, and Complete Protease Inhibitor Cocktail Tablets [Roche]). .. The beads were washed three times with 0.5 mL coimmunoprecipitation buffer and were subsequently used for the SDS-PAGE and/or for the RNA extraction.

Dot Blot:

Article Title: HIGH CHLOROPHYLL FLUORESCENCE145 Binds to and Stabilizes the psaA 5′ UTR via a Newly Defined Repeat Motif in Embryophyta
Article Snippet: Paragraph title: RNA Coimmunoprecipitation and Slot-Blot Hybridization ... The chloroplast pellet was lysed by incubation for 15 min on ice in coimmunoprecipitation buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% [v/v] Nonidet P‑40, and Complete Protease Inhibitor Cocktail Tablets [Roche]).

Activation Assay:

Article Title: Defining the functional binding sites of interleukin 12 receptor β1 and interleukin 23 receptor to Janus kinases
Article Snippet: Stimulation assays For analysis of STAT3 activation in cotransfected fibrosarcoma cell lines, cells were starved for 16 h in serum-free medium. .. Subsequently cells were harvested and lysed in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1 mM NaF, 1 mM Na3 VO4 , 1% Nonidet P‑40, and 1% Triton X-100 supplemented with complete protease inhibitor cocktail tablets (Roche Diagnostics, Mannheim, Germany).

Incubation:

Article Title: HIGH CHLOROPHYLL FLUORESCENCE145 Binds to and Stabilizes the psaA 5′ UTR via a Newly Defined Repeat Motif in Embryophyta
Article Snippet: .. The chloroplast pellet was lysed by incubation for 15 min on ice in coimmunoprecipitation buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% [v/v] Nonidet P‑40, and Complete Protease Inhibitor Cocktail Tablets [Roche]). .. Membranes were then pelleted by centrifugation at 36,000 g for 30 min. One milligram of protein was incubated with 25 μL coimmunoprecipitation buffer-washed GFP-Trap-M beads (Chromotek) for 1 h at 4°C with rotation.

Article Title: Defining the functional binding sites of interleukin 12 receptor β1 and interleukin 23 receptor to Janus kinases
Article Snippet: Subsequently cells were harvested and lysed in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1 mM NaF, 1 mM Na3 VO4 , 1% Nonidet P‑40, and 1% Triton X-100 supplemented with complete protease inhibitor cocktail tablets (Roche Diagnostics, Mannheim, Germany). .. Ba/F3-gp130 cell lines were washed three times with sterile PBS and incubated in serum-free DMEM for at least 4 h. Cells were stimulated with HIL‑23, HIL‑12, or HIL-6 as indicated, harvested, frozen in liquid nitrogen, and lysed as described.

Ethanol Precipitation:

Article Title: HIGH CHLOROPHYLL FLUORESCENCE145 Binds to and Stabilizes the psaA 5′ UTR via a Newly Defined Repeat Motif in Embryophyta
Article Snippet: The chloroplast pellet was lysed by incubation for 15 min on ice in coimmunoprecipitation buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% [v/v] Nonidet P‑40, and Complete Protease Inhibitor Cocktail Tablets [Roche]). .. For the slot-blot hybridization, RNA from the pellet und supernatant was extracted by phenol-chloroform treatment and subsequent ethanol precipitation.

SDS Page:

Article Title: HIGH CHLOROPHYLL FLUORESCENCE145 Binds to and Stabilizes the psaA 5′ UTR via a Newly Defined Repeat Motif in Embryophyta
Article Snippet: The chloroplast pellet was lysed by incubation for 15 min on ice in coimmunoprecipitation buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% [v/v] Nonidet P‑40, and Complete Protease Inhibitor Cocktail Tablets [Roche]). .. The beads were washed three times with 0.5 mL coimmunoprecipitation buffer and were subsequently used for the SDS-PAGE and/or for the RNA extraction.

Isolation:

Article Title: HIGH CHLOROPHYLL FLUORESCENCE145 Binds to and Stabilizes the psaA 5′ UTR via a Newly Defined Repeat Motif in Embryophyta
Article Snippet: Chloroplasts were isolated from the wild-type and hcf145-2comgfp plants as described previously ( ). .. The chloroplast pellet was lysed by incubation for 15 min on ice in coimmunoprecipitation buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% [v/v] Nonidet P‑40, and Complete Protease Inhibitor Cocktail Tablets [Roche]).

Hybridization:

Article Title: HIGH CHLOROPHYLL FLUORESCENCE145 Binds to and Stabilizes the psaA 5′ UTR via a Newly Defined Repeat Motif in Embryophyta
Article Snippet: Paragraph title: RNA Coimmunoprecipitation and Slot-Blot Hybridization ... The chloroplast pellet was lysed by incubation for 15 min on ice in coimmunoprecipitation buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% [v/v] Nonidet P‑40, and Complete Protease Inhibitor Cocktail Tablets [Roche]).

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  • 99
    Roche nonidet p 40 buffer
    Steady-state ubiquitination of TRAIL-R1 on lysine residue 273 by an endogenous machinery. A , MCF-7 Casp-3  cells were transfected to express FLAG-ubiquitin, together with mRFP only (−), or with mRFP-chimeras of WT TRAIL-R1 ( WT ) or its K273A lysine mutant ( K / A ). MARCH-8.HA cDNA (+) or an empty control vector (−) were additionally transfected as indicated. Cells were lysed in Nonidet P-40 buffer, TRAIL-R1 was isolated with anti(α)-mRFP antibody and immunoprecipitates ( IP ) were analyzed by immunoblotting ( IB ) with α-mRFP antibody to detect TRAIL-R1, α-FLAG antibody to detect ubiquitin and α-HA antibody to detect MARCH-8.  Asterisk  denotes the heavy chain of the antibody used for IP.  Solid  and  open arrowheads  indicate, respectively, TRAIL-R1.mRFP and mRFP only. Blot is representative of 4 independent experiments.  B , MCF-7 Casp-3  cells were transfected to express FLAG-ubiquitin, together with either mRFP only (−), with mRFP chimeras of WT TRAIL-R1 ( WT ) or the K273A TRAIL-R1 mutant ( K / A ), or with a truncated TRAIL-R1 lacking the C-terminal 116 residues (ΔWT). TRAIL-R1 was isolated with α-mRFP antibody and immunoprecipitates were analyzed by immunoblotting with α-mRFP antibody to detect TRAIL-R1 and with α-FLAG antibody to detect ubiquitin. Data shown are representative of two independent experiments.  C , MCF-7 Casp-3  cells were transfected to express FLAG-ubiquitin, together with mRFP only (−), or with mRFP-chimeras of WT TRAIL-R1 ( WT ) or its K273A lysine mutant ( K / A ). Cells were lysed by boiling in SDS, Nonidet P-40 buffer was added in excess and immunoprecipitation of TRAIL-R1 and analysis were performed as outlined for  panel A. Asterisk  denotes the heavy chain of the antibody used for IP.  Solid  and  open arrowheads  indicate, respectively, TRAIL-R1.mRFP and mRFP only. The blot is representative of 2 independent experiments.  D , alignment of primary amino acid sequence of part of the transmembrane segment ( italic ) and the remaining 14 residues of the cytoplasmic tail of the truncated TRAIL-R1 mutants used in  E . Relevant potential ubiquitination sites are shown in  bold. E , MCF-7 Casp-3  cells were transfected to express FLAG-ubiquitin, together with mRFP-tagged TRAIL-R1 WT or mutants shown in  D . TRAIL-R1 was isolated with α-mRFP antibody and immunoprecipitates were analyzed by immunoblotting with α-mRFP antibody to detect TRAIL-R1 and α-FLAG antibody to detect ubiquitin. The blot is representative of 2 independent experiments.  Asterisk  denotes the heavy chain of the antibody used for IP.
    Nonidet P 40 Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nonidet p 40 buffer/product/Roche
    Average 99 stars, based on 38 article reviews
    Price from $9.99 to $1999.99
    nonidet p 40 buffer - by Bioz Stars, 2020-03
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    92
    Roche te np 40 buffer
    Cdc48 and the ubiquilins reduce the detergent-insoluble fraction of Cps1. (A) The detergent-insoluble fraction of Cps1 decreases substantially in WT but less so cdc48-10 ddi1Δ cells. WT and cdc48-10 ddi1Δ cells expressing endogenous myc-Cps1 from its genomic locus were subjected to a cycloheximide (CHX)-chase degradation/sedimentation assay to resolve the amount of Triton X-100 detergent-insoluble Cps1 in the pellet fraction, as described under Materials and Methods . Samples of were removed after 0, 15, and 30 min of CHX treatment before processing to determine the relative amounts of myc-Cps1 in the total, pellet, and supernatant (see Supplemental Figure S6A) fractions by Western analysis with anti-myc antibodies. Anti-Snf7 antibodies were employed to assess the levels of total protein loaded and in subsequent quantitative analyses used for normalization of the results (unlike actin, Snf7 is not degraded upon CHX treatment). The amount of insoluble Cps1 in the pellet was normalized to the loading control, after which the percentage was calculated relative to the amount at 0 h. A representative experiment is shown in the top panel. A histogram of the quantification of three repetitive experiments is shown beneath. kDa = kilodaltons. (B) The detergent-insoluble fraction of eisosome protein, Pil1, does not change in cdc48-10 ddi1Δ cells. WT and cdc48-10 ddi1Δ yeast expressing myc-Pil1 from its genomic locus were subjected to the cycloheximide-chase degradation/sedimentation assay in A to resolve the amount of Triton X-100 detergent-insoluble Pil1 by Western analysis using anti-myc antibodies as in A. A histogram of showing the results of three repetitive experiments is shown beneath. (C) The level of <t>NP-40</t> detergent-insoluble Cps1 increases in ddi1Δ and ddi1Δ dsk2Δ rad23Δ cells. WT control cells (W303), ddi1Δ , and ddi1Δ dsk2Δ rad23Δ cells expressing HA-Cps1 were subjected to the same procedure as in A, and the percentage of insoluble Cps1 in the pellet fraction after 1 h was calculated after the normalization for gel loading. In the representative experiment, the level of NP-40-insoluble Cps1 increased in the ddi1Δ and ddi1Δ dsk2Δ rad23Δ pellet fractions by 34 and 78%, respectively. A histogram of three repetitive experiments is shown at the bottom.
    Te Np 40 Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/te np 40 buffer/product/Roche
    Average 92 stars, based on 2 article reviews
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    99
    Roche ripa buffer
    Sustained activation of N211Q DDR1b. COS1 cells expressing WT or N211Q DDR1b were serum-starved (18 h) before stimulation (2 h) with (+) 10 μg/ml rat tail collagen I ( Col. I ) or vehicle control (−), as described under “Experimental Procedures.” After stimulation, the media were aspirated, and the cells were washed thoroughly with warm <t>PBS.</t> The dishes were then supplemented with serum-free media and incubated at 37 °C for the indicated times. The cells were lysed with <t>RIPA</t> buffer, and the lysates were analyzed for receptor activation ( A ) and total receptor expression ( B ), as described in Fig. 2 . Black arrow in A indicates phosphorylated DDR1b, and white arrow in B indicates total DDR1b. Anti-Tyr(P) (α- pTyr ).
    Ripa Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 4719 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ripa buffer/product/Roche
    Average 99 stars, based on 4719 article reviews
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    99
    Roche nonidet p 40
    Analyses of HA/CD44-mediated HOXD10 and RhoGTPase expression in MDA-MB-231 cells.  Detection of HA/CD44-induced HOXD10 and RhoGTPase (RhoA/RhoC) expression in MDA-MB-231 cells was performed by solubilizing cells with 1% Nonidet P-40 (Nonidet P-40) buffer
    Nonidet P 40, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 488 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Steady-state ubiquitination of TRAIL-R1 on lysine residue 273 by an endogenous machinery. A , MCF-7 Casp-3  cells were transfected to express FLAG-ubiquitin, together with mRFP only (−), or with mRFP-chimeras of WT TRAIL-R1 ( WT ) or its K273A lysine mutant ( K / A ). MARCH-8.HA cDNA (+) or an empty control vector (−) were additionally transfected as indicated. Cells were lysed in Nonidet P-40 buffer, TRAIL-R1 was isolated with anti(α)-mRFP antibody and immunoprecipitates ( IP ) were analyzed by immunoblotting ( IB ) with α-mRFP antibody to detect TRAIL-R1, α-FLAG antibody to detect ubiquitin and α-HA antibody to detect MARCH-8.  Asterisk  denotes the heavy chain of the antibody used for IP.  Solid  and  open arrowheads  indicate, respectively, TRAIL-R1.mRFP and mRFP only. Blot is representative of 4 independent experiments.  B , MCF-7 Casp-3  cells were transfected to express FLAG-ubiquitin, together with either mRFP only (−), with mRFP chimeras of WT TRAIL-R1 ( WT ) or the K273A TRAIL-R1 mutant ( K / A ), or with a truncated TRAIL-R1 lacking the C-terminal 116 residues (ΔWT). TRAIL-R1 was isolated with α-mRFP antibody and immunoprecipitates were analyzed by immunoblotting with α-mRFP antibody to detect TRAIL-R1 and with α-FLAG antibody to detect ubiquitin. Data shown are representative of two independent experiments.  C , MCF-7 Casp-3  cells were transfected to express FLAG-ubiquitin, together with mRFP only (−), or with mRFP-chimeras of WT TRAIL-R1 ( WT ) or its K273A lysine mutant ( K / A ). Cells were lysed by boiling in SDS, Nonidet P-40 buffer was added in excess and immunoprecipitation of TRAIL-R1 and analysis were performed as outlined for  panel A. Asterisk  denotes the heavy chain of the antibody used for IP.  Solid  and  open arrowheads  indicate, respectively, TRAIL-R1.mRFP and mRFP only. The blot is representative of 2 independent experiments.  D , alignment of primary amino acid sequence of part of the transmembrane segment ( italic ) and the remaining 14 residues of the cytoplasmic tail of the truncated TRAIL-R1 mutants used in  E . Relevant potential ubiquitination sites are shown in  bold. E , MCF-7 Casp-3  cells were transfected to express FLAG-ubiquitin, together with mRFP-tagged TRAIL-R1 WT or mutants shown in  D . TRAIL-R1 was isolated with α-mRFP antibody and immunoprecipitates were analyzed by immunoblotting with α-mRFP antibody to detect TRAIL-R1 and α-FLAG antibody to detect ubiquitin. The blot is representative of 2 independent experiments.  Asterisk  denotes the heavy chain of the antibody used for IP.

    Journal: The Journal of Biological Chemistry

    Article Title: Ubiquitination by the Membrane-associated RING-CH-8 (MARCH-8) Ligase Controls Steady-state Cell Surface Expression of Tumor Necrosis Factor-related Apoptosis Inducing Ligand (TRAIL) Receptor 1 *

    doi: 10.1074/jbc.M112.448209

    Figure Lengend Snippet: Steady-state ubiquitination of TRAIL-R1 on lysine residue 273 by an endogenous machinery. A , MCF-7 Casp-3 cells were transfected to express FLAG-ubiquitin, together with mRFP only (−), or with mRFP-chimeras of WT TRAIL-R1 ( WT ) or its K273A lysine mutant ( K / A ). MARCH-8.HA cDNA (+) or an empty control vector (−) were additionally transfected as indicated. Cells were lysed in Nonidet P-40 buffer, TRAIL-R1 was isolated with anti(α)-mRFP antibody and immunoprecipitates ( IP ) were analyzed by immunoblotting ( IB ) with α-mRFP antibody to detect TRAIL-R1, α-FLAG antibody to detect ubiquitin and α-HA antibody to detect MARCH-8. Asterisk denotes the heavy chain of the antibody used for IP. Solid and open arrowheads indicate, respectively, TRAIL-R1.mRFP and mRFP only. Blot is representative of 4 independent experiments. B , MCF-7 Casp-3 cells were transfected to express FLAG-ubiquitin, together with either mRFP only (−), with mRFP chimeras of WT TRAIL-R1 ( WT ) or the K273A TRAIL-R1 mutant ( K / A ), or with a truncated TRAIL-R1 lacking the C-terminal 116 residues (ΔWT). TRAIL-R1 was isolated with α-mRFP antibody and immunoprecipitates were analyzed by immunoblotting with α-mRFP antibody to detect TRAIL-R1 and with α-FLAG antibody to detect ubiquitin. Data shown are representative of two independent experiments. C , MCF-7 Casp-3 cells were transfected to express FLAG-ubiquitin, together with mRFP only (−), or with mRFP-chimeras of WT TRAIL-R1 ( WT ) or its K273A lysine mutant ( K / A ). Cells were lysed by boiling in SDS, Nonidet P-40 buffer was added in excess and immunoprecipitation of TRAIL-R1 and analysis were performed as outlined for panel A. Asterisk denotes the heavy chain of the antibody used for IP. Solid and open arrowheads indicate, respectively, TRAIL-R1.mRFP and mRFP only. The blot is representative of 2 independent experiments. D , alignment of primary amino acid sequence of part of the transmembrane segment ( italic ) and the remaining 14 residues of the cytoplasmic tail of the truncated TRAIL-R1 mutants used in E . Relevant potential ubiquitination sites are shown in bold. E , MCF-7 Casp-3 cells were transfected to express FLAG-ubiquitin, together with mRFP-tagged TRAIL-R1 WT or mutants shown in D . TRAIL-R1 was isolated with α-mRFP antibody and immunoprecipitates were analyzed by immunoblotting with α-mRFP antibody to detect TRAIL-R1 and α-FLAG antibody to detect ubiquitin. The blot is representative of 2 independent experiments. Asterisk denotes the heavy chain of the antibody used for IP.

    Article Snippet: Western Blotting and Immunoprecipitation Cells were harvested and lysed in Nonidet P-40 buffer consisting of 50 mm Tris-HCl, pH 7.4, 1% Nonidet P-40, 150 mm NaCl, 1 mm PMSF and Complete Protease Inhibitors (Roche).

    Techniques: Transfection, Mutagenesis, Plasmid Preparation, Isolation, Immunoprecipitation, Sequencing

    Cdc48 and the ubiquilins reduce the detergent-insoluble fraction of Cps1. (A) The detergent-insoluble fraction of Cps1 decreases substantially in WT but less so cdc48-10 ddi1Δ cells. WT and cdc48-10 ddi1Δ cells expressing endogenous myc-Cps1 from its genomic locus were subjected to a cycloheximide (CHX)-chase degradation/sedimentation assay to resolve the amount of Triton X-100 detergent-insoluble Cps1 in the pellet fraction, as described under Materials and Methods . Samples of were removed after 0, 15, and 30 min of CHX treatment before processing to determine the relative amounts of myc-Cps1 in the total, pellet, and supernatant (see Supplemental Figure S6A) fractions by Western analysis with anti-myc antibodies. Anti-Snf7 antibodies were employed to assess the levels of total protein loaded and in subsequent quantitative analyses used for normalization of the results (unlike actin, Snf7 is not degraded upon CHX treatment). The amount of insoluble Cps1 in the pellet was normalized to the loading control, after which the percentage was calculated relative to the amount at 0 h. A representative experiment is shown in the top panel. A histogram of the quantification of three repetitive experiments is shown beneath. kDa = kilodaltons. (B) The detergent-insoluble fraction of eisosome protein, Pil1, does not change in cdc48-10 ddi1Δ cells. WT and cdc48-10 ddi1Δ yeast expressing myc-Pil1 from its genomic locus were subjected to the cycloheximide-chase degradation/sedimentation assay in A to resolve the amount of Triton X-100 detergent-insoluble Pil1 by Western analysis using anti-myc antibodies as in A. A histogram of showing the results of three repetitive experiments is shown beneath. (C) The level of NP-40 detergent-insoluble Cps1 increases in ddi1Δ and ddi1Δ dsk2Δ rad23Δ cells. WT control cells (W303), ddi1Δ , and ddi1Δ dsk2Δ rad23Δ cells expressing HA-Cps1 were subjected to the same procedure as in A, and the percentage of insoluble Cps1 in the pellet fraction after 1 h was calculated after the normalization for gel loading. In the representative experiment, the level of NP-40-insoluble Cps1 increased in the ddi1Δ and ddi1Δ dsk2Δ rad23Δ pellet fractions by 34 and 78%, respectively. A histogram of three repetitive experiments is shown at the bottom.

    Journal: Molecular Biology of the Cell

    Article Title: Cdc48 and ubiquilins confer selective anterograde protein sorting and entry into the multivesicular body in yeast

    doi: 10.1091/mbc.E17-11-0652

    Figure Lengend Snippet: Cdc48 and the ubiquilins reduce the detergent-insoluble fraction of Cps1. (A) The detergent-insoluble fraction of Cps1 decreases substantially in WT but less so cdc48-10 ddi1Δ cells. WT and cdc48-10 ddi1Δ cells expressing endogenous myc-Cps1 from its genomic locus were subjected to a cycloheximide (CHX)-chase degradation/sedimentation assay to resolve the amount of Triton X-100 detergent-insoluble Cps1 in the pellet fraction, as described under Materials and Methods . Samples of were removed after 0, 15, and 30 min of CHX treatment before processing to determine the relative amounts of myc-Cps1 in the total, pellet, and supernatant (see Supplemental Figure S6A) fractions by Western analysis with anti-myc antibodies. Anti-Snf7 antibodies were employed to assess the levels of total protein loaded and in subsequent quantitative analyses used for normalization of the results (unlike actin, Snf7 is not degraded upon CHX treatment). The amount of insoluble Cps1 in the pellet was normalized to the loading control, after which the percentage was calculated relative to the amount at 0 h. A representative experiment is shown in the top panel. A histogram of the quantification of three repetitive experiments is shown beneath. kDa = kilodaltons. (B) The detergent-insoluble fraction of eisosome protein, Pil1, does not change in cdc48-10 ddi1Δ cells. WT and cdc48-10 ddi1Δ yeast expressing myc-Pil1 from its genomic locus were subjected to the cycloheximide-chase degradation/sedimentation assay in A to resolve the amount of Triton X-100 detergent-insoluble Pil1 by Western analysis using anti-myc antibodies as in A. A histogram of showing the results of three repetitive experiments is shown beneath. (C) The level of NP-40 detergent-insoluble Cps1 increases in ddi1Δ and ddi1Δ dsk2Δ rad23Δ cells. WT control cells (W303), ddi1Δ , and ddi1Δ dsk2Δ rad23Δ cells expressing HA-Cps1 were subjected to the same procedure as in A, and the percentage of insoluble Cps1 in the pellet fraction after 1 h was calculated after the normalization for gel loading. In the representative experiment, the level of NP-40-insoluble Cps1 increased in the ddi1Δ and ddi1Δ dsk2Δ rad23Δ pellet fractions by 34 and 78%, respectively. A histogram of three repetitive experiments is shown at the bottom.

    Article Snippet: Next, 25 O.D.600 units were taken for coimmunoprecipitation (co-IP) in 1 ml of TE/NP-40 buffer with 10 μl of monoclonal anti-HA antibody (Roche) by incubation on rotator overnight at 4°C.

    Techniques: Expressing, Sedimentation, Western Blot

    Sustained activation of N211Q DDR1b. COS1 cells expressing WT or N211Q DDR1b were serum-starved (18 h) before stimulation (2 h) with (+) 10 μg/ml rat tail collagen I ( Col. I ) or vehicle control (−), as described under “Experimental Procedures.” After stimulation, the media were aspirated, and the cells were washed thoroughly with warm PBS. The dishes were then supplemented with serum-free media and incubated at 37 °C for the indicated times. The cells were lysed with RIPA buffer, and the lysates were analyzed for receptor activation ( A ) and total receptor expression ( B ), as described in Fig. 2 . Black arrow in A indicates phosphorylated DDR1b, and white arrow in B indicates total DDR1b. Anti-Tyr(P) (α- pTyr ).

    Journal: The Journal of Biological Chemistry

    Article Title: Glycosylation at Asn211 Regulates the Activation State of the Discoidin Domain Receptor 1 (DDR1) *

    doi: 10.1074/jbc.M113.541102

    Figure Lengend Snippet: Sustained activation of N211Q DDR1b. COS1 cells expressing WT or N211Q DDR1b were serum-starved (18 h) before stimulation (2 h) with (+) 10 μg/ml rat tail collagen I ( Col. I ) or vehicle control (−), as described under “Experimental Procedures.” After stimulation, the media were aspirated, and the cells were washed thoroughly with warm PBS. The dishes were then supplemented with serum-free media and incubated at 37 °C for the indicated times. The cells were lysed with RIPA buffer, and the lysates were analyzed for receptor activation ( A ) and total receptor expression ( B ), as described in Fig. 2 . Black arrow in A indicates phosphorylated DDR1b, and white arrow in B indicates total DDR1b. Anti-Tyr(P) (α- pTyr ).

    Article Snippet: To obtain the cell lysates, the cells were washed twice with cold PBS and then lysed in RIPA buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS) supplemented with protease inhibitors (Roche Applied Science, complete, Mini, EDTA-free), 10 mm NaF, and 1 mm sodium orthovanadate.

    Techniques: Activation Assay, Expressing, Incubation

    Analyses of HA/CD44-mediated HOXD10 and RhoGTPase expression in MDA-MB-231 cells.  Detection of HA/CD44-induced HOXD10 and RhoGTPase (RhoA/RhoC) expression in MDA-MB-231 cells was performed by solubilizing cells with 1% Nonidet P-40 (Nonidet P-40) buffer

    Journal: The Journal of Biological Chemistry

    Article Title: Hyaluronan-CD44 Interaction Promotes c-Src-mediated Twist Signaling, MicroRNA-10b Expression, and RhoA/RhoC Up-regulation, Leading to Rho-kinase-associated Cytoskeleton Activation and Breast Tumor Cell Invasion *

    doi: 10.1074/jbc.M110.162305

    Figure Lengend Snippet: Analyses of HA/CD44-mediated HOXD10 and RhoGTPase expression in MDA-MB-231 cells. Detection of HA/CD44-induced HOXD10 and RhoGTPase (RhoA/RhoC) expression in MDA-MB-231 cells was performed by solubilizing cells with 1% Nonidet P-40 (Nonidet P-40) buffer

    Article Snippet: These cells were then immediately lysed in Nonidet P-40 buffer (50 m m HEPES (pH 7.5), 150 m m NaCl, 20 m m MgCl2 , 1% Nonidet P-40, 1 m m Na3 VO4 , 1 m m NaF, Complete protease inhibitor mixture (Roche Applied Science), 1 m m PMSF, 1× HaltTM phosphatase inhibitor mixture (Pierce)) at 4 °C and centrifuged to obtain the lysates.

    Techniques: Expressing, Multiple Displacement Amplification