ripa buffer  (Roche)


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    Structured Review

    Roche ripa buffer
    The effect of KPT-330 on gene expression, and binding of CRM1 to HOX regions in MEGAL and HEL cells. ( A-B ) Protein expression of Nup214, SET-Nup214, CRM1, and GAPDH. ( A ) MEGAL and ( B ) HEL cells were cultured in the absence or presence of DMSO (vehicle control) or KPT-330 (100 nM or 1000 nM) for 24 hr; cell lysates were prepared using <t>RIPA</t> buffer and analyzed by <t>immunoblotting</t> using anti-Nup214, anti-CRM1, or anti-GAPDH antibodies. ( C ) qPCR analysis of HOX cluster genes ( HOXA9 , HOXA11 , HOXB4 , and HOXB9 ) in MEGAL and HEL cells treated without or with DMSO (vehicle control) or KPT-330 (100 nM or 1000 nM) for 24 hr. GAPDH was used as a reference gene. Data are presented as mean values ± SEM of three independent experiments (n = 3). ( D ) The data in ( C ) were reanalyzed to obtain a ratio by comparing with the values for DMSO-treated samples. Note that low expression genes ( HOXA9 in HEL and HOXB4 in MEGAL) were omitted in ( D ). Data are presented as mean values ± SEM. Asterisks indicate statistical significance determined by Student’s t -test; *p
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    Images

    1) Product Images from "Chromatin-bound CRM1 recruits SET-Nup214 and NPM1c onto HOX clusters causing aberrant HOX expression in leukemia cells"

    Article Title: Chromatin-bound CRM1 recruits SET-Nup214 and NPM1c onto HOX clusters causing aberrant HOX expression in leukemia cells

    Journal: eLife

    doi: 10.7554/eLife.46667

    The effect of KPT-330 on gene expression, and binding of CRM1 to HOX regions in MEGAL and HEL cells. ( A-B ) Protein expression of Nup214, SET-Nup214, CRM1, and GAPDH. ( A ) MEGAL and ( B ) HEL cells were cultured in the absence or presence of DMSO (vehicle control) or KPT-330 (100 nM or 1000 nM) for 24 hr; cell lysates were prepared using RIPA buffer and analyzed by immunoblotting using anti-Nup214, anti-CRM1, or anti-GAPDH antibodies. ( C ) qPCR analysis of HOX cluster genes ( HOXA9 , HOXA11 , HOXB4 , and HOXB9 ) in MEGAL and HEL cells treated without or with DMSO (vehicle control) or KPT-330 (100 nM or 1000 nM) for 24 hr. GAPDH was used as a reference gene. Data are presented as mean values ± SEM of three independent experiments (n = 3). ( D ) The data in ( C ) were reanalyzed to obtain a ratio by comparing with the values for DMSO-treated samples. Note that low expression genes ( HOXA9 in HEL and HOXB4 in MEGAL) were omitted in ( D ). Data are presented as mean values ± SEM. Asterisks indicate statistical significance determined by Student’s t -test; *p
    Figure Legend Snippet: The effect of KPT-330 on gene expression, and binding of CRM1 to HOX regions in MEGAL and HEL cells. ( A-B ) Protein expression of Nup214, SET-Nup214, CRM1, and GAPDH. ( A ) MEGAL and ( B ) HEL cells were cultured in the absence or presence of DMSO (vehicle control) or KPT-330 (100 nM or 1000 nM) for 24 hr; cell lysates were prepared using RIPA buffer and analyzed by immunoblotting using anti-Nup214, anti-CRM1, or anti-GAPDH antibodies. ( C ) qPCR analysis of HOX cluster genes ( HOXA9 , HOXA11 , HOXB4 , and HOXB9 ) in MEGAL and HEL cells treated without or with DMSO (vehicle control) or KPT-330 (100 nM or 1000 nM) for 24 hr. GAPDH was used as a reference gene. Data are presented as mean values ± SEM of three independent experiments (n = 3). ( D ) The data in ( C ) were reanalyzed to obtain a ratio by comparing with the values for DMSO-treated samples. Note that low expression genes ( HOXA9 in HEL and HOXB4 in MEGAL) were omitted in ( D ). Data are presented as mean values ± SEM. Asterisks indicate statistical significance determined by Student’s t -test; *p

    Techniques Used: Expressing, Binding Assay, Cell Culture, Real-time Polymerase Chain Reaction

    Protein expression and CRM1 binding profiles in HL60 cells. ( A ) Protein expression of Nup214, SET-Nup214, CRM1, and GAPDH. LOUCY and HL60 cell lines were cultured in the absence or presence of DMSO (vehicle control) or KPT-330 (100 nM or 1000 nM) for 24 hr; cell lysates were prepared using RIPA buffer and analyzed by immunoblotting with anti-Nup214, anti-CRM1, or anti-GAPDH antibodies. ( B–D ) CRM1 ChIP-seq for HL60 cells was performed as described in Figure 1 . ( B : whole genome; C : whole chromosome 17 and HOXB cluster, D : whole chromosome seven and HOXA cluster).
    Figure Legend Snippet: Protein expression and CRM1 binding profiles in HL60 cells. ( A ) Protein expression of Nup214, SET-Nup214, CRM1, and GAPDH. LOUCY and HL60 cell lines were cultured in the absence or presence of DMSO (vehicle control) or KPT-330 (100 nM or 1000 nM) for 24 hr; cell lysates were prepared using RIPA buffer and analyzed by immunoblotting with anti-Nup214, anti-CRM1, or anti-GAPDH antibodies. ( B–D ) CRM1 ChIP-seq for HL60 cells was performed as described in Figure 1 . ( B : whole genome; C : whole chromosome 17 and HOXB cluster, D : whole chromosome seven and HOXA cluster).

    Techniques Used: Expressing, Binding Assay, Cell Culture, Chromatin Immunoprecipitation

    The effect of KPT-330 on the protein and HOX gene expression levels in OCI-AML3 cells. ( A ) Protein expression of CRM1, NPM1c and GAPDH in OCI-AML3 cells cultured in the absence or presence of DMSO (vehicle control) or KPT-330 (100 nM or 1000 nM) for 24 hr. The cell lysates were prepared in RIPA buffer and analyzed by immunoblotting using anti-CRM1, anti-NPM1c, or anti-GAPDH antibodies. ( B ) qPCR analysis of HOX cluster genes ( HOXA9 , HOXA11 , HOXB4 , and HOXB9 ) in OCI-AML3 cells treated with DMSO (vehicle control) or KPT-330 (100 nM or 1000 nM) for 24 hr. GAPDH was used as a reference gene. Data are presented as mean values ± SEM of three independent experiments (n = 3). ( C ) The data in ( B ) were reanalyzed to obtain a ratio by comparing the values of the KPT-330-treated samples with the values for DMSO-treated samples. Data are presented as mean values ± SEM. Asterisks indicate statistical significance determined by Student’s t -test; **p
    Figure Legend Snippet: The effect of KPT-330 on the protein and HOX gene expression levels in OCI-AML3 cells. ( A ) Protein expression of CRM1, NPM1c and GAPDH in OCI-AML3 cells cultured in the absence or presence of DMSO (vehicle control) or KPT-330 (100 nM or 1000 nM) for 24 hr. The cell lysates were prepared in RIPA buffer and analyzed by immunoblotting using anti-CRM1, anti-NPM1c, or anti-GAPDH antibodies. ( B ) qPCR analysis of HOX cluster genes ( HOXA9 , HOXA11 , HOXB4 , and HOXB9 ) in OCI-AML3 cells treated with DMSO (vehicle control) or KPT-330 (100 nM or 1000 nM) for 24 hr. GAPDH was used as a reference gene. Data are presented as mean values ± SEM of three independent experiments (n = 3). ( C ) The data in ( B ) were reanalyzed to obtain a ratio by comparing the values of the KPT-330-treated samples with the values for DMSO-treated samples. Data are presented as mean values ± SEM. Asterisks indicate statistical significance determined by Student’s t -test; **p

    Techniques Used: Expressing, Cell Culture, Real-time Polymerase Chain Reaction

    2) Product Images from "RanBP2/Nup358 enhances miRNA activity by sumoylating and stabilizing Argonaute 1"

    Article Title: RanBP2/Nup358 enhances miRNA activity by sumoylating and stabilizing Argonaute 1

    Journal: bioRxiv

    doi: 10.1101/555896

    RanBP2 promotes the sumoylation and inhibits the ubiquitination of AGO1. (A) WT U2OS, and RanBP2-dE3 cells were co-transfected with FH-AGO1 , V5-Ubc9 , and His-tagged SUMO2 ( His6-SUMO2 “+”) or control vector ( His6-SUMO2 “-”). 24 h post-transfection cells were lysed in 6 M guanidinium chloride, and the His6-SUMO2 conjugates were isolated on Nickel beads (“Ni2+ NTA PD”) or the lysates were directly analyzed (“input”) and separated by SDS-PAGE. Conjugates were analyzed for the presence of FH-AGO1 by immunoblotting for HA (IB: HA), and for total His6-SUMO2 conjugates by immunoblotting for His (IB: His). Relative levels of each signal, as analyzed by densitometry, are indicated below each blot. Input lysates were immunoblotted for FH-AGO1, V5-Ubc9, RanBP2 and α-tubulin. (B) WT U2OS, and RanBP2-dE3 cells were co-transfected with FH-AGO1, and His-Myc-tagged ubiquitin (His-Myc-Ub “+”) or as V5-Ubc9 as a control (His-Myc-Ub “-”). 18 h post-transfection, cells were treated with MG132 (10 µM) for an additional 7 h to preserve ubiquitinated conjugates. Cells were lysed in 6 M guanidinium chloride, and the His-Myc-Ub conjugates were isolated on Nickel beads (“Ni2+ NTA PD”) or the lysates were directly analyzed (“input”) and separated by SDS-PAGE. Conjugates were analyzed for the presence of FH-AGO1 by immunoblotting for HA (IB: HA), and for total His-Myc-Ub conjugates by immunoblotting for His (IB: His). Relative levels of each signal, as analyzed by densitometry, are indicated below each blot. Input lysates were immunoblotted for FH-AGO1, RanBP2 and α-tubulin. (C) WT U2OS, and RanBP2-dE3 cells were transfected with FH-AGO1 or FH-EYFP. 18 h post-transfection, cells were treated with MG132 (10 µM) for an additional 7 h to preserve ubiquitinated conjugates. Cells were lysed in RIPA buffer, and the FH-AGO1/FH-EYFP and associated proteins were isolated by immunoprecipitation using anti-HA antibodies (“IP HA”) or the lysates were directly analyzed (“input”) and separated by SDS-PAGE. The immuoprecipitates were analyzed for ubiquitinated proteins by immunoblotting against ubiquitin (IB: Ub). They were also analyzed for the immunoprecipitated FH-AGO1/FH-EYFP by immunoblotting against HA, and for co-immunoprecipitated RanBP2. Input lysates were immunoblotted for RanBP2, FH-AGO1 and FH-EYFP (IB: HA) and α-tubulin. (D) WT U2OS, and RanBP2-dE3 cells were transfected with FH-AGO1 and either V5-Ubc9 to enhance sumoylation or control plasmid. 18 h post-transfection, cells were treated with MG132 (10 µM) for an additional 7 h to preserve ubiquitinated conjugates. Cells were lysed in RIPA buffer, and the FH-AGO1/FH-EYFP were isolated by immunoprecipitation using anti-HA antibodies (“IP HA”) or the lysates were directly analyzed (“input”) and separated by SDS-PAGE. The immuoprecipitates were analyzed for ubiquitinated proteins by immunoblotting against ubiquitin (IB: Ub). Input lysates were immunoblotted for FH-AGO1 (IB: HA), RanBP2, V5-Ubc9, RanGAP1 and α-tubulin. Note that Ubc9 overexpression, rescues RanGAP1-sumoylation in RanBP2-dE3 cells and decreases the amount of ubiquitinated FH-AGO1.(E) General model for the regulation of IL6 by RanBP2.
    Figure Legend Snippet: RanBP2 promotes the sumoylation and inhibits the ubiquitination of AGO1. (A) WT U2OS, and RanBP2-dE3 cells were co-transfected with FH-AGO1 , V5-Ubc9 , and His-tagged SUMO2 ( His6-SUMO2 “+”) or control vector ( His6-SUMO2 “-”). 24 h post-transfection cells were lysed in 6 M guanidinium chloride, and the His6-SUMO2 conjugates were isolated on Nickel beads (“Ni2+ NTA PD”) or the lysates were directly analyzed (“input”) and separated by SDS-PAGE. Conjugates were analyzed for the presence of FH-AGO1 by immunoblotting for HA (IB: HA), and for total His6-SUMO2 conjugates by immunoblotting for His (IB: His). Relative levels of each signal, as analyzed by densitometry, are indicated below each blot. Input lysates were immunoblotted for FH-AGO1, V5-Ubc9, RanBP2 and α-tubulin. (B) WT U2OS, and RanBP2-dE3 cells were co-transfected with FH-AGO1, and His-Myc-tagged ubiquitin (His-Myc-Ub “+”) or as V5-Ubc9 as a control (His-Myc-Ub “-”). 18 h post-transfection, cells were treated with MG132 (10 µM) for an additional 7 h to preserve ubiquitinated conjugates. Cells were lysed in 6 M guanidinium chloride, and the His-Myc-Ub conjugates were isolated on Nickel beads (“Ni2+ NTA PD”) or the lysates were directly analyzed (“input”) and separated by SDS-PAGE. Conjugates were analyzed for the presence of FH-AGO1 by immunoblotting for HA (IB: HA), and for total His-Myc-Ub conjugates by immunoblotting for His (IB: His). Relative levels of each signal, as analyzed by densitometry, are indicated below each blot. Input lysates were immunoblotted for FH-AGO1, RanBP2 and α-tubulin. (C) WT U2OS, and RanBP2-dE3 cells were transfected with FH-AGO1 or FH-EYFP. 18 h post-transfection, cells were treated with MG132 (10 µM) for an additional 7 h to preserve ubiquitinated conjugates. Cells were lysed in RIPA buffer, and the FH-AGO1/FH-EYFP and associated proteins were isolated by immunoprecipitation using anti-HA antibodies (“IP HA”) or the lysates were directly analyzed (“input”) and separated by SDS-PAGE. The immuoprecipitates were analyzed for ubiquitinated proteins by immunoblotting against ubiquitin (IB: Ub). They were also analyzed for the immunoprecipitated FH-AGO1/FH-EYFP by immunoblotting against HA, and for co-immunoprecipitated RanBP2. Input lysates were immunoblotted for RanBP2, FH-AGO1 and FH-EYFP (IB: HA) and α-tubulin. (D) WT U2OS, and RanBP2-dE3 cells were transfected with FH-AGO1 and either V5-Ubc9 to enhance sumoylation or control plasmid. 18 h post-transfection, cells were treated with MG132 (10 µM) for an additional 7 h to preserve ubiquitinated conjugates. Cells were lysed in RIPA buffer, and the FH-AGO1/FH-EYFP were isolated by immunoprecipitation using anti-HA antibodies (“IP HA”) or the lysates were directly analyzed (“input”) and separated by SDS-PAGE. The immuoprecipitates were analyzed for ubiquitinated proteins by immunoblotting against ubiquitin (IB: Ub). Input lysates were immunoblotted for FH-AGO1 (IB: HA), RanBP2, V5-Ubc9, RanGAP1 and α-tubulin. Note that Ubc9 overexpression, rescues RanGAP1-sumoylation in RanBP2-dE3 cells and decreases the amount of ubiquitinated FH-AGO1.(E) General model for the regulation of IL6 by RanBP2.

    Techniques Used: Transfection, Plasmid Preparation, Isolation, SDS Page, Immunoprecipitation, Over Expression

    3) Product Images from "Role of the transcriptional regulator SP140 in resistance to bacterial infections via repression of type I interferons"

    Article Title: Role of the transcriptional regulator SP140 in resistance to bacterial infections via repression of type I interferons

    Journal: bioRxiv

    doi: 10.1101/2020.01.07.897553

    Sp110 −/− mice are not susceptible to M. tuberculosis infections. ( A ), BMMs were treated with 10U/ml of IFNγ for 24 hours and cells were lysed with RIPA buffer. 5μg of total protein was loaded on each lane, and immunoblot was performed with respective antibodies as shown. Molecular weight standards are shown on the left of each blot in kDa. Individual membranes were imaged separately. Three independent lines of Sp100 −/− mice were analyzed (denoted lines 61, 65, and 71). ( B-D ), Lung of mice infected with M. tuberculosis were stained with hematoxylin and eosin (H E) for histology ( B ), measured for CFU at 25 days post-infection (Mann-Whitney test) ( C ) or, monitored for survival ( D ). All except B6 mice were bred in-house, and combined results from the three independent Sp110 −/− lines are shown. Representative of 2 experiments ( B , D ); combined results of 3 infections ( C ). *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.005.
    Figure Legend Snippet: Sp110 −/− mice are not susceptible to M. tuberculosis infections. ( A ), BMMs were treated with 10U/ml of IFNγ for 24 hours and cells were lysed with RIPA buffer. 5μg of total protein was loaded on each lane, and immunoblot was performed with respective antibodies as shown. Molecular weight standards are shown on the left of each blot in kDa. Individual membranes were imaged separately. Three independent lines of Sp100 −/− mice were analyzed (denoted lines 61, 65, and 71). ( B-D ), Lung of mice infected with M. tuberculosis were stained with hematoxylin and eosin (H E) for histology ( B ), measured for CFU at 25 days post-infection (Mann-Whitney test) ( C ) or, monitored for survival ( D ). All except B6 mice were bred in-house, and combined results from the three independent Sp110 −/− lines are shown. Representative of 2 experiments ( B , D ); combined results of 3 infections ( C ). *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.005.

    Techniques Used: Mouse Assay, Molecular Weight, Infection, Staining, MANN-WHITNEY

    4) Product Images from "Deletion of the First Cysteine-Rich Region of the Varicella-Zoster Virus Glycoprotein E Ectodomain Abolishes the gE and gI Interaction and Differentially Affects Cell-Cell Spread and Viral Entry ▿"

    Article Title: Deletion of the First Cysteine-Rich Region of the Varicella-Zoster Virus Glycoprotein E Ectodomain Abolishes the gE and gI Interaction and Differentially Affects Cell-Cell Spread and Viral Entry ▿

    Journal:

    doi: 10.1128/JVI.00913-08

    Analysis of gE and gI interaction and glycosylation in melanoma cells. (A) Coimmunoprecipitation of gE and gI in infected cells. Melanoma cells were inoculated with the rOka-ΔCys mutant or rOka control, and lysates were collected in RIPA buffer.
    Figure Legend Snippet: Analysis of gE and gI interaction and glycosylation in melanoma cells. (A) Coimmunoprecipitation of gE and gI in infected cells. Melanoma cells were inoculated with the rOka-ΔCys mutant or rOka control, and lysates were collected in RIPA buffer.

    Techniques Used: Infection, Mutagenesis

    5) Product Images from "Stable depletion of RUNX1-ETO in Kasumi-1 cells induces expression and enhanced proteolytic activity of Cathepsin G and Neutrophil Elastase"

    Article Title: Stable depletion of RUNX1-ETO in Kasumi-1 cells induces expression and enhanced proteolytic activity of Cathepsin G and Neutrophil Elastase

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0225977

    Mapping of protease cleavage sites by mass spectrometry. A) Whole cell lysates of Kasumi-1/ctrl and Kasumi-1/shRE cells were prepared in RIPA buffer supplemented with cOmplete Protease Inhibitor Cocktail (Roche) on day twelve after transduction. Proteins were separated by SDS PAGE, stained with Coomassie blue (n = 3) and subjected to LC-MS analysis. B) The identified peptides were categorized as tryptic peptides, resulting from trypsin digestion during the MS sample preparation procedure, or non-tryptic peptides, resulting from proteolytic cleavage during cell lysis. C) IceLogo showing amino acids at positions P4-P4´, which are enriched or depleted in peptides from Kasumi-1/shRE compared to Kasumi-1/ctrl with p
    Figure Legend Snippet: Mapping of protease cleavage sites by mass spectrometry. A) Whole cell lysates of Kasumi-1/ctrl and Kasumi-1/shRE cells were prepared in RIPA buffer supplemented with cOmplete Protease Inhibitor Cocktail (Roche) on day twelve after transduction. Proteins were separated by SDS PAGE, stained with Coomassie blue (n = 3) and subjected to LC-MS analysis. B) The identified peptides were categorized as tryptic peptides, resulting from trypsin digestion during the MS sample preparation procedure, or non-tryptic peptides, resulting from proteolytic cleavage during cell lysis. C) IceLogo showing amino acids at positions P4-P4´, which are enriched or depleted in peptides from Kasumi-1/shRE compared to Kasumi-1/ctrl with p

    Techniques Used: Mass Spectrometry, Protease Inhibitor, Transduction, SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy, Sample Prep, Lysis

    Related Articles

    Transduction:

    Article Title: Stable depletion of RUNX1-ETO in Kasumi-1 cells induces expression and enhanced proteolytic activity of Cathepsin G and Neutrophil Elastase
    Article Snippet: .. Sample preparation for MS analysis Whole cell lysates from Kasumi-1/ctrl and Kasumi-1/shRE were prepared in RIPA buffer (50mM Tris HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS), supplemented with complete Protease Inhibitor Cocktail (Roche), on day twelve after lentiviral transduction. .. Protein concentration was determined by DC-Assay (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions and 50 μg protein per sample were boiled for five minutes in 2x NuPage LDS sample buffer (Invitrogen).

    Transfection:

    Article Title: RanBP2/Nup358 enhances miRNA activity by sumoylating and stabilizing Argonaute 1
    Article Snippet: .. 24 h after transfection, cells were lysed in RIPA buffer (50 mM Tris-HCL, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, and complete protease inhibitor cocktail (Roche), pH 7.4) for immunoprecipitation. .. Cell lysates were incubated with Protein-G Sepharose beads coupled to the anti-HA antibody for 2-3 h at 4 °C.

    Sample Prep:

    Article Title: Stable depletion of RUNX1-ETO in Kasumi-1 cells induces expression and enhanced proteolytic activity of Cathepsin G and Neutrophil Elastase
    Article Snippet: .. Sample preparation for MS analysis Whole cell lysates from Kasumi-1/ctrl and Kasumi-1/shRE were prepared in RIPA buffer (50mM Tris HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS), supplemented with complete Protease Inhibitor Cocktail (Roche), on day twelve after lentiviral transduction. .. Protein concentration was determined by DC-Assay (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions and 50 μg protein per sample were boiled for five minutes in 2x NuPage LDS sample buffer (Invitrogen).

    Mutagenesis:

    Article Title: Deletion of the First Cysteine-Rich Region of the Varicella-Zoster Virus Glycoprotein E Ectodomain Abolishes the gE and gI Interaction and Differentially Affects Cell-Cell Spread and Viral Entry ▿
    Article Snippet: .. Melanoma cells were infected with the recombinant rOka-ΔCys mutant, along with the rOka control, and protein lysates were collected in RIPA buffer for analyzing the gE/gI interaction or in 25 mM Tris-HCl, pH 7.4, 5 mM EDTA, 15 mM NaCl, and 0.1% NP-40 for studying the gE/IDE interaction ( ); lysis buffer was supplemented with protease inhibitor cocktail (Complete minitablet; Roche Inc., Indianapolis, IN). .. Protein lysates from uninfected melanoma cells were used as a negative control.

    Protease Inhibitor:

    Article Title: Cancer cells depend on environmental lipids for proliferation when electron acceptors are limited
    Article Snippet: .. ImmunoblottingCells washed with ice-cold PBS, and scraped into cold RIPA buffer containing cOmplete Mini protease inhibitor (Roche 11836170001) and PhosStop Phosphatase Inhibitor Cocktail Tablets (Roche 04906845001). .. Protein concentration was calculated using the BCA Protein Assay (Pierce 23225) with BSA as a standard.

    Article Title: Stable depletion of RUNX1-ETO in Kasumi-1 cells induces expression and enhanced proteolytic activity of Cathepsin G and Neutrophil Elastase
    Article Snippet: .. Sample preparation for MS analysis Whole cell lysates from Kasumi-1/ctrl and Kasumi-1/shRE were prepared in RIPA buffer (50mM Tris HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS), supplemented with complete Protease Inhibitor Cocktail (Roche), on day twelve after lentiviral transduction. .. Protein concentration was determined by DC-Assay (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions and 50 μg protein per sample were boiled for five minutes in 2x NuPage LDS sample buffer (Invitrogen).

    Article Title: Role of the transcriptional regulator SP140 in resistance to bacterial infections via repression of type I interferons
    Article Snippet: .. Immunoblot Samples were lysed in RIPA buffer with protease inhibitor cocktail (Roche) to obtain total protein lysate and were clarified by spinning at ~16,000×g for 30 min at 4°C. .. Clarified lysates were analyzed with Pierce BCA protein assay kit (Thermo Fisher Scientific) according to manufacturer specification and diluted to the same concentration and denatured with SDS-loading buffer.

    Article Title: RanBP2/Nup358 enhances miRNA activity by sumoylating and stabilizing Argonaute 1
    Article Snippet: .. 24 h after transfection, cells were lysed in RIPA buffer (50 mM Tris-HCL, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, and complete protease inhibitor cocktail (Roche), pH 7.4) for immunoprecipitation. .. Cell lysates were incubated with Protein-G Sepharose beads coupled to the anti-HA antibody for 2-3 h at 4 °C.

    Article Title: Deletion of the First Cysteine-Rich Region of the Varicella-Zoster Virus Glycoprotein E Ectodomain Abolishes the gE and gI Interaction and Differentially Affects Cell-Cell Spread and Viral Entry ▿
    Article Snippet: .. Melanoma cells were infected with the recombinant rOka-ΔCys mutant, along with the rOka control, and protein lysates were collected in RIPA buffer for analyzing the gE/gI interaction or in 25 mM Tris-HCl, pH 7.4, 5 mM EDTA, 15 mM NaCl, and 0.1% NP-40 for studying the gE/IDE interaction ( ); lysis buffer was supplemented with protease inhibitor cocktail (Complete minitablet; Roche Inc., Indianapolis, IN). .. Protein lysates from uninfected melanoma cells were used as a negative control.

    Article Title: Chromatin-bound CRM1 recruits SET-Nup214 and NPM1c onto HOX clusters causing aberrant HOX expression in leukemia cells
    Article Snippet: .. Immunoblotting Cells were collected by centrifugation and RIPA buffer containing a protease inhibitor cocktail (cOmplete ULTRA, Roche) was added. .. Protein concentration of each sample was measured by BCA protein assay kit (Pierce).

    Centrifugation:

    Article Title: Chromatin-bound CRM1 recruits SET-Nup214 and NPM1c onto HOX clusters causing aberrant HOX expression in leukemia cells
    Article Snippet: .. Immunoblotting Cells were collected by centrifugation and RIPA buffer containing a protease inhibitor cocktail (cOmplete ULTRA, Roche) was added. .. Protein concentration of each sample was measured by BCA protein assay kit (Pierce).

    Infection:

    Article Title: Deletion of the First Cysteine-Rich Region of the Varicella-Zoster Virus Glycoprotein E Ectodomain Abolishes the gE and gI Interaction and Differentially Affects Cell-Cell Spread and Viral Entry ▿
    Article Snippet: .. Melanoma cells were infected with the recombinant rOka-ΔCys mutant, along with the rOka control, and protein lysates were collected in RIPA buffer for analyzing the gE/gI interaction or in 25 mM Tris-HCl, pH 7.4, 5 mM EDTA, 15 mM NaCl, and 0.1% NP-40 for studying the gE/IDE interaction ( ); lysis buffer was supplemented with protease inhibitor cocktail (Complete minitablet; Roche Inc., Indianapolis, IN). .. Protein lysates from uninfected melanoma cells were used as a negative control.

    Immunoprecipitation:

    Article Title: RanBP2/Nup358 enhances miRNA activity by sumoylating and stabilizing Argonaute 1
    Article Snippet: .. 24 h after transfection, cells were lysed in RIPA buffer (50 mM Tris-HCL, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, and complete protease inhibitor cocktail (Roche), pH 7.4) for immunoprecipitation. .. Cell lysates were incubated with Protein-G Sepharose beads coupled to the anti-HA antibody for 2-3 h at 4 °C.

    Article Title: HPV16 E5 Mediates Resistance to PD-L1 Blockade and can be targeted with Rimantadine in Head and Neck Cancer
    Article Snippet: .. Immunoprecipitation and immunoblottingCells were lysed in RIPA buffer composed of 25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, and protease inhibitors (Roche). .. The cell lysates were centrifuged (15,000 × g) at 4°C for 5 min. For immunoprecipitation, soluble fractions were precleared using a Protein G/A-Agarose Suspension (EMD Millopore, Billerica, MA) at 4 °C for 15 min. Precleared cell lysates were immunoprecipitated for 2 hours with anti-FLAG M2 antibody (Sigma).

    Mass Spectrometry:

    Article Title: Stable depletion of RUNX1-ETO in Kasumi-1 cells induces expression and enhanced proteolytic activity of Cathepsin G and Neutrophil Elastase
    Article Snippet: .. Sample preparation for MS analysis Whole cell lysates from Kasumi-1/ctrl and Kasumi-1/shRE were prepared in RIPA buffer (50mM Tris HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS), supplemented with complete Protease Inhibitor Cocktail (Roche), on day twelve after lentiviral transduction. .. Protein concentration was determined by DC-Assay (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions and 50 μg protein per sample were boiled for five minutes in 2x NuPage LDS sample buffer (Invitrogen).

    Lysis:

    Article Title: Deletion of the First Cysteine-Rich Region of the Varicella-Zoster Virus Glycoprotein E Ectodomain Abolishes the gE and gI Interaction and Differentially Affects Cell-Cell Spread and Viral Entry ▿
    Article Snippet: .. Melanoma cells were infected with the recombinant rOka-ΔCys mutant, along with the rOka control, and protein lysates were collected in RIPA buffer for analyzing the gE/gI interaction or in 25 mM Tris-HCl, pH 7.4, 5 mM EDTA, 15 mM NaCl, and 0.1% NP-40 for studying the gE/IDE interaction ( ); lysis buffer was supplemented with protease inhibitor cocktail (Complete minitablet; Roche Inc., Indianapolis, IN). .. Protein lysates from uninfected melanoma cells were used as a negative control.

    Western Blot:

    Article Title: Small‐molecule inhibition of prostaglandin E receptor 2 impairs cyclooxygenase‐associated malignant glioma growth, et al. Small‐molecule inhibition of prostaglandin E receptor 2 impairs cyclooxygenase‐associated malignant glioma growth
    Article Snippet: .. Western blot analysis Cells were homogenized on ice in RIPA buffer (25 mM Tris·HCl pH 7.6, 150 mM NaCl, 1% NP‐40, 1% sodium deoxycholate, 0.1% SDS) containing a mixture of protease and phosphatase inhibitors (Roche Applied Science, Penzberg, Germany). .. The homogenates were centrifuged (12,000× g, 15 min, 4°C), and protein concentration in the supernate was measured by BCA Protein Assay Kit (Pierce, Rockford, lL, USA).

    Recombinant:

    Article Title: Deletion of the First Cysteine-Rich Region of the Varicella-Zoster Virus Glycoprotein E Ectodomain Abolishes the gE and gI Interaction and Differentially Affects Cell-Cell Spread and Viral Entry ▿
    Article Snippet: .. Melanoma cells were infected with the recombinant rOka-ΔCys mutant, along with the rOka control, and protein lysates were collected in RIPA buffer for analyzing the gE/gI interaction or in 25 mM Tris-HCl, pH 7.4, 5 mM EDTA, 15 mM NaCl, and 0.1% NP-40 for studying the gE/IDE interaction ( ); lysis buffer was supplemented with protease inhibitor cocktail (Complete minitablet; Roche Inc., Indianapolis, IN). .. Protein lysates from uninfected melanoma cells were used as a negative control.

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    Roche ripa doc buffer
    Low dose of apelin-36 attenuates cerebral I/R injury-induced caspase-3 activation in rats. (A) The MCA territory of sham treated rats, I/R model rats and I/R model rats treated with apelin-36 (labeled I/R-Apelin) were lysed in <t>RIPA-DOC</t> buffer. Cell lysates were resolved on 12% Tris–Glycine sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cleaved caspase-3 was detected by cleaved caspase-3 antibody and β-actin served as an internal control was detected by β-actin antibody. (B) Quantification of cleaved caspase-3 expression. The ratio of cleaved caspase-3 to β-actin was further normalized to the sham treated rats. Values represent mean ± SEM. N = 5, * p
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    Low dose of apelin-36 attenuates cerebral I/R injury-induced caspase-3 activation in rats. (A) The MCA territory of sham treated rats, I/R model rats and I/R model rats treated with apelin-36 (labeled I/R-Apelin) were lysed in RIPA-DOC buffer. Cell lysates were resolved on 12% Tris–Glycine sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cleaved caspase-3 was detected by cleaved caspase-3 antibody and β-actin served as an internal control was detected by β-actin antibody. (B) Quantification of cleaved caspase-3 expression. The ratio of cleaved caspase-3 to β-actin was further normalized to the sham treated rats. Values represent mean ± SEM. N = 5, * p

    Journal: Frontiers in Neurology

    Article Title: Low Dose of Apelin-36 Attenuates ER Stress-Associated Apoptosis in Rats with Ischemic Stroke

    doi: 10.3389/fneur.2017.00556

    Figure Lengend Snippet: Low dose of apelin-36 attenuates cerebral I/R injury-induced caspase-3 activation in rats. (A) The MCA territory of sham treated rats, I/R model rats and I/R model rats treated with apelin-36 (labeled I/R-Apelin) were lysed in RIPA-DOC buffer. Cell lysates were resolved on 12% Tris–Glycine sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cleaved caspase-3 was detected by cleaved caspase-3 antibody and β-actin served as an internal control was detected by β-actin antibody. (B) Quantification of cleaved caspase-3 expression. The ratio of cleaved caspase-3 to β-actin was further normalized to the sham treated rats. Values represent mean ± SEM. N = 5, * p

    Article Snippet: Western Blot Analysis Brain tissues were homogenized in the RIPA-DOC buffer supplemented with PMSF and phosphatase inhibitor (Roche).

    Techniques: Activation Assay, Labeling, Polyacrylamide Gel Electrophoresis, Expressing

    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

    Glucose starvation increases exosome secretion in H9C2 cells. (A-B) Representative electron microscopy images of isolated U and P exosomes collected from 90 ml of conditioned medium from H9C2 cells grown for 48 h under glucose-starved (+St) or glucose-replete (-St) conditions. Scale bars, 200 nm. (C) Detection of tetraspanins by western blotting of U and P exosome extracts from 90 ml of culture medium from H9C2 cultured as in (A). All exosome fraction obtained from both experimental condition were resuspended in equal amount of RIPA buffer and the same amount of RIPA-proteins were loaded in each lane. Graph shows the densitometric analysis of western blot data (n = 3 for U exosomes and n = 1 for P exosomes). (D) WB of CD81, CD9 and Calnexin for 20 μg of exosomal protein isolated by standard ultracentrifugation protocol or 30% sucrose cushion protocol. We didn’t found Calnexin contamination signal for both protocols. Lys: cell lysate (E) Quantification of acetylcholinesterase (Ac Co) activity of exosomes obtained with Exoquick-TC from equal amounts (20 ml) of conditioned medium from H9C2 cells cultured as in (A) (n = 3). A.U. arbitrary units, * P

    Journal: PLoS ONE

    Article Title: Glucose Starvation in Cardiomyocytes Enhances Exosome Secretion and Promotes Angiogenesis in Endothelial Cells

    doi: 10.1371/journal.pone.0138849

    Figure Lengend Snippet: Glucose starvation increases exosome secretion in H9C2 cells. (A-B) Representative electron microscopy images of isolated U and P exosomes collected from 90 ml of conditioned medium from H9C2 cells grown for 48 h under glucose-starved (+St) or glucose-replete (-St) conditions. Scale bars, 200 nm. (C) Detection of tetraspanins by western blotting of U and P exosome extracts from 90 ml of culture medium from H9C2 cultured as in (A). All exosome fraction obtained from both experimental condition were resuspended in equal amount of RIPA buffer and the same amount of RIPA-proteins were loaded in each lane. Graph shows the densitometric analysis of western blot data (n = 3 for U exosomes and n = 1 for P exosomes). (D) WB of CD81, CD9 and Calnexin for 20 μg of exosomal protein isolated by standard ultracentrifugation protocol or 30% sucrose cushion protocol. We didn’t found Calnexin contamination signal for both protocols. Lys: cell lysate (E) Quantification of acetylcholinesterase (Ac Co) activity of exosomes obtained with Exoquick-TC from equal amounts (20 ml) of conditioned medium from H9C2 cells cultured as in (A) (n = 3). A.U. arbitrary units, * P

    Article Snippet: Western blot analysis Cells and exosomes were lysed in RIPA buffer (1% NP40, 0.5% deoxycholate, 0.1% sodium dodecyl sulphate in Tris-buffered saline) with complete protease inhibitors (Roche Diagnostics).

    Techniques: Electron Microscopy, Isolation, Western Blot, Cell Culture, Activity Assay

    Proteomic analysis of rat neonatal CM-derived U exosomes. (A) Representative electron microscopy images of isolated U exosomes collected from 90 ml of conditioned medium from rat neonatal CM grown for 48 h under glucose-starved (+St) or glucose-replete (-St) conditions. Scale bars, 200 nm. (B) Detection of tetraspanins by western blotting of U exosome extracts from 90 ml of conditioned medium from rat neonatal CM cultured as in (A). All U exosome fraction obtained from both experimental condition were resuspended in equal amount of RIPA buffer and the same volume of RIPA-proteins were loaded in each lane. (C) SDS-PAGE electrophoresis and Coomassie blue staining of U exosomal proteins from conditioned medium from rat neonatal CM cultured. U exosome pellets obtained from 90 ml of cultures media were resuspended in RIPA buffer and 30 μg of U exosomal protein from both experimental conditions (+/- St) were loaded in each lane. CD9 and CD81 WB for the same experiment shows equals tetraspanins signaling in both lanes. (D) Protein-protein interaction network obtained using STRING software in U exosomes from rat neonatal CM conditioned medium (+/-St). The images show the confidence view ( http://string-db.org/ ). Stronger associations are represented by thicker lines. (E) Biological processes common or unique to -St or +St treatment group as analyzed using Gene Ontology String software.

    Journal: PLoS ONE

    Article Title: Glucose Starvation in Cardiomyocytes Enhances Exosome Secretion and Promotes Angiogenesis in Endothelial Cells

    doi: 10.1371/journal.pone.0138849

    Figure Lengend Snippet: Proteomic analysis of rat neonatal CM-derived U exosomes. (A) Representative electron microscopy images of isolated U exosomes collected from 90 ml of conditioned medium from rat neonatal CM grown for 48 h under glucose-starved (+St) or glucose-replete (-St) conditions. Scale bars, 200 nm. (B) Detection of tetraspanins by western blotting of U exosome extracts from 90 ml of conditioned medium from rat neonatal CM cultured as in (A). All U exosome fraction obtained from both experimental condition were resuspended in equal amount of RIPA buffer and the same volume of RIPA-proteins were loaded in each lane. (C) SDS-PAGE electrophoresis and Coomassie blue staining of U exosomal proteins from conditioned medium from rat neonatal CM cultured. U exosome pellets obtained from 90 ml of cultures media were resuspended in RIPA buffer and 30 μg of U exosomal protein from both experimental conditions (+/- St) were loaded in each lane. CD9 and CD81 WB for the same experiment shows equals tetraspanins signaling in both lanes. (D) Protein-protein interaction network obtained using STRING software in U exosomes from rat neonatal CM conditioned medium (+/-St). The images show the confidence view ( http://string-db.org/ ). Stronger associations are represented by thicker lines. (E) Biological processes common or unique to -St or +St treatment group as analyzed using Gene Ontology String software.

    Article Snippet: Western blot analysis Cells and exosomes were lysed in RIPA buffer (1% NP40, 0.5% deoxycholate, 0.1% sodium dodecyl sulphate in Tris-buffered saline) with complete protease inhibitors (Roche Diagnostics).

    Techniques: Derivative Assay, Electron Microscopy, Isolation, Western Blot, Cell Culture, SDS Page, Electrophoresis, Staining, Software

    Effect of C. sinica or α-viniferin on expression of Tyro gene. B16-F0 cells were pretreated with C. sinica or α-viniferin for 2 h and stimulated with α-MSH for 48 h (A-C) or 20 h (D, E) in the presence of C. sinica or α-viniferin. Cell lysates were prepared with phosphate buffer, and cell extracts with RIPA buffer. (A) Cell lysates were reacted with 1 mM L-dopa, and the velocity of increasing absorbance values at 475 nm was immediately measured. Tyro activity is represented as the initial velocity of L-dopa oxidation (nmol/min). (B) Cell lysates were resolved on non-denaturing acrylamide gels (without 2-mercaptoethanol) by electrophoresis, and subjected to zymography with soaking of the gels in 1 mM L-dopa. (C) Cell extracts were resolved on SDS-acrylamide gels by electrophoresis, and subjected to Western blot (WB) analysis with anti-Tyro or anti-GAPDH antibody. (D, E) Total RNAs were subjected to RT-PCR analysis of Tyro, TYRP1 or DCT with β-actin as an internal control, and resolved on agarose gels by electrophoresis. (F) B16-F0 cells were transfected with Tyro (-2236/+59)-Luc reporter construct in combination with Renilla control vector. The transfected cells were pretreated with C. sinica or α-viniferin for 2 h and stimulated with α-MSH for 18 h in the presence of C. sinica or α-viniferin. Firefly luciferase activity, a reporter of the promoter activity of Tyro gene, is represented as a relative fold after normalizing to Renilla activity, a reference of transfection efficiency. Data are mean ± SEM. # p

    Journal: Theranostics

    Article Title: α-Viniferin Improves Facial Hyperpigmentation via Accelerating Feedback Termination of cAMP/PKA-Signaled Phosphorylation Circuit in Facultative Melanogenesis

    doi: 10.7150/thno.24385

    Figure Lengend Snippet: Effect of C. sinica or α-viniferin on expression of Tyro gene. B16-F0 cells were pretreated with C. sinica or α-viniferin for 2 h and stimulated with α-MSH for 48 h (A-C) or 20 h (D, E) in the presence of C. sinica or α-viniferin. Cell lysates were prepared with phosphate buffer, and cell extracts with RIPA buffer. (A) Cell lysates were reacted with 1 mM L-dopa, and the velocity of increasing absorbance values at 475 nm was immediately measured. Tyro activity is represented as the initial velocity of L-dopa oxidation (nmol/min). (B) Cell lysates were resolved on non-denaturing acrylamide gels (without 2-mercaptoethanol) by electrophoresis, and subjected to zymography with soaking of the gels in 1 mM L-dopa. (C) Cell extracts were resolved on SDS-acrylamide gels by electrophoresis, and subjected to Western blot (WB) analysis with anti-Tyro or anti-GAPDH antibody. (D, E) Total RNAs were subjected to RT-PCR analysis of Tyro, TYRP1 or DCT with β-actin as an internal control, and resolved on agarose gels by electrophoresis. (F) B16-F0 cells were transfected with Tyro (-2236/+59)-Luc reporter construct in combination with Renilla control vector. The transfected cells were pretreated with C. sinica or α-viniferin for 2 h and stimulated with α-MSH for 18 h in the presence of C. sinica or α-viniferin. Firefly luciferase activity, a reporter of the promoter activity of Tyro gene, is represented as a relative fold after normalizing to Renilla activity, a reference of transfection efficiency. Data are mean ± SEM. # p

    Article Snippet: Cell extracts were prepared with RIPA buffer (iNtRON, IBS-BR002), resolved on SDS-acrylamide gels by electrophoresis, and transferred to polyvinylidene difluoride membranes (Roche 03010040001).

    Techniques: Expressing, Activity Assay, Electrophoresis, Zymography, Western Blot, Reverse Transcription Polymerase Chain Reaction, Transfection, Construct, Plasmid Preparation, Luciferase