np 40 substitute  (Millipore)

 
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    Name:
    Nonidet P 40 Substitute solution
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    Catalog Number:
    98379
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    Structured Review

    Millipore np 40 substitute
    Nonidet P 40 Substitute solution

    https://www.bioz.com/result/np 40 substitute/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    np 40 substitute - by Bioz Stars, 2021-05
    97/100 stars

    Images

    1) Product Images from "Lack of specificity of antibodies raised against CLN3, the lysosomal/endosomal transmembrane protein mutated in juvenile Batten disease"

    Article Title: Lack of specificity of antibodies raised against CLN3, the lysosomal/endosomal transmembrane protein mutated in juvenile Batten disease

    Journal: Bioscience Reports

    doi: 10.1042/BSR20171229

    Anti-CLN3 antibodies detect the same protein bands in wild-type (WT) and Cln3 −/− mouse brain extracts Three different anti-CLN3 antibodies [Abnova rabbit anti-CLN3 (D01P), Abnova mouse anti-CLN3 (M03), and Abcam rabbit anti-CLN3 (ab75959)] were tested in immunoblot experiments using protein extracts of surface cross-linked cerebellum and cortex tissue samples from 1-month-old WT and Cln3 −/− male mice. Protein extracts were prepared by sonication in a lysis buffer containing 500 mM NaCl, 0.1% NP-40 substitute and protease, and phosphatase inhibitor cocktails. Sixty micrograms of protein were loaded in each lane of SDS-containing 10% polyacrylamide gels. Prior to loading, samples were treated as indicated in reducing sample buffer without or with 4 M urea. After the electrophoretic separation, proteins were transferred onto nitrocellulose membranes and probed with the anti-CLN3 antibodies (Abnova rabbit, 1:500; Abnova mouse, 1:500; Abcam rabbit, 1:700); M.W. marker: PageRuler Plus (Thermo Fisher Scientific). The immunoblots shown are representative of two separate experiments.
    Figure Legend Snippet: Anti-CLN3 antibodies detect the same protein bands in wild-type (WT) and Cln3 −/− mouse brain extracts Three different anti-CLN3 antibodies [Abnova rabbit anti-CLN3 (D01P), Abnova mouse anti-CLN3 (M03), and Abcam rabbit anti-CLN3 (ab75959)] were tested in immunoblot experiments using protein extracts of surface cross-linked cerebellum and cortex tissue samples from 1-month-old WT and Cln3 −/− male mice. Protein extracts were prepared by sonication in a lysis buffer containing 500 mM NaCl, 0.1% NP-40 substitute and protease, and phosphatase inhibitor cocktails. Sixty micrograms of protein were loaded in each lane of SDS-containing 10% polyacrylamide gels. Prior to loading, samples were treated as indicated in reducing sample buffer without or with 4 M urea. After the electrophoretic separation, proteins were transferred onto nitrocellulose membranes and probed with the anti-CLN3 antibodies (Abnova rabbit, 1:500; Abnova mouse, 1:500; Abcam rabbit, 1:700); M.W. marker: PageRuler Plus (Thermo Fisher Scientific). The immunoblots shown are representative of two separate experiments.

    Techniques Used: Mouse Assay, Sonication, Lysis, Marker, Western Blot

    Related Articles

    Lysis:

    Article Title: Association of Phosphorylated Serine/Arginine (SR) Splicing Factors With The U1-Small Ribonucleoprotein (snRNP) Autoantigen Complex Accompanies Apoptotic Cell Death
    Article Snippet: .. After the third NP-40 lysis buffer wash, the immunoprecipitate was digested in a volume of 300 μl for 1 h at 37°C in a solution containing 50 μg/ml proteinase K ( Sigma Chemical Co. ), 10 mM Tris, pH 7.8, 10 mM EDTA, and 0.5% SDS. .. The RNA was isolated after two extractions with a phenol/chloroform/isoamyl alcohol (25:24:1) mixture ( GIBCO BRL ).

    Article Title: Crude subcellular fractionation of cultured mammalian cell lines
    Article Snippet: .. Briefly, following the NP40 lysis step the nucleus was solubilized and its contents released using RIPA buffer supplemented with Benzonase (SIGMA) to digest DNA and RNA. ..

    Article Title: Divergent roles of BECN1 in LC3 lipidation and autophagosomal function
    Article Snippet: Immunoblotting analysis and coimmunoprecipitation (coIP) For immunoblotting, cells were treated by H2 O2 exposure or amino acid starvation (amino acid-free Earle's Balanced Salt Solution, HyClone, SH30029.02) for different time periods, and then lysed for immunoblotting analysis according to standard protocol. .. For CoIP assay, plasmids were transfected into HeLa cells prior to different treatment and the cells were lysed by NP40 lysis buffer (1% NP40, 150 mM NaCl, 50 mM Tris-Cl, pH 7.4) and subjected for immunoprecipitation analysis according to the manufacturer's protocol of Sigma anti-Flag M2 affinity gel (Sigma, A2220). .. Electron microscopy The cells were fixed using 2.5% glutaraldehyde and 100 mM phosphate buffer, pH 7.2, one to 1.5 h at room temperature.

    Article Title: Mitochondrial SSBP1 protects cells from proteotoxic stresses by potentiating stress-induced HSF1 transcriptional activity
    Article Snippet: HEK293 cells in a 10-cm dish were transfected with a pShuttle-CMV expression vector for Flag-tagged hHSF1 or an hHSF1 point mutant, and were lysed with NP-40 lysis buffer. .. After centrifugation, the supernatant (400 μl) was incubated with 3 μl of rabbit antiserum for SSBP1 (αmSSBP1-1) at 4 °C for 1 h, and mixed with 40 μl protein A- or protein G-Sepharose beads (GE Healthcare) by rotating at 4 °C for 1 h. The complexes were washed with NP-40 lysis buffer, and were subjected to western blotting using mouse monoclonal IgG for Flag peptide (M2, Sigma-Aldrich). .. Immunofluorescence HeLa cells cultured on glass coverslips in 6 cm dishes at 37 °C for 24 h were infected for 2 h with adenovirus expressing scrambled RNA or short hairpin RNA for HSF1 or each VDAC protein (1 × 107 p.f.u. per ml), and then maintained with normal medium for 70 h. After being heat-shocked at 42 °C for 60 min, the cells were fixed with 4% paraformaldehyde in medium at room temperature for 10 min.

    Article Title: Determinants of GBP Recruitment to Toxoplasma gondii Vacuoles and the Parasitic Factors That Control It
    Article Snippet: RAW264.7 cells were stimulated with 200 U/ml IFN-γ overnight, starved for 1 h, metabolically labeled with [35 S] for 10 min, and chased for the indicated time. .. At the end of each time point, cells were lysed in 0.5% NP-40 lysis buffer and IPs and re-IPs were performed with a rabbit polyclonal anti-mGBP1 antiserum and a mouse monoclonal anti-FLAG antibody (Sigma-Aldrich). ..

    Incubation:

    Article Title: Deficiency of Huntingtin Has Pleiotropic Effects in the Social Amoeba Dictyostelium discoideum
    Article Snippet: Spores from mature fruiting bodies were harvested using sterile pipette tips containing 10 µL of spore buffer (40 mM KH2 PO4 , 20 mM KCl, 2.5 mM MgCl2 ), washed twice by centrifugation at 12,500 g for 2 minutes at room temperature and counted with a hemocytometer. .. Aliquots of 100 spores were heated to 45°C for 10 minutes, incubated with 0.5% NP40 detergent (Sigma-Aldrich, St Louis, MO) for 5 minutes or incubated with spore buffer alone for 5 minutes as a control. ..

    Article Title: Mitochondrial SSBP1 protects cells from proteotoxic stresses by potentiating stress-induced HSF1 transcriptional activity
    Article Snippet: HEK293 cells in a 10-cm dish were transfected with a pShuttle-CMV expression vector for Flag-tagged hHSF1 or an hHSF1 point mutant, and were lysed with NP-40 lysis buffer. .. After centrifugation, the supernatant (400 μl) was incubated with 3 μl of rabbit antiserum for SSBP1 (αmSSBP1-1) at 4 °C for 1 h, and mixed with 40 μl protein A- or protein G-Sepharose beads (GE Healthcare) by rotating at 4 °C for 1 h. The complexes were washed with NP-40 lysis buffer, and were subjected to western blotting using mouse monoclonal IgG for Flag peptide (M2, Sigma-Aldrich). .. Immunofluorescence HeLa cells cultured on glass coverslips in 6 cm dishes at 37 °C for 24 h were infected for 2 h with adenovirus expressing scrambled RNA or short hairpin RNA for HSF1 or each VDAC protein (1 × 107 p.f.u. per ml), and then maintained with normal medium for 70 h. After being heat-shocked at 42 °C for 60 min, the cells were fixed with 4% paraformaldehyde in medium at room temperature for 10 min.

    Co-Immunoprecipitation Assay:

    Article Title: Divergent roles of BECN1 in LC3 lipidation and autophagosomal function
    Article Snippet: Immunoblotting analysis and coimmunoprecipitation (coIP) For immunoblotting, cells were treated by H2 O2 exposure or amino acid starvation (amino acid-free Earle's Balanced Salt Solution, HyClone, SH30029.02) for different time periods, and then lysed for immunoblotting analysis according to standard protocol. .. For CoIP assay, plasmids were transfected into HeLa cells prior to different treatment and the cells were lysed by NP40 lysis buffer (1% NP40, 150 mM NaCl, 50 mM Tris-Cl, pH 7.4) and subjected for immunoprecipitation analysis according to the manufacturer's protocol of Sigma anti-Flag M2 affinity gel (Sigma, A2220). .. Electron microscopy The cells were fixed using 2.5% glutaraldehyde and 100 mM phosphate buffer, pH 7.2, one to 1.5 h at room temperature.

    Transfection:

    Article Title: Divergent roles of BECN1 in LC3 lipidation and autophagosomal function
    Article Snippet: Immunoblotting analysis and coimmunoprecipitation (coIP) For immunoblotting, cells were treated by H2 O2 exposure or amino acid starvation (amino acid-free Earle's Balanced Salt Solution, HyClone, SH30029.02) for different time periods, and then lysed for immunoblotting analysis according to standard protocol. .. For CoIP assay, plasmids were transfected into HeLa cells prior to different treatment and the cells were lysed by NP40 lysis buffer (1% NP40, 150 mM NaCl, 50 mM Tris-Cl, pH 7.4) and subjected for immunoprecipitation analysis according to the manufacturer's protocol of Sigma anti-Flag M2 affinity gel (Sigma, A2220). .. Electron microscopy The cells were fixed using 2.5% glutaraldehyde and 100 mM phosphate buffer, pH 7.2, one to 1.5 h at room temperature.

    Article Title: Epstein-Barr Virus Nuclear Protein 2 Has at Least Two N-Terminal Domains That Mediate Self-Association
    Article Snippet: The interaction of the Flag epitope-tagged EBNA-2 amino-terminal 194-amino-acid fusion protein (F194) with wild-type EBNA-2 was then investigated by transfection of BJAB cells with pSGF194 and with pSG5 expressing wild-type EBNA-2 (pSGWE2). .. After 24 h, the transfected BJAB cells were lysed in 1% NP-40 buffer (10 mM Tris-Cl [pH 7.4], 1 mM EDTA, 150 mM NaCl, 3% glycerol, 1 mM phenylmethylsulfonyl fluoride, 5-μg/ml leupeptin, 10-μg/ml aprotinin), and half was used to immunoprecipitate F194 with M2 anti-Flag monoclonal antibody (Sigma Chemical Co.) and protein G-Sepharose (Pharmacia Corporation), while the other half was used to immunoprecipitate wild-type EBNA-2 with PE2 monoclonal antibody. ..

    Immunoprecipitation:

    Article Title: Divergent roles of BECN1 in LC3 lipidation and autophagosomal function
    Article Snippet: Immunoblotting analysis and coimmunoprecipitation (coIP) For immunoblotting, cells were treated by H2 O2 exposure or amino acid starvation (amino acid-free Earle's Balanced Salt Solution, HyClone, SH30029.02) for different time periods, and then lysed for immunoblotting analysis according to standard protocol. .. For CoIP assay, plasmids were transfected into HeLa cells prior to different treatment and the cells were lysed by NP40 lysis buffer (1% NP40, 150 mM NaCl, 50 mM Tris-Cl, pH 7.4) and subjected for immunoprecipitation analysis according to the manufacturer's protocol of Sigma anti-Flag M2 affinity gel (Sigma, A2220). .. Electron microscopy The cells were fixed using 2.5% glutaraldehyde and 100 mM phosphate buffer, pH 7.2, one to 1.5 h at room temperature.

    Article Title: G-protein-coupled receptors regulate autophagy by ZBTB16-mediated ubiquitination and proteasomal degradation of Atg14L
    Article Snippet: Antibodies Anti-ATG14L (MBL, PD026,Lot.003), anti-ZBTB16 (ab39354, abcam), anti-Beclin1 (sc-H-300, Santa Cruz), anti-Vps34 (12452-1-AP, PTG), anti LC3B (L7543, Sigma), anti-P62 (PM045, MBL), anti-P-GSK3β (ser9) (#9323, CST), anti-GSK3β (#9832S, CST), anti-p44/42 MAPK kinase (#9102, CST), anti-p-p44/42 MAPK (T202/Y204) (#9101S, CST), anti-pAKT (S473) (#4060S, CST), anti-AKT (sc-H136, Santa Cruz), anti-Tubulin (PM054, MBL), anti-Myc (M4439, Sigma), anti-Flag M2 (F1804, Sigma), anti-Cullin3 (ab75851, abcam), anti-Xpress (R910-25, Invitrogen), anti-phosphoserine (44911M, Invitrogen), anti-p-Threonine-proline (#9391S, CST), Gαq/11 antibody (C-19) (sc-392, Santa Cruz), Gαs antibody (A-16) (sc-26766, Santa Cruz), anti-Gα13 (sc-410, Santa Cruz), anti-Ubiquitin (Z0458, Dako), mouse EM48 (MAB5374, Millipore). .. Immunoprecipitation Cells were lysed with NP-40 buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% NP-40, protease inhibitors cocktail [Sigma], 5% glycerol, 10 mM NaF, 1 mM PMSF), or Buffer II (1 mM EDTA, 0.1% NP-40, 10 mM Tris-HCl pH 7.5). .. Whole cell lysates obtained by centrifugation were incubated with 5 mg of antibody and protein G agarose beads (Invitrogen) overnight at 4°C.

    Centrifugation:

    Article Title: Mitochondrial SSBP1 protects cells from proteotoxic stresses by potentiating stress-induced HSF1 transcriptional activity
    Article Snippet: HEK293 cells in a 10-cm dish were transfected with a pShuttle-CMV expression vector for Flag-tagged hHSF1 or an hHSF1 point mutant, and were lysed with NP-40 lysis buffer. .. After centrifugation, the supernatant (400 μl) was incubated with 3 μl of rabbit antiserum for SSBP1 (αmSSBP1-1) at 4 °C for 1 h, and mixed with 40 μl protein A- or protein G-Sepharose beads (GE Healthcare) by rotating at 4 °C for 1 h. The complexes were washed with NP-40 lysis buffer, and were subjected to western blotting using mouse monoclonal IgG for Flag peptide (M2, Sigma-Aldrich). .. Immunofluorescence HeLa cells cultured on glass coverslips in 6 cm dishes at 37 °C for 24 h were infected for 2 h with adenovirus expressing scrambled RNA or short hairpin RNA for HSF1 or each VDAC protein (1 × 107 p.f.u. per ml), and then maintained with normal medium for 70 h. After being heat-shocked at 42 °C for 60 min, the cells were fixed with 4% paraformaldehyde in medium at room temperature for 10 min.

    Western Blot:

    Article Title: Mitochondrial SSBP1 protects cells from proteotoxic stresses by potentiating stress-induced HSF1 transcriptional activity
    Article Snippet: HEK293 cells in a 10-cm dish were transfected with a pShuttle-CMV expression vector for Flag-tagged hHSF1 or an hHSF1 point mutant, and were lysed with NP-40 lysis buffer. .. After centrifugation, the supernatant (400 μl) was incubated with 3 μl of rabbit antiserum for SSBP1 (αmSSBP1-1) at 4 °C for 1 h, and mixed with 40 μl protein A- or protein G-Sepharose beads (GE Healthcare) by rotating at 4 °C for 1 h. The complexes were washed with NP-40 lysis buffer, and were subjected to western blotting using mouse monoclonal IgG for Flag peptide (M2, Sigma-Aldrich). .. Immunofluorescence HeLa cells cultured on glass coverslips in 6 cm dishes at 37 °C for 24 h were infected for 2 h with adenovirus expressing scrambled RNA or short hairpin RNA for HSF1 or each VDAC protein (1 × 107 p.f.u. per ml), and then maintained with normal medium for 70 h. After being heat-shocked at 42 °C for 60 min, the cells were fixed with 4% paraformaldehyde in medium at room temperature for 10 min.

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    Millipore non ionic detergent triton x 114
    Triton X-114 solubility of different cell-associated and secreted PPAD species.  P .  gingivalis  isolates were cultured in BHI, and the cell and growth medium fractions were separated by centrifugation. Subsequently, both fractions were extracted with Triton X-114. Upon phase separation at 37 °C, proteins in the different detergent-rich (T) and aqueous (A) phases were analyzed by LDS-PAGE and Western blotting with PPAD-specific antibodies. Names of PPAD sorting type I isolates are underlined. Molecular weights of marker proteins and different PPAD species are indicated. The full-length blot is presented in Figure   S1 .
    Non Ionic Detergent Triton X 114, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/non ionic detergent triton x 114/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    97
    Millipore igepal ca 630
    Giant magnetoresistance biosensor showed higher sensitivity than ELISA for detection of IAV. Swine IAV strain H3N2v or control (mock) were treated with 1% <t>IGEPAL</t> <t>CA-630</t> to disrupt virus particle and used for detection by GMR biosensor and ELISA. (A) Binding curves in real-time on GMR biosensor; (B) Signals averaged over the last 10 data points from different concentrations of IAV and negative control (mock) in GMR biosensor and; (C) Antigen capture ELISA with different concentrations of IAV. Dotted line indicates the cut off value. Error bars represent SEM.
    Igepal Ca 630, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/igepal ca 630/product/Millipore
    Average 97 stars, based on 1 article reviews
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    igepal ca 630 - by Bioz Stars, 2021-05
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    Triton X-114 solubility of different cell-associated and secreted PPAD species.  P .  gingivalis  isolates were cultured in BHI, and the cell and growth medium fractions were separated by centrifugation. Subsequently, both fractions were extracted with Triton X-114. Upon phase separation at 37 °C, proteins in the different detergent-rich (T) and aqueous (A) phases were analyzed by LDS-PAGE and Western blotting with PPAD-specific antibodies. Names of PPAD sorting type I isolates are underlined. Molecular weights of marker proteins and different PPAD species are indicated. The full-length blot is presented in Figure   S1 .

    Journal: Scientific Reports

    Article Title: Dropping anchor: attachment of peptidylarginine deiminase via A-LPS to secreted outer membrane vesicles of Porphyromonas gingivalis

    doi: 10.1038/s41598-018-27223-5

    Figure Lengend Snippet: Triton X-114 solubility of different cell-associated and secreted PPAD species. P . gingivalis isolates were cultured in BHI, and the cell and growth medium fractions were separated by centrifugation. Subsequently, both fractions were extracted with Triton X-114. Upon phase separation at 37 °C, proteins in the different detergent-rich (T) and aqueous (A) phases were analyzed by LDS-PAGE and Western blotting with PPAD-specific antibodies. Names of PPAD sorting type I isolates are underlined. Molecular weights of marker proteins and different PPAD species are indicated. The full-length blot is presented in Figure  S1 .

    Article Snippet: Aqueous two-phase system protein purification To assess modification of the ~75–85-kDa PPAD species with A-LPS, a protein phase separation assay was applied using the non-ionic detergent Triton X-114 (Sigma-Aldrich, St. Louis, USA) .

    Techniques: Solubility, Cell Culture, Centrifugation, Polyacrylamide Gel Electrophoresis, Western Blot, Marker

    A-LPS modification of PPAD in sorting type I and II isolates of  P .  gingivalis . Cells of  P .  gingivalis  sorting type I and II isolates were cultured in BHI and, subsequently, extracted with Triton X-114. Upon phase separation at 37 °C, proteins in the detergent-rich (T) and aqueous (A) phases were analyzed by LDS-PAGE and Western blotting. In panel A immunodetection was performed with an A-LPS-specific monoclonal antibody. Panel B shows a dual-labeling image upon immunodetection with the A-LPS-specific monoclonal antibody (green signal) and a PPAD-specific rabbit antibody (red signal). Bands representing the A-LPS-modified PPAD species of ~75–85-kDa PPAD are boxed in both panels. Names of sorting type I isolates are underlined. Molecular weights of marker proteins are indicated. The full-length blot is presented in Figure   S1 . Please note that the order of ‘A’- and ‘T’-labeled lanes is inversed compared to Fig.   2  as samples were loaded in a different order.

    Journal: Scientific Reports

    Article Title: Dropping anchor: attachment of peptidylarginine deiminase via A-LPS to secreted outer membrane vesicles of Porphyromonas gingivalis

    doi: 10.1038/s41598-018-27223-5

    Figure Lengend Snippet: A-LPS modification of PPAD in sorting type I and II isolates of P . gingivalis . Cells of P . gingivalis sorting type I and II isolates were cultured in BHI and, subsequently, extracted with Triton X-114. Upon phase separation at 37 °C, proteins in the detergent-rich (T) and aqueous (A) phases were analyzed by LDS-PAGE and Western blotting. In panel A immunodetection was performed with an A-LPS-specific monoclonal antibody. Panel B shows a dual-labeling image upon immunodetection with the A-LPS-specific monoclonal antibody (green signal) and a PPAD-specific rabbit antibody (red signal). Bands representing the A-LPS-modified PPAD species of ~75–85-kDa PPAD are boxed in both panels. Names of sorting type I isolates are underlined. Molecular weights of marker proteins are indicated. The full-length blot is presented in Figure  S1 . Please note that the order of ‘A’- and ‘T’-labeled lanes is inversed compared to Fig.  2 as samples were loaded in a different order.

    Article Snippet: Aqueous two-phase system protein purification To assess modification of the ~75–85-kDa PPAD species with A-LPS, a protein phase separation assay was applied using the non-ionic detergent Triton X-114 (Sigma-Aldrich, St. Louis, USA) .

    Techniques: Modification, Cell Culture, Polyacrylamide Gel Electrophoresis, Western Blot, Immunodetection, Labeling, Marker

    Hsp90 is required for TNF-triggered necroptosis. ( a ,  b ) 17AAG treatment inhibits TNF-induced necroptosis. HT29 cells were pretreated with or without different concentrations of 17AAG for 12 h, and then treated with the indicated stimuli for 24 h. Cell images were taken with a Nikon-TE2000 microscope ( a ). Scale bar, 200  μ m. Cell viability was determined by an MTS assay ( b ). Results are the means±S.D. of triplicate measurements. The final concentrations of 20 ng/ml TNF, 100 nM Smac mimetic, 20  μ M z-VAD and 10  μ M necrostatin-1 were used. T, TNF; S, Smac mimetic; Z, z-VAD; Nec-1, necrostatin-1. ( c ) 17AAG treatment reduces the phosphorylation of MLKL. HT29 cells were pretreated with or without different concentrations of 17AAG for 12 h, and then treated with the indicated stimuli for 8 h. The activation of MLKL was analyzed by immunoblotting with a T357/S358 phospho-specific MLKL antibody. Protein levels were detected by western blotting using anti-RIP1, anti-RIP3, anti-MLKL and anti-actin antibodies. ( d ) 17AAG inhibits TNF-induced oligomerization of phosphorylated MLKL. HT29 cells were treated as in ( c ). The cells were harvested and lysed, and non-reducing samples (without  β -mercaptoethanol) of whole-cell lysate were analyzed by immunoblotting with a T357/S358 phospho-specific MLKL antibody. ( e ) 17AAG inhibits plasma membrane translocation of phosphorylated MLKL. HT29 cells were pretreated with or without 125 nM 17AAG for 12 h, and then treated with the indicated stimuli for 8 h. The cells were harvested and then separated into the aqueous phase (Aq) and detergent phase (Det) using Triton X-114 lysis buffer as described in the experimental procedures. The samples were analyzed by western blotting with the indicated antibodies. ( f ) The effect of 17AAG on TNF-induced necrosome formation. Ten-centimetre plates of HT29 cells were pretreated with or without 125 nM 17AAG for 12 h and then stimulated with TNF alone or TSZ for 8 h. Cells were then harvested and whole-cell extracts were immunoprecipitated with anti-RIP3 antibody or anti-IgG antibody and subsequently analyzed by western blotting for the indicated proteins. The asterisks denote nonspecific IgG bands

    Journal: Cell Death & Disease

    Article Title: Hsp90 modulates the stability of MLKL and is required for TNF-induced necroptosis

    doi: 10.1038/cddis.2015.390

    Figure Lengend Snippet: Hsp90 is required for TNF-triggered necroptosis. ( a , b ) 17AAG treatment inhibits TNF-induced necroptosis. HT29 cells were pretreated with or without different concentrations of 17AAG for 12 h, and then treated with the indicated stimuli for 24 h. Cell images were taken with a Nikon-TE2000 microscope ( a ). Scale bar, 200  μ m. Cell viability was determined by an MTS assay ( b ). Results are the means±S.D. of triplicate measurements. The final concentrations of 20 ng/ml TNF, 100 nM Smac mimetic, 20  μ M z-VAD and 10  μ M necrostatin-1 were used. T, TNF; S, Smac mimetic; Z, z-VAD; Nec-1, necrostatin-1. ( c ) 17AAG treatment reduces the phosphorylation of MLKL. HT29 cells were pretreated with or without different concentrations of 17AAG for 12 h, and then treated with the indicated stimuli for 8 h. The activation of MLKL was analyzed by immunoblotting with a T357/S358 phospho-specific MLKL antibody. Protein levels were detected by western blotting using anti-RIP1, anti-RIP3, anti-MLKL and anti-actin antibodies. ( d ) 17AAG inhibits TNF-induced oligomerization of phosphorylated MLKL. HT29 cells were treated as in ( c ). The cells were harvested and lysed, and non-reducing samples (without β -mercaptoethanol) of whole-cell lysate were analyzed by immunoblotting with a T357/S358 phospho-specific MLKL antibody. ( e ) 17AAG inhibits plasma membrane translocation of phosphorylated MLKL. HT29 cells were pretreated with or without 125 nM 17AAG for 12 h, and then treated with the indicated stimuli for 8 h. The cells were harvested and then separated into the aqueous phase (Aq) and detergent phase (Det) using Triton X-114 lysis buffer as described in the experimental procedures. The samples were analyzed by western blotting with the indicated antibodies. ( f ) The effect of 17AAG on TNF-induced necrosome formation. Ten-centimetre plates of HT29 cells were pretreated with or without 125 nM 17AAG for 12 h and then stimulated with TNF alone or TSZ for 8 h. Cells were then harvested and whole-cell extracts were immunoprecipitated with anti-RIP3 antibody or anti-IgG antibody and subsequently analyzed by western blotting for the indicated proteins. The asterisks denote nonspecific IgG bands

    Article Snippet: The following reagents were used: Hsp90 inhibitor 17AAG (Selleckchem, Huston, TX, USA, S1141); z-VAD-fmk (ApexBio, Huston, TX, USA, A1902); Triton X-114 (Sigma-Aldrich, St. Louis, MO, USA); anti-HA (sc-805), anti-Myc (sc-40), anti-RIP3 (sc-374639) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-Hsp90 (ab178854), anti-phospho-MLKL (ab187091) (Abcam, Cambridge, MA, USA); anti-MLKL (Merck Millipore, Billerica, MA, USA, MABC604); anti-Flag M2 (Sigma-Aldrich); anti-RIP1 (BD Pharmingen, Franklin Lakes, NJ, USA, 51-6559GR).

    Techniques: Microscopy, MTS Assay, Activation Assay, Western Blot, Translocation Assay, Lysis, Immunoprecipitation

    Coexpression of Hsp90 enhances MLKL-mediated necroptosis. ( a ) The 293T cells were transfected with vector, with Flag-Hsp90 α , with MLKL-Myc alone, or with Flag-Hsp90 α  and MLKL-Myc. After 24 h of transfection, cell images were taken with a Nikon-TE2000 microscope. Scale bar, 100  μ m. ( b ) The 293T cells were transfected as in ( a ). After 24 h of transfection, cell death was quantified by propidium iodide (PI) staining. Cell death data are the means±S.D. of three independent experiments. ( c ) Hsp90 α  increases MLKL oligomerization. The 293T cells were transfected with the indicated plasmids. The cells were harvested 24 h after transfection, and non-reducing samples (without  β -mercaptoethanol) of whole-cell lysate were analyzed by immunoblotting with anti-HA antibody. ( d ) Hsp90 α  increases the plasma membrane translocation of MLKL. The 293T cells were transfected with the indicated expression vectors. After 24 h of transfection, the cells were harvested and lysed in Triton X-114 lysis buffer and then separated into aqueous phase (Aq) and detergent phase (Det) as described in the experimental procedures. The samples were resolved and probed with the indicated antibodies.  β -actin and Cox4 were used as loading controls for soluble protein and membrane protein, respectively

    Journal: Cell Death & Disease

    Article Title: Hsp90 modulates the stability of MLKL and is required for TNF-induced necroptosis

    doi: 10.1038/cddis.2015.390

    Figure Lengend Snippet: Coexpression of Hsp90 enhances MLKL-mediated necroptosis. ( a ) The 293T cells were transfected with vector, with Flag-Hsp90 α , with MLKL-Myc alone, or with Flag-Hsp90 α and MLKL-Myc. After 24 h of transfection, cell images were taken with a Nikon-TE2000 microscope. Scale bar, 100  μ m. ( b ) The 293T cells were transfected as in ( a ). After 24 h of transfection, cell death was quantified by propidium iodide (PI) staining. Cell death data are the means±S.D. of three independent experiments. ( c ) Hsp90 α increases MLKL oligomerization. The 293T cells were transfected with the indicated plasmids. The cells were harvested 24 h after transfection, and non-reducing samples (without β -mercaptoethanol) of whole-cell lysate were analyzed by immunoblotting with anti-HA antibody. ( d ) Hsp90 α increases the plasma membrane translocation of MLKL. The 293T cells were transfected with the indicated expression vectors. After 24 h of transfection, the cells were harvested and lysed in Triton X-114 lysis buffer and then separated into aqueous phase (Aq) and detergent phase (Det) as described in the experimental procedures. The samples were resolved and probed with the indicated antibodies. β -actin and Cox4 were used as loading controls for soluble protein and membrane protein, respectively

    Article Snippet: The following reagents were used: Hsp90 inhibitor 17AAG (Selleckchem, Huston, TX, USA, S1141); z-VAD-fmk (ApexBio, Huston, TX, USA, A1902); Triton X-114 (Sigma-Aldrich, St. Louis, MO, USA); anti-HA (sc-805), anti-Myc (sc-40), anti-RIP3 (sc-374639) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-Hsp90 (ab178854), anti-phospho-MLKL (ab187091) (Abcam, Cambridge, MA, USA); anti-MLKL (Merck Millipore, Billerica, MA, USA, MABC604); anti-Flag M2 (Sigma-Aldrich); anti-RIP1 (BD Pharmingen, Franklin Lakes, NJ, USA, 51-6559GR).

    Techniques: Transfection, Plasmid Preparation, Microscopy, Staining, Translocation Assay, Expressing, Lysis

    CopB and CopB2 partition differently in Triton X-114 extracts. Integral membrane association was tested by extracting  C. trachomatis  L2-infected HeLa cultures with Triton X-114 and separating proteins into aqueous and detergent soluble fractions. (A) Cultures were harvested at 20 h postinfection, and a parallel, mock-treated HeLa culture was harvested as a specificity control. Equivalent quantities of total protein from aqueous (Aq)- and detergent (Det)-soluble samples were resolved by SDS-PAGE in 12% polyacrylamide gels. Immunoblots were probed with anti-MOMP, anti-GroEL, anti-CopN, or anti-IncG as a fractionation control and with antibody specific for CopB or CopB2. Proteins were visualized by probing with secondary antibodies conjugated to alkaline phosphatase and development with NBT-BCIP. (B) HeLa cultures were mock treated or  C. trachomatis  L2 infected at an MOI of 10. Cultures were harvested at 2 h postinfection, extracted with Triton X-114, and probed by immunoblotting with the indicated antibodies. Proteins were visualized by probing with secondary antibodies conjugated to horseradish peroxidase and development with ECL Plus chemiluminescence reagent.

    Journal: Infection and Immunity

    Article Title: Biochemical and Localization Analyses of Putative Type III Secretion Translocator Proteins CopB and CopB2 of Chlamydia trachomatis Reveal Significant Distinctions ▿

    doi: 10.1128/IAI.00159-11

    Figure Lengend Snippet: CopB and CopB2 partition differently in Triton X-114 extracts. Integral membrane association was tested by extracting C. trachomatis L2-infected HeLa cultures with Triton X-114 and separating proteins into aqueous and detergent soluble fractions. (A) Cultures were harvested at 20 h postinfection, and a parallel, mock-treated HeLa culture was harvested as a specificity control. Equivalent quantities of total protein from aqueous (Aq)- and detergent (Det)-soluble samples were resolved by SDS-PAGE in 12% polyacrylamide gels. Immunoblots were probed with anti-MOMP, anti-GroEL, anti-CopN, or anti-IncG as a fractionation control and with antibody specific for CopB or CopB2. Proteins were visualized by probing with secondary antibodies conjugated to alkaline phosphatase and development with NBT-BCIP. (B) HeLa cultures were mock treated or C. trachomatis L2 infected at an MOI of 10. Cultures were harvested at 2 h postinfection, extracted with Triton X-114, and probed by immunoblotting with the indicated antibodies. Proteins were visualized by probing with secondary antibodies conjugated to horseradish peroxidase and development with ECL Plus chemiluminescence reagent.

    Article Snippet: For Triton X-114 extractions, soluble and integral membrane proteins were separated based on solubility in Triton X-114 (Sigma) to examine the partitioning of selected proteins during chlamydial infection.

    Techniques: Infection, SDS Page, Western Blot, Fractionation

    Giant magnetoresistance biosensor showed higher sensitivity than ELISA for detection of IAV. Swine IAV strain H3N2v or control (mock) were treated with 1% IGEPAL CA-630 to disrupt virus particle and used for detection by GMR biosensor and ELISA. (A) Binding curves in real-time on GMR biosensor; (B) Signals averaged over the last 10 data points from different concentrations of IAV and negative control (mock) in GMR biosensor and; (C) Antigen capture ELISA with different concentrations of IAV. Dotted line indicates the cut off value. Error bars represent SEM.

    Journal: Frontiers in Microbiology

    Article Title: Giant Magnetoresistance-based Biosensor for Detection of Influenza A Virus

    doi: 10.3389/fmicb.2016.00400

    Figure Lengend Snippet: Giant magnetoresistance biosensor showed higher sensitivity than ELISA for detection of IAV. Swine IAV strain H3N2v or control (mock) were treated with 1% IGEPAL CA-630 to disrupt virus particle and used for detection by GMR biosensor and ELISA. (A) Binding curves in real-time on GMR biosensor; (B) Signals averaged over the last 10 data points from different concentrations of IAV and negative control (mock) in GMR biosensor and; (C) Antigen capture ELISA with different concentrations of IAV. Dotted line indicates the cut off value. Error bars represent SEM.

    Article Snippet: For immunoassays the virus was inactivated at 60°C for 1 h. To disrupt the virus particles, the mock and virus preparation were treated with 1% IGEPAL CA-630 (Sigma-Aldrich, Product No. I8896) for 10 min at 37°C.

    Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Negative Control