nonidet p 40  (Millipore)


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    Nonidet P 40
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    Structured Review

    Millipore nonidet p 40
    Nonidet P 40

    https://www.bioz.com/result/nonidet p 40/product/Millipore
    Average 99 stars, based on 205 article reviews
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    nonidet p 40 - by Bioz Stars, 2020-11
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    Images

    1) Product Images from "Identification of Novel in Vivo Phosphorylation Sites of the Human Proapoptotic Protein BAD"

    Article Title: Identification of Novel in Vivo Phosphorylation Sites of the Human Proapoptotic Protein BAD

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.010702

    In vitro  phosphorylation of recombinant GST-BAD by kinases overexpressed in HEK293 cells.  HEK293 cells were transiently transfected with the indicated plasmids. Inactive mutants of PAK1 and Akt/PKB (K299R and K179A, respectively) were used as negative controls. 16 h post-transfection, cells were cultivated for an additional 30 h in medium supplemented with 0.3% serum. Afterward, cells were washed once in PBS and lysed by the direct addition of Nonidet P-40 buffer. For the kinase assay, 35 μg of the protein lysates containing the desired kinases were mixed with 1 μg of recombinant GST-BAD (purified from  E. coli ) in kinase buffer. Proteins were separated on a 12% SDS-polyacrylamide gel and blotted. Phosphorylation of BAD was visualized with phosphospecific BAD antibodies. Expression levels of RAF kinases, PAK1, and Akt/PKB are shown in the  lower panels . This experiment was repeated three times with the same results.
    Figure Legend Snippet: In vitro phosphorylation of recombinant GST-BAD by kinases overexpressed in HEK293 cells. HEK293 cells were transiently transfected with the indicated plasmids. Inactive mutants of PAK1 and Akt/PKB (K299R and K179A, respectively) were used as negative controls. 16 h post-transfection, cells were cultivated for an additional 30 h in medium supplemented with 0.3% serum. Afterward, cells were washed once in PBS and lysed by the direct addition of Nonidet P-40 buffer. For the kinase assay, 35 μg of the protein lysates containing the desired kinases were mixed with 1 μg of recombinant GST-BAD (purified from E. coli ) in kinase buffer. Proteins were separated on a 12% SDS-polyacrylamide gel and blotted. Phosphorylation of BAD was visualized with phosphospecific BAD antibodies. Expression levels of RAF kinases, PAK1, and Akt/PKB are shown in the lower panels . This experiment was repeated three times with the same results.

    Techniques Used: In Vitro, Recombinant, Transfection, Kinase Assay, Purification, Expressing

    2) Product Images from "Krp1 (Sarcosin) Promotes Lateral Fusion of Myofibril Assembly Intermediates in Cultured Mouse Cardiomyocytes"

    Article Title: Krp1 (Sarcosin) Promotes Lateral Fusion of Myofibril Assembly Intermediates in Cultured Mouse Cardiomyocytes

    Journal: Experimental cell research

    doi: 10.1016/j.yexcr.2007.12.009

    Immunoblot analysis of Nonidet P-40 extracted and insoluble fractions. Equal volumes of total cell lysate, extracted and insoluble fractions were analyzed by immunoblot (left) and densitometric analysis (right). Representative immunoblots are shown. The graph displays the means and SEM of three independent experiments. A large proportion of Krp1 remains in the insoluble pellet along with most of the cytoskeletal components (myosin, actin, N-RAP) and caveolin-3, although the majority of Krp1 is present in the extracted supernatant along with the cytosolic marker GAPDH and the integral membrane marker ß1-integrin.
    Figure Legend Snippet: Immunoblot analysis of Nonidet P-40 extracted and insoluble fractions. Equal volumes of total cell lysate, extracted and insoluble fractions were analyzed by immunoblot (left) and densitometric analysis (right). Representative immunoblots are shown. The graph displays the means and SEM of three independent experiments. A large proportion of Krp1 remains in the insoluble pellet along with most of the cytoskeletal components (myosin, actin, N-RAP) and caveolin-3, although the majority of Krp1 is present in the extracted supernatant along with the cytosolic marker GAPDH and the integral membrane marker ß1-integrin.

    Techniques Used: Western Blot, Marker

    3) Product Images from "The Modulation of CD40 Ligand Signaling by Transmembrane CD28 Splice Variant in Human T Cells"

    Article Title: The Modulation of CD40 Ligand Signaling by Transmembrane CD28 Splice Variant in Human T Cells

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20031705

    (a) Tyrosine phosphorylation of CD28i induced by CD40L stimulation. CD28-HA–transfected D1.1 cells were serum starved for 6 h and incubated with anti-CD40L Ab for 30 min on ice. Anti–goat IgG was added to start stimulation at 37°C for 2, 5, and 10 min. Nonidet P-40 cell lysates of the stimulated cells were immunoprecipitated with anti-HA Ab. Immunoprecipitates were analyzed on Western blots. The top panel was blotted with anti-Phosphotyrosine (RC20; indicated as p-Tyr). The same membrane was reprobed with anti-HA Ab (indicated as CD28i-HA). L, Ig light chain; lane 1, nonstimulated control; lane 2, CD40L stimulated for 2 min; lane 3, CD40L stimulated for 5 min; lane 4, CD40L stimulated for 10 min. (b) Induction of CD28i-HA expression by doxicyclin in D1.1 transfectant cells. CD28i-HA (∼23 kD) was expressed in D1.1 cells by doxicyclin-inducible promoter and whole cell lysates were assayed by HA-specific Western blotting. (−), doxicyclin-nontreated cells; (+), doxicyclin-treated cells (0.5 μg/ml for 48 h). (c) Overexpression of CD28i-HA enhances the activation of JNK and PAK2 in CD40L-stimulated D1.1 cells. Cells were stimulated with anti-CD40L Ab for periods of time indicated on top. Whole cell lysates were characterized with Western blotting specific for active-form JNK (represented by p46), PAK2 (∼64 kD), or Akt (∼60 kD), and indicated as pJNK, pPAK2, and pAkt on the left of each panel. To measure the level of proteins, the assay membranes were reprobed with JNK-, PAK-, or Akt-specific Abs and indicated as JNK, PAK2, or Akt on the left of each panel. Dox (−), cells not treated with doxicyclin; Dox (+), cells treated with doxicyclin.
    Figure Legend Snippet: (a) Tyrosine phosphorylation of CD28i induced by CD40L stimulation. CD28-HA–transfected D1.1 cells were serum starved for 6 h and incubated with anti-CD40L Ab for 30 min on ice. Anti–goat IgG was added to start stimulation at 37°C for 2, 5, and 10 min. Nonidet P-40 cell lysates of the stimulated cells were immunoprecipitated with anti-HA Ab. Immunoprecipitates were analyzed on Western blots. The top panel was blotted with anti-Phosphotyrosine (RC20; indicated as p-Tyr). The same membrane was reprobed with anti-HA Ab (indicated as CD28i-HA). L, Ig light chain; lane 1, nonstimulated control; lane 2, CD40L stimulated for 2 min; lane 3, CD40L stimulated for 5 min; lane 4, CD40L stimulated for 10 min. (b) Induction of CD28i-HA expression by doxicyclin in D1.1 transfectant cells. CD28i-HA (∼23 kD) was expressed in D1.1 cells by doxicyclin-inducible promoter and whole cell lysates were assayed by HA-specific Western blotting. (−), doxicyclin-nontreated cells; (+), doxicyclin-treated cells (0.5 μg/ml for 48 h). (c) Overexpression of CD28i-HA enhances the activation of JNK and PAK2 in CD40L-stimulated D1.1 cells. Cells were stimulated with anti-CD40L Ab for periods of time indicated on top. Whole cell lysates were characterized with Western blotting specific for active-form JNK (represented by p46), PAK2 (∼64 kD), or Akt (∼60 kD), and indicated as pJNK, pPAK2, and pAkt on the left of each panel. To measure the level of proteins, the assay membranes were reprobed with JNK-, PAK-, or Akt-specific Abs and indicated as JNK, PAK2, or Akt on the left of each panel. Dox (−), cells not treated with doxicyclin; Dox (+), cells treated with doxicyclin.

    Techniques Used: Transfection, Incubation, Immunoprecipitation, Western Blot, Expressing, Over Expression, Activation Assay

    4) Product Images from "Oda5p, a Novel Axonemal Protein Required for Assembly of the Outer Dynein Arm and an Associated Adenylate Kinase D⃞"

    Article Title: Oda5p, a Novel Axonemal Protein Required for Assembly of the Outer Dynein Arm and an Associated Adenylate Kinase D⃞

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E03-11-0820

    Western blot analysis indicates Oda5p is a salt-extractable,  M r  76,000 axonemal protein that sediments at ∼5S in sucrose density gradients. (A) The Oda5 antibody recognizes an  M r  76,000 band in wild-type whole cells that is absent from the  oda5-2  whole cells, confirming the antibody recognizes the correct protein. This band is not detected in cell bodies lacking flagella (middle), yet it is readily detected in whole flagella (right). (B) Oda5p remains associated with the axoneme after Nonidet P-40 detergent extraction (demembranated axonemes) and is not detected in the Nonidet P-40 detergent-soluble membrane + matrix fraction (membrane + matrix). Extraction of demembranated axonemes with 0.6 M KCl releases Oda5p into the KCl extract; none remains in the KCl-extracted axonemes. (C) Sucrose gradient fractions were probed with antibodies to outer dynein arm components IC2 and γ-DHC, and with the Oda5p-antibody. The outer dynein arm/ODA-DC complex sediments at ∼23S as expected; however, Oda5p sediments at ∼5S.
    Figure Legend Snippet: Western blot analysis indicates Oda5p is a salt-extractable, M r 76,000 axonemal protein that sediments at ∼5S in sucrose density gradients. (A) The Oda5 antibody recognizes an M r 76,000 band in wild-type whole cells that is absent from the oda5-2 whole cells, confirming the antibody recognizes the correct protein. This band is not detected in cell bodies lacking flagella (middle), yet it is readily detected in whole flagella (right). (B) Oda5p remains associated with the axoneme after Nonidet P-40 detergent extraction (demembranated axonemes) and is not detected in the Nonidet P-40 detergent-soluble membrane + matrix fraction (membrane + matrix). Extraction of demembranated axonemes with 0.6 M KCl releases Oda5p into the KCl extract; none remains in the KCl-extracted axonemes. (C) Sucrose gradient fractions were probed with antibodies to outer dynein arm components IC2 and γ-DHC, and with the Oda5p-antibody. The outer dynein arm/ODA-DC complex sediments at ∼23S as expected; however, Oda5p sediments at ∼5S.

    Techniques Used: Western Blot

    5) Product Images from "Novel Filaments 5 nm in Diameter Constitute the Cytosolic Ring of the Plastid Division Apparatus"

    Article Title: Novel Filaments 5 nm in Diameter Constitute the Cytosolic Ring of the Plastid Division Apparatus

    Journal: The Plant Cell

    doi:

    SDS-PAGE Analysis of Isolated Chloroplasts and Fractions in Which the Outer PD Rings Are Enriched. In the synchronous culture, cells were in interphase during light periods (L) and in M phase during dark periods (D). Nondividing and dividing chloroplasts were isolated from interphase (I) and M phase (M) synchronous cultures, respectively, and chloroplasts containing 5 μg of protein were analyzed (lanes 1 and 2). The chloroplasts were then lysed in hypotonic medium containing 0.5% Nonidet P-40 and 100 μg/mL DNase I. Pellets obtained from chloroplasts containing 1 mg of protein were analyzed (lanes 3 and 4). Proteins of the pellet were further extracted with 1 M NaCl, and the insoluble pellets were analyzed (lanes 5 and 6). To exclude differences between light and dark conditions, nondividing chloroplasts were isolated from a concentrated culture during the dark period, in which cells were in interphase. Then, the chloroplasts were lysed using the same treatment as was used for lanes 3 and 4 (lanes 7 and 8). Dividing chloroplasts were isolated from M phase culture synchronized without 5-fluorodeoxyuridine (−FdU; lanes 9 and 10). Arrowheads indicate specific bands in the dividing phase.
    Figure Legend Snippet: SDS-PAGE Analysis of Isolated Chloroplasts and Fractions in Which the Outer PD Rings Are Enriched. In the synchronous culture, cells were in interphase during light periods (L) and in M phase during dark periods (D). Nondividing and dividing chloroplasts were isolated from interphase (I) and M phase (M) synchronous cultures, respectively, and chloroplasts containing 5 μg of protein were analyzed (lanes 1 and 2). The chloroplasts were then lysed in hypotonic medium containing 0.5% Nonidet P-40 and 100 μg/mL DNase I. Pellets obtained from chloroplasts containing 1 mg of protein were analyzed (lanes 3 and 4). Proteins of the pellet were further extracted with 1 M NaCl, and the insoluble pellets were analyzed (lanes 5 and 6). To exclude differences between light and dark conditions, nondividing chloroplasts were isolated from a concentrated culture during the dark period, in which cells were in interphase. Then, the chloroplasts were lysed using the same treatment as was used for lanes 3 and 4 (lanes 7 and 8). Dividing chloroplasts were isolated from M phase culture synchronized without 5-fluorodeoxyuridine (−FdU; lanes 9 and 10). Arrowheads indicate specific bands in the dividing phase.

    Techniques Used: SDS Page, Isolation

    Immunoblot of FtsZ by Using Anti-CmFtsZ2 Antiserum. (A)  Specificity of anti-CmFtsZ2 antiserum. Total protein (100 μg) of  C. merolae  were separated by SDS-PAGE and reacted with the antiserum raised against recombinant CmFtsZ2 protein (lanes 1 and 2) or anti-Hsp60 antibody (lanes 3 and 4), respectively. Antibodies were preincubated with purified recombinant CmFtsZ2 protein (lanes 2 and 4). (B)  Localization of FtsZ. Isolated dividing chloroplasts containing 100 μg of protein (cp), pellet (ppt), and the supernatant (sup) obtained from the same amount of chloroplasts by 0.1% Nonidet P-40 treatment were separated by SDS-PAGE. FtsZ (60-kD band) was detected in the chloroplasts and the supernatant but not in the pellet.
    Figure Legend Snippet: Immunoblot of FtsZ by Using Anti-CmFtsZ2 Antiserum. (A) Specificity of anti-CmFtsZ2 antiserum. Total protein (100 μg) of C. merolae were separated by SDS-PAGE and reacted with the antiserum raised against recombinant CmFtsZ2 protein (lanes 1 and 2) or anti-Hsp60 antibody (lanes 3 and 4), respectively. Antibodies were preincubated with purified recombinant CmFtsZ2 protein (lanes 2 and 4). (B) Localization of FtsZ. Isolated dividing chloroplasts containing 100 μg of protein (cp), pellet (ppt), and the supernatant (sup) obtained from the same amount of chloroplasts by 0.1% Nonidet P-40 treatment were separated by SDS-PAGE. FtsZ (60-kD band) was detected in the chloroplasts and the supernatant but not in the pellet.

    Techniques Used: SDS Page, Recombinant, Purification, Isolation

    Comparison of the Morphology of the Outer, Middle, and Inner PD Rings after Nonidet P-40 Treatment with That in Isolated Chloroplasts in Thin Sections. (A)  to  (C)  Serial thin sections of an isolated chloroplast showing a tangential section  (A)  and a cross-section  (B)  of the PD ring. The cross-sectional image of the PD ring is magnified in  (C) . (D)  to  (F)  Serial thin sections of the insoluble portion after 0.1% Nonidet P-40 treatment showing a tangential section  (D)  and a cross-section  (E)  of the PD ring. The cross-sectional image of the PD ring is magnified in  (F) . Arrows, arrowhead, and double-arrowhead indicate the outer, inner, and middle PD rings, respectively. Bars in  (A)  and  (B)  = 500 nm; bars in  (C)  and  (F)  = 50 nm; bars in  (D)  and  (E)  = 200 nm.
    Figure Legend Snippet: Comparison of the Morphology of the Outer, Middle, and Inner PD Rings after Nonidet P-40 Treatment with That in Isolated Chloroplasts in Thin Sections. (A) to (C) Serial thin sections of an isolated chloroplast showing a tangential section (A) and a cross-section (B) of the PD ring. The cross-sectional image of the PD ring is magnified in (C) . (D) to (F) Serial thin sections of the insoluble portion after 0.1% Nonidet P-40 treatment showing a tangential section (D) and a cross-section (E) of the PD ring. The cross-sectional image of the PD ring is magnified in (F) . Arrows, arrowhead, and double-arrowhead indicate the outer, inner, and middle PD rings, respectively. Bars in (A) and (B) = 500 nm; bars in (C) and (F) = 50 nm; bars in (D) and (E) = 200 nm.

    Techniques Used: Isolation

    Visualization of the Outer PD Ring by Negative Staining after Treatment with Nonidet P-40. (A)  Thin section of a  C .  merolae  cell containing a dividing chloroplast and mitochondrion showing the PD ring and the mitochondrion-dividing ring. The section cut the cell at the center perpendicular to the equator. (B)  Magnified cross-sectional view of the PD ring showing that the PD ring is composed of three rings. (C)  Phase-contrast image of isolated dividing chloroplasts from a synchronized culture. (D)  The outer PD ring of an isolated dividing chloroplast was visualized as green fluorescence by labeling surface proteins of the chloroplast with NHS-biotin and detecting them with FITC-avidin. (E)  Chloroplasts shown in  (D)  were lysed by the addition of solution containing 0.5% Nonidet P-40 from the edge of the cover glass. The outer PD ring remained insoluble. (F)  and  (G)  Negative staining images of isolated chloroplasts before  (F)  and after  (G)  0.1% Nonidet P-40 treatment. (H)  The outer PD ring was clearly observed as a closed ring by negative staining after Nonidet P-40 treatment in oblique samples. (I)  Low magnification images frequently showed that the outer PD rings are attached to the outer envelopes of chloroplasts. Small arrows indicate the mitochondrion-dividing ring. Large arrows, arrowhead, and double arrowhead indicate the outer, inner, and middle PD rings, respectively. cp, chloroplast; mb, microbody; mt, mitochondrion; n, nucleus. Bars in  (A) ,  (F) , and  (G)  = 500 nm; bars in  (B)  and  (H)  = 100 nm; bar in  (C)  = 5 μm; bars in  (D)  and  (E)  = 2 μm; bar in  (I)  = 1 μm.
    Figure Legend Snippet: Visualization of the Outer PD Ring by Negative Staining after Treatment with Nonidet P-40. (A) Thin section of a C . merolae cell containing a dividing chloroplast and mitochondrion showing the PD ring and the mitochondrion-dividing ring. The section cut the cell at the center perpendicular to the equator. (B) Magnified cross-sectional view of the PD ring showing that the PD ring is composed of three rings. (C) Phase-contrast image of isolated dividing chloroplasts from a synchronized culture. (D) The outer PD ring of an isolated dividing chloroplast was visualized as green fluorescence by labeling surface proteins of the chloroplast with NHS-biotin and detecting them with FITC-avidin. (E) Chloroplasts shown in (D) were lysed by the addition of solution containing 0.5% Nonidet P-40 from the edge of the cover glass. The outer PD ring remained insoluble. (F) and (G) Negative staining images of isolated chloroplasts before (F) and after (G) 0.1% Nonidet P-40 treatment. (H) The outer PD ring was clearly observed as a closed ring by negative staining after Nonidet P-40 treatment in oblique samples. (I) Low magnification images frequently showed that the outer PD rings are attached to the outer envelopes of chloroplasts. Small arrows indicate the mitochondrion-dividing ring. Large arrows, arrowhead, and double arrowhead indicate the outer, inner, and middle PD rings, respectively. cp, chloroplast; mb, microbody; mt, mitochondrion; n, nucleus. Bars in (A) , (F) , and (G) = 500 nm; bars in (B) and (H) = 100 nm; bar in (C) = 5 μm; bars in (D) and (E) = 2 μm; bar in (I) = 1 μm.

    Techniques Used: Negative Staining, Isolation, Fluorescence, Labeling, Avidin-Biotin Assay

    6) Product Images from "Oda5p, a Novel Axonemal Protein Required for Assembly of the Outer Dynein Arm and an Associated Adenylate Kinase D⃞"

    Article Title: Oda5p, a Novel Axonemal Protein Required for Assembly of the Outer Dynein Arm and an Associated Adenylate Kinase D⃞

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E03-11-0820

    Western blot analysis indicates Oda5p is a salt-extractable,  M r  76,000 axonemal protein that sediments at ∼5S in sucrose density gradients. (A) The Oda5 antibody recognizes an  M r  76,000 band in wild-type whole cells that is absent from the  oda5-2  whole cells, confirming the antibody recognizes the correct protein. This band is not detected in cell bodies lacking flagella (middle), yet it is readily detected in whole flagella (right). (B) Oda5p remains associated with the axoneme after Nonidet P-40 detergent extraction (demembranated axonemes) and is not detected in the Nonidet P-40 detergent-soluble membrane + matrix fraction (membrane + matrix). Extraction of demembranated axonemes with 0.6 M KCl releases Oda5p into the KCl extract; none remains in the KCl-extracted axonemes. (C) Sucrose gradient fractions were probed with antibodies to outer dynein arm components IC2 and γ-DHC, and with the Oda5p-antibody. The outer dynein arm/ODA-DC complex sediments at ∼23S as expected; however, Oda5p sediments at ∼5S.
    Figure Legend Snippet: Western blot analysis indicates Oda5p is a salt-extractable, M r 76,000 axonemal protein that sediments at ∼5S in sucrose density gradients. (A) The Oda5 antibody recognizes an M r 76,000 band in wild-type whole cells that is absent from the oda5-2 whole cells, confirming the antibody recognizes the correct protein. This band is not detected in cell bodies lacking flagella (middle), yet it is readily detected in whole flagella (right). (B) Oda5p remains associated with the axoneme after Nonidet P-40 detergent extraction (demembranated axonemes) and is not detected in the Nonidet P-40 detergent-soluble membrane + matrix fraction (membrane + matrix). Extraction of demembranated axonemes with 0.6 M KCl releases Oda5p into the KCl extract; none remains in the KCl-extracted axonemes. (C) Sucrose gradient fractions were probed with antibodies to outer dynein arm components IC2 and γ-DHC, and with the Oda5p-antibody. The outer dynein arm/ODA-DC complex sediments at ∼23S as expected; however, Oda5p sediments at ∼5S.

    Techniques Used: Western Blot

    7) Product Images from "Kaposi's sarcoma-associated herpesvirus encodes two proteins that block cell surface display of MHC class I chains by enhancing their endocytosis"

    Article Title: Kaposi's sarcoma-associated herpesvirus encodes two proteins that block cell surface display of MHC class I chains by enhancing their endocytosis

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    Intracellular transport and stability of MHC I molecules. ( A ) HeLa cells stably transfected with pCEP4-K3 ( Lower ) or the pCEP4 vector alone ( Upper ) were metabolically labeled for 20 min and chased for the indicated time periods. After being lysed in 1% Nonidet P-40, lysates were divided into three aliquots. Two were kept on ice and one was incubated at 37°C for 1 h. Heterodimeric MHC I molecules were immunoprecipitated with mAb W6/32 and treated with endo H where indicated. ( B ) BJAB cells stably transfected with pcdef3-K5 or the vector alone were radiolabeled for 30 min and chased for the indicated time periods. MHC I molecules in cell lysates were immunoprecipitated with mAb W6/32. Quantitation of the data is presented as a fraction of initial MHC I levels; ⋄, K5 expressing cells; ●, control cells. ( C ) BJAB cells stably transfected with pcdef3-K5 were radiolabeled for 30 min and chased for 3 h in the presence or absence of the indicated inhibitors. MHC I molecules were immunoprecipitated by using mAb W6/32.
    Figure Legend Snippet: Intracellular transport and stability of MHC I molecules. ( A ) HeLa cells stably transfected with pCEP4-K3 ( Lower ) or the pCEP4 vector alone ( Upper ) were metabolically labeled for 20 min and chased for the indicated time periods. After being lysed in 1% Nonidet P-40, lysates were divided into three aliquots. Two were kept on ice and one was incubated at 37°C for 1 h. Heterodimeric MHC I molecules were immunoprecipitated with mAb W6/32 and treated with endo H where indicated. ( B ) BJAB cells stably transfected with pcdef3-K5 or the vector alone were radiolabeled for 30 min and chased for the indicated time periods. MHC I molecules in cell lysates were immunoprecipitated with mAb W6/32. Quantitation of the data is presented as a fraction of initial MHC I levels; ⋄, K5 expressing cells; ●, control cells. ( C ) BJAB cells stably transfected with pcdef3-K5 were radiolabeled for 30 min and chased for 3 h in the presence or absence of the indicated inhibitors. MHC I molecules were immunoprecipitated by using mAb W6/32.

    Techniques Used: Stable Transfection, Transfection, Plasmid Preparation, Metabolic Labelling, Labeling, Incubation, Immunoprecipitation, Quantitation Assay, Expressing

    8) Product Images from "Cell-cycle arrest and apoptosis hypersusceptibility as a consequence of Lck deficiency in nontransformed T lymphocytes"

    Article Title: Cell-cycle arrest and apoptosis hypersusceptibility as a consequence of Lck deficiency in nontransformed T lymphocytes

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    Lck-deficient T cells express increased levels of predominantly tyrosine-phosphorylated cdc2 kinase. ( A ) T cells were activated by anti-CD3 cross-linking for the indicated times. Whole-cell lysates were prepared, and ≈50 μg of total protein was run per lane on 9% SDS/PAGE and immunoblotted with anti-cdc2 antibodies ( Upper ). Subsequently, the membrane was stripped and reprobed with 4G10 (anti-phosphotyrosine) mAb ( Lower ). The position of the 30-kDa molecular-mass standard is indicated. ( B ) Resting D10 and D8 cells were lysed in Nonidet P-40 containing lysis buffer, and ≈500 μg of total protein was immunoprecipitated with either normal rabbit serum (NRS) or cdc2-specific serum. The precipitates were resolved on 12.5% SDS/PAGE, transferred to poly(vinylidene difluoride) membrane, and immunoblotted wth 4G10 anti-phosphotyrosine(α-pY) mAb ( Upper ). Subsequently, the blot was stripped and reprobed with anti-cdc2 antiserum (α-cdc2;  Lower ). The molecular mass of standards is given in kDa. The cdc2 band is indicated by an arrow. The results are representative of five ( A ) and two ( B ) independent experiments.
    Figure Legend Snippet: Lck-deficient T cells express increased levels of predominantly tyrosine-phosphorylated cdc2 kinase. ( A ) T cells were activated by anti-CD3 cross-linking for the indicated times. Whole-cell lysates were prepared, and ≈50 μg of total protein was run per lane on 9% SDS/PAGE and immunoblotted with anti-cdc2 antibodies ( Upper ). Subsequently, the membrane was stripped and reprobed with 4G10 (anti-phosphotyrosine) mAb ( Lower ). The position of the 30-kDa molecular-mass standard is indicated. ( B ) Resting D10 and D8 cells were lysed in Nonidet P-40 containing lysis buffer, and ≈500 μg of total protein was immunoprecipitated with either normal rabbit serum (NRS) or cdc2-specific serum. The precipitates were resolved on 12.5% SDS/PAGE, transferred to poly(vinylidene difluoride) membrane, and immunoblotted wth 4G10 anti-phosphotyrosine(α-pY) mAb ( Upper ). Subsequently, the blot was stripped and reprobed with anti-cdc2 antiserum (α-cdc2; Lower ). The molecular mass of standards is given in kDa. The cdc2 band is indicated by an arrow. The results are representative of five ( A ) and two ( B ) independent experiments.

    Techniques Used: SDS Page, Lysis, Immunoprecipitation

    Defective cdc2-associated histone H1 kinase activity in D8 T cells. Resting T cells were grown for 16 h in medium containing IL-2 (20 units/ml) and IL-4 (100 units/ml) lymphokines in the presence (lanes 2 and 4) or absence (lanes 1 and 3) of nocodazole (100 ng/ml). Cell lysates were prepared in Nonidet P-40 lysis buffer, and equal protein amounts (≈500 μg per sample) were immunoprecipitated with antiserum specific to cdc2. Immune complexes were used in a kinase reaction in the presence of histone H1 as the substrate. The results are representative of three independent experiments.
    Figure Legend Snippet: Defective cdc2-associated histone H1 kinase activity in D8 T cells. Resting T cells were grown for 16 h in medium containing IL-2 (20 units/ml) and IL-4 (100 units/ml) lymphokines in the presence (lanes 2 and 4) or absence (lanes 1 and 3) of nocodazole (100 ng/ml). Cell lysates were prepared in Nonidet P-40 lysis buffer, and equal protein amounts (≈500 μg per sample) were immunoprecipitated with antiserum specific to cdc2. Immune complexes were used in a kinase reaction in the presence of histone H1 as the substrate. The results are representative of three independent experiments.

    Techniques Used: Activity Assay, Lysis, Immunoprecipitation

    9) Product Images from "Cathepsin L Is Responsible for Processing and Activation of Proheparanase through Multiple Cleavages of a Linker Segment *"

    Article Title: Cathepsin L Is Responsible for Processing and Activation of Proheparanase through Multiple Cleavages of a Linker Segment *

    Journal:

    doi: 10.1074/jbc.M801327200

    Cathepsin L knock-out ( KO ) fibroblasts and tissues are unable  to process proheparanase.  Fibroblasts derived from either cathepsin L  knock-out RT2 tumors (○) or wild-type RT2 tumors (•) were lysed (1%  Nonidet P-40, 10 m m  EDTA in PBS
    Figure Legend Snippet: Cathepsin L knock-out ( KO ) fibroblasts and tissues are unable to process proheparanase. Fibroblasts derived from either cathepsin L knock-out RT2 tumors (○) or wild-type RT2 tumors (•) were lysed (1% Nonidet P-40, 10 m m EDTA in PBS

    Techniques Used: Knock-Out, Derivative Assay

    10) Product Images from "A Plant-Specific Dynamin-Related Protein Forms a Ring at the Chloroplast Division Site"

    Article Title: A Plant-Specific Dynamin-Related Protein Forms a Ring at the Chloroplast Division Site

    Journal: The Plant Cell

    doi: 10.1105/tpc.009373

    CmDnm2 Associates with Chloroplasts Only during the Division Phase. Immunoblot analyses using anti-CmDnm2 antibodies. Twenty micrograms of protein in each sample was separated in each lane, except for in  (C) , in which samples were obtained from isolated chloroplasts containing 100 μg of protein. (A)  Total protein from synchronized M-phase cells was blotted with preimmune antisera or anti-CmDnm2 antibodies. (B)  and  (C)  Dividing chloroplasts were isolated from M-phase synchronous culture  (B)  and fractionated further into the pellet (P) and supernatant (S) by osmotic bursting or treatment with 0.5% Nonidet P-40 and 0.1 mg/mL DNaseI and then centrifugation  (C) . (D)  Aliquots of the synchronous culture were collected at the indicated times, and total proteins were separated. 5-Flurodeoxyuridine (FdU) was added to the culture at a concentration of 10 μg/mL at 2 h before the onset of the second dark period. The cell cycle was arrested at S-phase by 5-flurodeoxyuridine, whereas chloroplast division occurred continuously, producing four or eight chloroplasts per cell.
    Figure Legend Snippet: CmDnm2 Associates with Chloroplasts Only during the Division Phase. Immunoblot analyses using anti-CmDnm2 antibodies. Twenty micrograms of protein in each sample was separated in each lane, except for in (C) , in which samples were obtained from isolated chloroplasts containing 100 μg of protein. (A) Total protein from synchronized M-phase cells was blotted with preimmune antisera or anti-CmDnm2 antibodies. (B) and (C) Dividing chloroplasts were isolated from M-phase synchronous culture (B) and fractionated further into the pellet (P) and supernatant (S) by osmotic bursting or treatment with 0.5% Nonidet P-40 and 0.1 mg/mL DNaseI and then centrifugation (C) . (D) Aliquots of the synchronous culture were collected at the indicated times, and total proteins were separated. 5-Flurodeoxyuridine (FdU) was added to the culture at a concentration of 10 μg/mL at 2 h before the onset of the second dark period. The cell cycle was arrested at S-phase by 5-flurodeoxyuridine, whereas chloroplast division occurred continuously, producing four or eight chloroplasts per cell.

    Techniques Used: Isolation, Centrifugation, Concentration Assay

    11) Product Images from "Receptor-interacting protein kinase 3 promotes platelet activation and thrombosis"

    Article Title: Receptor-interacting protein kinase 3 promotes platelet activation and thrombosis

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1610963114

    Coimmunoprecipitation of RIP3 with Gi or Gq in mouse platelets. Washed platelets (3 × 10 8  /mL) from WT mice were lysed with equal volumes of 2× Nonidet P-40 lysis buffer containing protease inhibitor mixture tablets on ice for 30 min.
    Figure Legend Snippet: Coimmunoprecipitation of RIP3 with Gi or Gq in mouse platelets. Washed platelets (3 × 10 8 /mL) from WT mice were lysed with equal volumes of 2× Nonidet P-40 lysis buffer containing protease inhibitor mixture tablets on ice for 30 min.

    Techniques Used: Mouse Assay, Lysis, Protease Inhibitor

    12) Product Images from "Measurement of soluble Fcγ receptor type IIIa derived from macrophages in plasma: increase in patients with rheumatoid arthritis"

    Article Title: Measurement of soluble Fcγ receptor type IIIa derived from macrophages in plasma: increase in patients with rheumatoid arthritis

    Journal: Clinical and Experimental Immunology

    doi: 10.1046/j.1365-2249.2003.02168.x

    Analysis of immunoprecipitates of MKGR14 by anti-Fc γ RIII immunoblot (a, b, c) and gel electrophoresis (d). (a, b, c) Immunoprecipitates of MKGR14 (MKGR) or CLBFcRgranI (CLB) prepared from 4-day-cultured monocytes (a), LGL (b: NK cells) or NA1NA2-neutrophils (c) were analysed by SDS-PAGE under non-reducing conditions and transferred to a nitrocellulose membrane. The Fc γ RIII were detected by immunoblotting with an anti-Fc γ RIII MoAb CLB-LM6·30. (d) Four-day-cultured monocytes were surface-labelled with biotin and lysed in 1% Nonidet P-40. The cell lysates were incubated with Sepharose-coupled MKGR14 (MKGR) or CLBFcRgranI (CLB), and the immunoprecipitates were then analysed by SDS-PAGE under non-reducing conditions and transferred to a nitrocellulose membrane. The biotin-labelled protein was detected by blotting with streptavidin. M r , relative molecular mass × 10 −3 .
    Figure Legend Snippet: Analysis of immunoprecipitates of MKGR14 by anti-Fc γ RIII immunoblot (a, b, c) and gel electrophoresis (d). (a, b, c) Immunoprecipitates of MKGR14 (MKGR) or CLBFcRgranI (CLB) prepared from 4-day-cultured monocytes (a), LGL (b: NK cells) or NA1NA2-neutrophils (c) were analysed by SDS-PAGE under non-reducing conditions and transferred to a nitrocellulose membrane. The Fc γ RIII were detected by immunoblotting with an anti-Fc γ RIII MoAb CLB-LM6·30. (d) Four-day-cultured monocytes were surface-labelled with biotin and lysed in 1% Nonidet P-40. The cell lysates were incubated with Sepharose-coupled MKGR14 (MKGR) or CLBFcRgranI (CLB), and the immunoprecipitates were then analysed by SDS-PAGE under non-reducing conditions and transferred to a nitrocellulose membrane. The biotin-labelled protein was detected by blotting with streptavidin. M r , relative molecular mass × 10 −3 .

    Techniques Used: Nucleic Acid Electrophoresis, Cell Culture, SDS Page, Incubation

    13) Product Images from "Cilium transition zone proteome reveals compartmentalization and differential dynamics of ciliopathy complexes"

    Article Title: Cilium transition zone proteome reveals compartmentalization and differential dynamics of ciliopathy complexes

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1604258113

    Isolation of transition zones. ( A ) Live cells expressing eYFP::FTZC at the TZ (green) were ( B ) treated with 1% Nonidet P-40 to extract the soluble material. ( C ) Washed cytoskeletons were sonicated to shear the TZs from other flagellar material. Tagged
    Figure Legend Snippet: Isolation of transition zones. ( A ) Live cells expressing eYFP::FTZC at the TZ (green) were ( B ) treated with 1% Nonidet P-40 to extract the soluble material. ( C ) Washed cytoskeletons were sonicated to shear the TZs from other flagellar material. Tagged

    Techniques Used: Isolation, Expressing, Sonication

    14) Product Images from "AMP-activated Protein Kinase and p38 MAPK ActivateO-GlcNAcylation of Neuronal Proteins during Glucose Deprivation *-GlcNAcylation of Neuronal Proteins during Glucose Deprivation * S⃞"

    Article Title: AMP-activated Protein Kinase and p38 MAPK ActivateO-GlcNAcylation of Neuronal Proteins during Glucose Deprivation *-GlcNAcylation of Neuronal Proteins during Glucose Deprivation * S⃞

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M801222200

    GST-OGT-(979–1036) overexpression disrupts the OGT-p38 interaction  and prevents the glucose deprivation-induced NF-H solubility change. A , lysates from Neuro-2a cells transfected with the indicated  constructs were immunoprecipitated ( IP ) for HA and immunoblotted  ( IB ) for OGT, GST, and HA.  B , Nonidet P-40-soluble  ( sol ) and -insoluble ( insol ) fractions prepared from  Neuro-2a cells transfected with GST or GST-OGT-(979–1036) and  glucose-deprived for the indicated times were immunoblotted for NF-H, GST,  tubulin, and actin.
    Figure Legend Snippet: GST-OGT-(979–1036) overexpression disrupts the OGT-p38 interaction and prevents the glucose deprivation-induced NF-H solubility change. A , lysates from Neuro-2a cells transfected with the indicated constructs were immunoprecipitated ( IP ) for HA and immunoblotted ( IB ) for OGT, GST, and HA. B , Nonidet P-40-soluble ( sol ) and -insoluble ( insol ) fractions prepared from Neuro-2a cells transfected with GST or GST-OGT-(979–1036) and glucose-deprived for the indicated times were immunoblotted for NF-H, GST, tubulin, and actin.

    Techniques Used: Over Expression, Solubility, Transfection, Construct, Immunoprecipitation

    15) Product Images from "Multivalent structure of an ??T cell receptor"

    Article Title: Multivalent structure of an ??T cell receptor

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    Identification of bispecific αβTCRs on the  surface of T cells from V β2 ×V β8  double  transgenic mice. Immunoprecipitations of TCRβ from transgenic T cells  were done with V β  domain-specific mAbs or control  reagents (−) and immunoblotted with mAbs to V β2 ,  V β8 , or C α . Cell sources were either spleen  T cells expanded in culture for 2–3 weeks with interleukin 2  ( A – C ) or freshly isolated single-cell  suspensions from spleen and thymus ( D ). Organs were  either from V β2  or V β8  single transgenic  mice or V β2 ×V β8 ), as indicated ( A ). In additional control  immunoprecipitations, lines labeled as C in  D ,  V β2  and V β8  single transgenic cells were  mixed in a 1:1 ratio before the immunoprecipitation with  TCRV β  mAb, which had the reciprocal specificity to those  used in immunoblotting. Double transgenic cells were used in the  remaining experiments ( B – D ).  Two-dimensional SDS/PAGE ( C ) of immunoprecipitates with  mAb to V β2  ( Left ) were blotted with mAb to  V β8  ( Upper ) and C α  ( Lower ), whereas immunoprecipitates with mAb to  V β8  ( Right ) where blotted with  anti-V β2  ( Upper ) and control  ( Lower ) mAbs. Immunoprecipitates were digested  ( B ) in the absence (−) or presence of endoglycosidase H (H)  or  N ). Sizes before and after the removal of N-linked sugars are  indicated by open arrowheads and asterisks, respectively. Cells were  lysed in 1% of either Nonidet P-40 (first three lines from the left in A ). Neat coprecipitations were attained in 30 independent  experiments, each with different transgenic mice. Ticks indicate  migration of molecular size markers (Bio-Rad), given in kDa: 101, 83,  50.6, and 35.5 kDa ( A ), 148, 60, 42, and 30 kDa  ( B ), 60 and 42 kDa ( C ), and 145, 83, 60, 50, and  35 kDa ( D ).
    Figure Legend Snippet: Identification of bispecific αβTCRs on the surface of T cells from V β2 ×V β8 double transgenic mice. Immunoprecipitations of TCRβ from transgenic T cells were done with V β domain-specific mAbs or control reagents (−) and immunoblotted with mAbs to V β2 , V β8 , or C α . Cell sources were either spleen T cells expanded in culture for 2–3 weeks with interleukin 2 ( A – C ) or freshly isolated single-cell suspensions from spleen and thymus ( D ). Organs were either from V β2 or V β8 single transgenic mice or V β2 ×V β8 ), as indicated ( A ). In additional control immunoprecipitations, lines labeled as C in D , V β2 and V β8 single transgenic cells were mixed in a 1:1 ratio before the immunoprecipitation with TCRV β mAb, which had the reciprocal specificity to those used in immunoblotting. Double transgenic cells were used in the remaining experiments ( B – D ). Two-dimensional SDS/PAGE ( C ) of immunoprecipitates with mAb to V β2 ( Left ) were blotted with mAb to V β8 ( Upper ) and C α ( Lower ), whereas immunoprecipitates with mAb to V β8 ( Right ) where blotted with anti-V β2 ( Upper ) and control ( Lower ) mAbs. Immunoprecipitates were digested ( B ) in the absence (−) or presence of endoglycosidase H (H) or N ). Sizes before and after the removal of N-linked sugars are indicated by open arrowheads and asterisks, respectively. Cells were lysed in 1% of either Nonidet P-40 (first three lines from the left in A ). Neat coprecipitations were attained in 30 independent experiments, each with different transgenic mice. Ticks indicate migration of molecular size markers (Bio-Rad), given in kDa: 101, 83, 50.6, and 35.5 kDa ( A ), 148, 60, 42, and 30 kDa ( B ), 60 and 42 kDa ( C ), and 145, 83, 60, 50, and 35 kDa ( D ).

    Techniques Used: Transgenic Assay, Mouse Assay, Isolation, Labeling, Immunoprecipitation, SDS Page, Migration

    16) Product Images from "Presynaptic Targeting of ?4?2 Nicotinic Acetylcholine Receptors Is Regulated by Neurexin-1? *"

    Article Title: Presynaptic Targeting of ?4?2 Nicotinic Acetylcholine Receptors Is Regulated by Neurexin-1? *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.017384

    Neurexin-1β forms complexes with α4β2 AChRs  in vivo . A , coimmunoprecipitation of NRX and α4β2 AChRs from whole rat brain lysates. Rat brains were homogenized, solubilized in 1% Nonidet P-40 lysis buffer, and incubated
    Figure Legend Snippet: Neurexin-1β forms complexes with α4β2 AChRs in vivo . A , coimmunoprecipitation of NRX and α4β2 AChRs from whole rat brain lysates. Rat brains were homogenized, solubilized in 1% Nonidet P-40 lysis buffer, and incubated

    Techniques Used: In Vivo, Lysis, Incubation

    17) Product Images from "Glycoprotein nonmetastatic melanoma protein b, a melanocytic cell marker, is a melanosome-specific and proteolytically released protein"

    Article Title: Glycoprotein nonmetastatic melanoma protein b, a melanocytic cell marker, is a melanosome-specific and proteolytically released protein

    Journal: The FASEB Journal

    doi: 10.1096/fj.09-151019

    GPNMB is not significantly buried in the TX-insoluble fraction. MNT-1 cells were solubilized with M-PER, 1% Nonidet P-40 or 1% TX containing lysis buffer. Soluble fractions (S) and insoluble fractions (I) were analyzed by immunoblotting using anti-GPNMB ( A ), αPEP13h ( B , left), and HMB45 ( B , right) antibodies.
    Figure Legend Snippet: GPNMB is not significantly buried in the TX-insoluble fraction. MNT-1 cells were solubilized with M-PER, 1% Nonidet P-40 or 1% TX containing lysis buffer. Soluble fractions (S) and insoluble fractions (I) were analyzed by immunoblotting using anti-GPNMB ( A ), αPEP13h ( B , left), and HMB45 ( B , right) antibodies.

    Techniques Used: Lysis

    18) Product Images from "Identification and Characterization of CD300H, a New Member of the Human CD300 Immunoreceptor Family *"

    Article Title: Identification and Characterization of CD300H, a New Member of the Human CD300 Immunoreceptor Family *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.643361

    Biochemical analyses of CD300H. A , BW5147 cells and transfectants expressing FLAG-tagged CD300H (5 × 10 6  cells/experiment) were lysed in 1% Nonidet P-40 buffer, immunoprecipitated ( IP ) with anti-FLAG, and immunoblotted ( IB ) with anti-FLAG.  B , BW5147 transfectants simultaneously expressing FLAG-CD300H and HA-CD300H were lysed in digitonin buffer, immunoprecipitated with anti-HA or anti-FLAG, and immunoblotted with anti-HA or anti-FLAG in reducing conditions.  C , culture supernatant from 293T cells transiently expressing FLAG-tagged CD300Hs or mock were immunoprecipitated with anti-FLAG and immunoblotted with anti-FLAG.  D , U937 cells and transfectants stably expressing FLAG-tagged CD300H were lysed in digitonin buffer, immunoprecipitated with anti-FLAG, anti-DAP12, anti-DAP10, or anti-FcϵRIγ, and immunoblotted with anti-FLAG, anti-DAP12, anti-DAP10, or anti-FcϵRIγ. Data are representative of two independent experiments.
    Figure Legend Snippet: Biochemical analyses of CD300H. A , BW5147 cells and transfectants expressing FLAG-tagged CD300H (5 × 10 6 cells/experiment) were lysed in 1% Nonidet P-40 buffer, immunoprecipitated ( IP ) with anti-FLAG, and immunoblotted ( IB ) with anti-FLAG. B , BW5147 transfectants simultaneously expressing FLAG-CD300H and HA-CD300H were lysed in digitonin buffer, immunoprecipitated with anti-HA or anti-FLAG, and immunoblotted with anti-HA or anti-FLAG in reducing conditions. C , culture supernatant from 293T cells transiently expressing FLAG-tagged CD300Hs or mock were immunoprecipitated with anti-FLAG and immunoblotted with anti-FLAG. D , U937 cells and transfectants stably expressing FLAG-tagged CD300H were lysed in digitonin buffer, immunoprecipitated with anti-FLAG, anti-DAP12, anti-DAP10, or anti-FcϵRIγ, and immunoblotted with anti-FLAG, anti-DAP12, anti-DAP10, or anti-FcϵRIγ. Data are representative of two independent experiments.

    Techniques Used: Expressing, Immunoprecipitation, Stable Transfection

    19) Product Images from "Oda5p, a Novel Axonemal Protein Required for Assembly of the Outer Dynein Arm and an Associated Adenylate Kinase D⃞"

    Article Title: Oda5p, a Novel Axonemal Protein Required for Assembly of the Outer Dynein Arm and an Associated Adenylate Kinase D⃞

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E03-11-0820

    Western blot analysis indicates Oda5p is a salt-extractable,  M r  76,000 axonemal protein that sediments at ∼5S in sucrose density gradients. (A) The Oda5 antibody recognizes an  M r  76,000 band in wild-type whole cells that is absent from the  oda5-2  whole cells, confirming the antibody recognizes the correct protein. This band is not detected in cell bodies lacking flagella (middle), yet it is readily detected in whole flagella (right). (B) Oda5p remains associated with the axoneme after Nonidet P-40 detergent extraction (demembranated axonemes) and is not detected in the Nonidet P-40 detergent-soluble membrane + matrix fraction (membrane + matrix). Extraction of demembranated axonemes with 0.6 M KCl releases Oda5p into the KCl extract; none remains in the KCl-extracted axonemes. (C) Sucrose gradient fractions were probed with antibodies to outer dynein arm components IC2 and γ-DHC, and with the Oda5p-antibody. The outer dynein arm/ODA-DC complex sediments at ∼23S as expected; however, Oda5p sediments at ∼5S.
    Figure Legend Snippet: Western blot analysis indicates Oda5p is a salt-extractable, M r 76,000 axonemal protein that sediments at ∼5S in sucrose density gradients. (A) The Oda5 antibody recognizes an M r 76,000 band in wild-type whole cells that is absent from the oda5-2 whole cells, confirming the antibody recognizes the correct protein. This band is not detected in cell bodies lacking flagella (middle), yet it is readily detected in whole flagella (right). (B) Oda5p remains associated with the axoneme after Nonidet P-40 detergent extraction (demembranated axonemes) and is not detected in the Nonidet P-40 detergent-soluble membrane + matrix fraction (membrane + matrix). Extraction of demembranated axonemes with 0.6 M KCl releases Oda5p into the KCl extract; none remains in the KCl-extracted axonemes. (C) Sucrose gradient fractions were probed with antibodies to outer dynein arm components IC2 and γ-DHC, and with the Oda5p-antibody. The outer dynein arm/ODA-DC complex sediments at ∼23S as expected; however, Oda5p sediments at ∼5S.

    Techniques Used: Western Blot

    20) Product Images from "Krp1 (Sarcosin) Promotes Lateral Fusion of Myofibril Assembly Intermediates in Cultured Mouse Cardiomyocytes"

    Article Title: Krp1 (Sarcosin) Promotes Lateral Fusion of Myofibril Assembly Intermediates in Cultured Mouse Cardiomyocytes

    Journal: Experimental cell research

    doi: 10.1016/j.yexcr.2007.12.009

    Immunoblot analysis of Nonidet P-40 extracted and insoluble fractions. Equal volumes of total cell lysate, extracted and insoluble fractions were analyzed by immunoblot (left) and densitometric analysis (right). Representative immunoblots are shown. The graph displays the means and SEM of three independent experiments. A large proportion of Krp1 remains in the insoluble pellet along with most of the cytoskeletal components (myosin, actin, N-RAP) and caveolin-3, although the majority of Krp1 is present in the extracted supernatant along with the cytosolic marker GAPDH and the integral membrane marker ß1-integrin.
    Figure Legend Snippet: Immunoblot analysis of Nonidet P-40 extracted and insoluble fractions. Equal volumes of total cell lysate, extracted and insoluble fractions were analyzed by immunoblot (left) and densitometric analysis (right). Representative immunoblots are shown. The graph displays the means and SEM of three independent experiments. A large proportion of Krp1 remains in the insoluble pellet along with most of the cytoskeletal components (myosin, actin, N-RAP) and caveolin-3, although the majority of Krp1 is present in the extracted supernatant along with the cytosolic marker GAPDH and the integral membrane marker ß1-integrin.

    Techniques Used: Western Blot, Marker

    21) Product Images from "The Mcm2–7-interacting domain of human mini-chromosome maintenance 10 (Mcm10) protein is important for stable chromatin association and origin firing"

    Article Title: The Mcm2–7-interacting domain of human mini-chromosome maintenance 10 (Mcm10) protein is important for stable chromatin association and origin firing

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.779371

    Subcellular distribution of Mcm10 and association with Mcm2–7 were studied. A,  HeLa cells were first fractionated into Nonidet P-40-soluble ( lane 2 ) and insoluble ( lane 3 ) fractions, and then chromatin-bound fractions solubilized with benzonase digestion ( lane 4 ) and benzonase-unextractable fraction ( lane 5 ) were obtained from the Nonidet P-40-insoluble fraction. Whole cell extract was loaded onto  lane 1 . Subcellular localization of Mcm10, as well as several replication proteins, was investigated by immunoblotting. Mcm10 was mainly localized in the benzonase-soluble fraction.  B,  the benzonase-soluble fraction was prepared from a HeLa cell line that stably expressed HA-tagged Mcm10 and immunoprecipitated with control antibody ( lane 2 ) or anti-HA antibody ( lane 3 ). Precipitates were subjected to immunoblot analysis using anti-Mcm10 or Mcm2–7 antibodies.  Lane 1  contained 12.5 or 3% of the input material for the detection of Mcm10 or Mcm2–7, respectively. Chromatin-associated human HA-Mcm10 was co-purified with the Mcm2–7 complex as well as a small amount of endogenous Mcm10.  C,  the benzonase-soluble fraction was prepared and immunoprecipitated with anti-HA antibody in the presence of ethidium bromide. Precipitates were subjected to immunoblot analysis using anti-Mcm10, Mcm2, and Mcm6 antibodies. The amount of co-purified Mcm2–7 was decreased substantially in the presence of ethidium bromide, suggesting that the DNA binding of either Mcm10 or Mcm2–7 facilitated the complex formation.  D,  the benzonase-soluble fractions from an asynchronous culture and the Nonidet P-40–soluble fractions from metaphase cells were prepared and immunoprecipitated with anti-HA antibody. Precipitates were subjected to immunoblot analysis. Mcm10 did not interact with Mcm2–7 at metaphase.
    Figure Legend Snippet: Subcellular distribution of Mcm10 and association with Mcm2–7 were studied. A, HeLa cells were first fractionated into Nonidet P-40-soluble ( lane 2 ) and insoluble ( lane 3 ) fractions, and then chromatin-bound fractions solubilized with benzonase digestion ( lane 4 ) and benzonase-unextractable fraction ( lane 5 ) were obtained from the Nonidet P-40-insoluble fraction. Whole cell extract was loaded onto lane 1 . Subcellular localization of Mcm10, as well as several replication proteins, was investigated by immunoblotting. Mcm10 was mainly localized in the benzonase-soluble fraction. B, the benzonase-soluble fraction was prepared from a HeLa cell line that stably expressed HA-tagged Mcm10 and immunoprecipitated with control antibody ( lane 2 ) or anti-HA antibody ( lane 3 ). Precipitates were subjected to immunoblot analysis using anti-Mcm10 or Mcm2–7 antibodies. Lane 1 contained 12.5 or 3% of the input material for the detection of Mcm10 or Mcm2–7, respectively. Chromatin-associated human HA-Mcm10 was co-purified with the Mcm2–7 complex as well as a small amount of endogenous Mcm10. C, the benzonase-soluble fraction was prepared and immunoprecipitated with anti-HA antibody in the presence of ethidium bromide. Precipitates were subjected to immunoblot analysis using anti-Mcm10, Mcm2, and Mcm6 antibodies. The amount of co-purified Mcm2–7 was decreased substantially in the presence of ethidium bromide, suggesting that the DNA binding of either Mcm10 or Mcm2–7 facilitated the complex formation. D, the benzonase-soluble fractions from an asynchronous culture and the Nonidet P-40–soluble fractions from metaphase cells were prepared and immunoprecipitated with anti-HA antibody. Precipitates were subjected to immunoblot analysis. Mcm10 did not interact with Mcm2–7 at metaphase.

    Techniques Used: Stable Transfection, Immunoprecipitation, Purification, Binding Assay

    22) Product Images from "MAPK15 is part of the ULK complex and controls its activity to regulate early phases of the autophagic process"

    Article Title: MAPK15 is part of the ULK complex and controls its activity to regulate early phases of the autophagic process

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.002527

    MAPK15 participates in the ULK1 complex. A,  HEK293 and NIH3T3 were harvested and lysed in 1% Nonidet P-40 lysis buffer. The proteins (10 mg per sample) were then subjected to immunoprecipitation ( IP ) with isotype control IgG ( IgG ) or MAPK15-specific antibody (custom preparation). Total lysates and immunoprecipitated complexes were subjected to Western blotting ( WB ) to detect endogenous ULK1 and MAPK15 protein levels. Representative images from three different experiments are shown ( n  = 3).  B , HEK293T cells were transfected with plasmid encoding for ULK1-FLAG in combination with HA-MAPK15 WT , HA-MAPK15 KD , or the empty vector. After 24 h, total lysates were subject to anti-FLAG immunoprecipitation and analyzed by Western blot analysis ( n  = 3).  C , HEK293T cells were transfected with different plasmids encoding for FLAG-tagged ULK1, ATG13, FIP200, or ATG101 proteins in combination with HA-MAPK15 or the empty vector. After 24 h, total lysates were subject to anti-FLAG immunoprecipitation and analyzed by Western blot analysis ( n  = 3).  D , HeLa cells stably expressing EGFP-GABARAP were transfected with HA-MAPK15 and ULK1-FLAG plasmids and subjected to immunofluorescence analysis after 48 h. Representative images are from three different experiments ( n  = 3). EGFP-GABARAP is visualized in  green , ULK1-FLAG in  red , and HA-MAPK15 in  blue  ( upper panels ).  White squares  indicate zoom areas.  Scale bars , 10 μm.  E,  colocalization rate was measured by LAS AF (Leica Microsystem) software, analyzing single cells that express both MAPK15 and ULK1 in addition to EGFP-GABARAP. Thresholds were set at 20% for each channel as suggested from the manufacturer's protocol. Reciprocal colocalization rate between ULK1 and GABARAP, ULK1 and MAPK15, and GABARAP and MAPK15 were evaluated. Measures were obtained by analyzing at least 100 cells/sample from three different experiments.  F , colocalization spots were analyzed for fluorescent signal intensity for each channel; the  yellow line  indicates the measured points on the  merged image .
    Figure Legend Snippet: MAPK15 participates in the ULK1 complex. A, HEK293 and NIH3T3 were harvested and lysed in 1% Nonidet P-40 lysis buffer. The proteins (10 mg per sample) were then subjected to immunoprecipitation ( IP ) with isotype control IgG ( IgG ) or MAPK15-specific antibody (custom preparation). Total lysates and immunoprecipitated complexes were subjected to Western blotting ( WB ) to detect endogenous ULK1 and MAPK15 protein levels. Representative images from three different experiments are shown ( n = 3). B , HEK293T cells were transfected with plasmid encoding for ULK1-FLAG in combination with HA-MAPK15 WT , HA-MAPK15 KD , or the empty vector. After 24 h, total lysates were subject to anti-FLAG immunoprecipitation and analyzed by Western blot analysis ( n = 3). C , HEK293T cells were transfected with different plasmids encoding for FLAG-tagged ULK1, ATG13, FIP200, or ATG101 proteins in combination with HA-MAPK15 or the empty vector. After 24 h, total lysates were subject to anti-FLAG immunoprecipitation and analyzed by Western blot analysis ( n = 3). D , HeLa cells stably expressing EGFP-GABARAP were transfected with HA-MAPK15 and ULK1-FLAG plasmids and subjected to immunofluorescence analysis after 48 h. Representative images are from three different experiments ( n = 3). EGFP-GABARAP is visualized in green , ULK1-FLAG in red , and HA-MAPK15 in blue ( upper panels ). White squares indicate zoom areas. Scale bars , 10 μm. E, colocalization rate was measured by LAS AF (Leica Microsystem) software, analyzing single cells that express both MAPK15 and ULK1 in addition to EGFP-GABARAP. Thresholds were set at 20% for each channel as suggested from the manufacturer's protocol. Reciprocal colocalization rate between ULK1 and GABARAP, ULK1 and MAPK15, and GABARAP and MAPK15 were evaluated. Measures were obtained by analyzing at least 100 cells/sample from three different experiments. F , colocalization spots were analyzed for fluorescent signal intensity for each channel; the yellow line indicates the measured points on the merged image .

    Techniques Used: Lysis, Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, Stable Transfection, Expressing, Immunofluorescence, Software

    23) Product Images from "Oda5p, a Novel Axonemal Protein Required for Assembly of the Outer Dynein Arm and an Associated Adenylate Kinase D⃞"

    Article Title: Oda5p, a Novel Axonemal Protein Required for Assembly of the Outer Dynein Arm and an Associated Adenylate Kinase D⃞

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E03-11-0820

    Western blot analysis indicates Oda5p is a salt-extractable,  M r  76,000 axonemal protein that sediments at ∼5S in sucrose density gradients. (A) The Oda5 antibody recognizes an  M r  76,000 band in wild-type whole cells that is absent from the  oda5-2  whole cells, confirming the antibody recognizes the correct protein. This band is not detected in cell bodies lacking flagella (middle), yet it is readily detected in whole flagella (right). (B) Oda5p remains associated with the axoneme after Nonidet P-40 detergent extraction (demembranated axonemes) and is not detected in the Nonidet P-40 detergent-soluble membrane + matrix fraction (membrane + matrix). Extraction of demembranated axonemes with 0.6 M KCl releases Oda5p into the KCl extract; none remains in the KCl-extracted axonemes. (C) Sucrose gradient fractions were probed with antibodies to outer dynein arm components IC2 and γ-DHC, and with the Oda5p-antibody. The outer dynein arm/ODA-DC complex sediments at ∼23S as expected; however, Oda5p sediments at ∼5S.
    Figure Legend Snippet: Western blot analysis indicates Oda5p is a salt-extractable, M r 76,000 axonemal protein that sediments at ∼5S in sucrose density gradients. (A) The Oda5 antibody recognizes an M r 76,000 band in wild-type whole cells that is absent from the oda5-2 whole cells, confirming the antibody recognizes the correct protein. This band is not detected in cell bodies lacking flagella (middle), yet it is readily detected in whole flagella (right). (B) Oda5p remains associated with the axoneme after Nonidet P-40 detergent extraction (demembranated axonemes) and is not detected in the Nonidet P-40 detergent-soluble membrane + matrix fraction (membrane + matrix). Extraction of demembranated axonemes with 0.6 M KCl releases Oda5p into the KCl extract; none remains in the KCl-extracted axonemes. (C) Sucrose gradient fractions were probed with antibodies to outer dynein arm components IC2 and γ-DHC, and with the Oda5p-antibody. The outer dynein arm/ODA-DC complex sediments at ∼23S as expected; however, Oda5p sediments at ∼5S.

    Techniques Used: Western Blot

    24) Product Images from "Oda5p, a Novel Axonemal Protein Required for Assembly of the Outer Dynein Arm and an Associated Adenylate Kinase D⃞"

    Article Title: Oda5p, a Novel Axonemal Protein Required for Assembly of the Outer Dynein Arm and an Associated Adenylate Kinase D⃞

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E03-11-0820

    Western blot analysis indicates Oda5p is a salt-extractable,  M r  76,000 axonemal protein that sediments at ∼5S in sucrose density gradients. (A) The Oda5 antibody recognizes an  M r  76,000 band in wild-type whole cells that is absent from the  oda5-2  whole cells, confirming the antibody recognizes the correct protein. This band is not detected in cell bodies lacking flagella (middle), yet it is readily detected in whole flagella (right). (B) Oda5p remains associated with the axoneme after Nonidet P-40 detergent extraction (demembranated axonemes) and is not detected in the Nonidet P-40 detergent-soluble membrane + matrix fraction (membrane + matrix). Extraction of demembranated axonemes with 0.6 M KCl releases Oda5p into the KCl extract; none remains in the KCl-extracted axonemes. (C) Sucrose gradient fractions were probed with antibodies to outer dynein arm components IC2 and γ-DHC, and with the Oda5p-antibody. The outer dynein arm/ODA-DC complex sediments at ∼23S as expected; however, Oda5p sediments at ∼5S.
    Figure Legend Snippet: Western blot analysis indicates Oda5p is a salt-extractable, M r 76,000 axonemal protein that sediments at ∼5S in sucrose density gradients. (A) The Oda5 antibody recognizes an M r 76,000 band in wild-type whole cells that is absent from the oda5-2 whole cells, confirming the antibody recognizes the correct protein. This band is not detected in cell bodies lacking flagella (middle), yet it is readily detected in whole flagella (right). (B) Oda5p remains associated with the axoneme after Nonidet P-40 detergent extraction (demembranated axonemes) and is not detected in the Nonidet P-40 detergent-soluble membrane + matrix fraction (membrane + matrix). Extraction of demembranated axonemes with 0.6 M KCl releases Oda5p into the KCl extract; none remains in the KCl-extracted axonemes. (C) Sucrose gradient fractions were probed with antibodies to outer dynein arm components IC2 and γ-DHC, and with the Oda5p-antibody. The outer dynein arm/ODA-DC complex sediments at ∼23S as expected; however, Oda5p sediments at ∼5S.

    Techniques Used: Western Blot

    25) Product Images from "CD69 Suppresses Sphingosine 1-Phosophate Receptor-1 (S1P1) Function through Interaction with Membrane Helix 4 *"

    Article Title: CD69 Suppresses Sphingosine 1-Phosophate Receptor-1 (S1P1) Function through Interaction with Membrane Helix 4 *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.123299

    CD69-mediated down-modulation of S1P 1  is associated with protein degradation. A , flow cytometric analysis of Flag-S1P 1  or Flag-S1P 3  when co-transduced and sorted for low or high levels of CD69 in WEHI-231 cells. The relative amount of CD69 expression (+ or ++) was determined by expression of an IRES GFP reporter. Histogram overlays on the right show GFP reporter and hCD4 reporter expression for cells in quadrant 1 and 2 of the  top left plot , indicating the relative expression of the CD69-IRES-GFP construct and the Flag-S1P 1 -IRES-hCD4 construct, respectively.  B , co-IP of S1P 1  or S1P 3  with CD69 from the cells shown in the flow cytometric analysis. Densitometry readings are indicated showing the intensity of Flag and HA signal and the calculation of the ratio of HA signal to Flag signal is shown beneath the  lower panel . These data are representative of two experiments using standard lysis buffer (see “Experimental Procedures”) and one experiment using 1% Triton X in place of Brij97 and Nonidet P-40 with similar results. Nonspecific bands are indicated with an  asterisk .
    Figure Legend Snippet: CD69-mediated down-modulation of S1P 1 is associated with protein degradation. A , flow cytometric analysis of Flag-S1P 1 or Flag-S1P 3 when co-transduced and sorted for low or high levels of CD69 in WEHI-231 cells. The relative amount of CD69 expression (+ or ++) was determined by expression of an IRES GFP reporter. Histogram overlays on the right show GFP reporter and hCD4 reporter expression for cells in quadrant 1 and 2 of the top left plot , indicating the relative expression of the CD69-IRES-GFP construct and the Flag-S1P 1 -IRES-hCD4 construct, respectively. B , co-IP of S1P 1 or S1P 3 with CD69 from the cells shown in the flow cytometric analysis. Densitometry readings are indicated showing the intensity of Flag and HA signal and the calculation of the ratio of HA signal to Flag signal is shown beneath the lower panel . These data are representative of two experiments using standard lysis buffer (see “Experimental Procedures”) and one experiment using 1% Triton X in place of Brij97 and Nonidet P-40 with similar results. Nonspecific bands are indicated with an asterisk .

    Techniques Used: Flow Cytometry, Expressing, Construct, Co-Immunoprecipitation Assay, Lysis

    26) Product Images from "Chlamydia trachomatis Relies on Autonomous Phospholipid Synthesis for Membrane Biogenesis * Relies on Autonomous Phospholipid Synthesis for Membrane Biogenesis * ♦"

    Article Title: Chlamydia trachomatis Relies on Autonomous Phospholipid Synthesis for Membrane Biogenesis * Relies on Autonomous Phospholipid Synthesis for Membrane Biogenesis * ♦

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.657148

    Phospholipid composition of isolated  C. trachomatis . A,  uninfected and  C. trachomatis -infected HeLa cells were labeled with [ 14 C]choline for 48 h. The two cell cultures were lysed, and the  C. trachomatis  fractions were isolated either using the Renografin density gradient centrifugation or the Nonidet P-40 extraction methods. The amount of [ 14 C]choline that was associated with the  C. trachomatis  fraction in uninfected cells ( blue ) compared with  C. trachomatis -infected cells ( red ) was measured by scintillation counting. Data were derived from triplicate biological experiments.  B,  PC molecular species profile of the  C. trachomatis  fraction isolated from  C. trachomatis -infected cells by Renografin density gradient centrifugation.  C,  PC molecular species profile of the  C. trachomatis  fraction isolated from  C. trachomatis -infected cells by the Nonidet P-40 method.  D,  PE molecular species profile of Renografin-purified  C. trachomatis. E,  PE molecular species profile of  C. trachomatis  isolated by the Nonidet P-40 method. The spectra shown are representative of duplicate biological experiments. The major molecular species are labeled in the figure panels.  F, C. trachomatis -infected HeLa cells were labeled with [ 14 C]glucose for 48 h, and the  C. trachomatis  was isolated using the Nonidet P-40 method. Lipids were extracted from the  C. trachomatis -containing fraction, and the PC/SM fractions were separated from the PE/PG/CL fraction by thin layer chromatography and quantified using the Bioscan Imaging Detector. A representative chromatogram is shown. The  inset  shows the quantification of the chromatograms from biological triplicates.
    Figure Legend Snippet: Phospholipid composition of isolated C. trachomatis . A, uninfected and C. trachomatis -infected HeLa cells were labeled with [ 14 C]choline for 48 h. The two cell cultures were lysed, and the C. trachomatis fractions were isolated either using the Renografin density gradient centrifugation or the Nonidet P-40 extraction methods. The amount of [ 14 C]choline that was associated with the C. trachomatis fraction in uninfected cells ( blue ) compared with C. trachomatis -infected cells ( red ) was measured by scintillation counting. Data were derived from triplicate biological experiments. B, PC molecular species profile of the C. trachomatis fraction isolated from C. trachomatis -infected cells by Renografin density gradient centrifugation. C, PC molecular species profile of the C. trachomatis fraction isolated from C. trachomatis -infected cells by the Nonidet P-40 method. D, PE molecular species profile of Renografin-purified C. trachomatis. E, PE molecular species profile of C. trachomatis isolated by the Nonidet P-40 method. The spectra shown are representative of duplicate biological experiments. The major molecular species are labeled in the figure panels. F, C. trachomatis -infected HeLa cells were labeled with [ 14 C]glucose for 48 h, and the C. trachomatis was isolated using the Nonidet P-40 method. Lipids were extracted from the C. trachomatis -containing fraction, and the PC/SM fractions were separated from the PE/PG/CL fraction by thin layer chromatography and quantified using the Bioscan Imaging Detector. A representative chromatogram is shown. The inset shows the quantification of the chromatograms from biological triplicates.

    Techniques Used: Isolation, Infection, Labeling, Gradient Centrifugation, Derivative Assay, Purification, Thin Layer Chromatography, Imaging

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    Stable Transfection:

    Article Title: SIVA1 directs the E3 ubiquitin ligase RAD18 for PCNA monoubiquitination
    Article Snippet: .. For affinity purification, HEK293T cells stably expressing tagged proteins were lysed with NETN buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, and 0.5% Nonidet P-40) containing Benzonase (EMD Millipore) for 20 min. .. The supernatants were cleared at 14,000 rpm to remove debris and then incubated with streptavidin-conjugated beads (GE Healthcare) for 2 h at 4°C.

    Protease Inhibitor:

    Article Title: The Epstein-Barr Virus G-Protein-Coupled Receptor Contributes to Immune Evasion by Targeting MHC Class I Molecules for Degradation
    Article Snippet: .. Samples containing 2×106 cells were lysed in 400 µl of NP-40 buffer (0.5% Nonidet P-40, 5 mM MgCl2 and 50 mM Tris-HCl, pH 7.5) with protease inhibitor cocktail (Sigma) at 4°C for 45 min. .. Nuclei and insoluble debris were removed by centrifugation, and the supernatants were precleared first with 1.2 µl of normal mouse serum and 20 µl Dynabeads Protein A (Invitrogen) for 2 hr at 4°C, and then again with 20 µl Dynabeads Protein A and 20 µl Dynabeads protein G at 4°C overnight.

    other:

    Article Title: Oda5p, a Novel Axonemal Protein Required for Assembly of the Outer Dynein Arm and an Associated Adenylate Kinase D⃞
    Article Snippet: Because the AK is not predicted to contain transmembrane domains, and because its presence in the flagellum is dependent upon Oda5p, which is an axonemal protein, it is probable that the AK also is an axonemal component and that it is associated with the axoneme via interactions that survive the Tergitol treatment but are disrupted by Nonidet P-40.

    Article Title: Recruitment of the cross-linked opsonic receptor CD32A (Fc?RIIA) to high-density detergent-resistant membrane domains in human neutrophils
    Article Snippet: iPr2 P -F (di-isopropyl fluorophosphate) was obtained from Helixx Technologies (Scarborough, ON, Canada) and NP40 (Nonidet P40) and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4- d ]pyrimidine (referred to as PP2) were obtained from Calbiochem (San Diego, CA, U.S.A.).

    Article Title: Krp1 (Sarcosin) Promotes Lateral Fusion of Myofibril Assembly Intermediates in Cultured Mouse Cardiomyocytes
    Article Snippet: Krp1 association with cellular compartments was further explored by separation of extracted and insoluble fractions following treatment with Nonidet P-40.

    Expressing:

    Article Title: SIVA1 directs the E3 ubiquitin ligase RAD18 for PCNA monoubiquitination
    Article Snippet: .. For affinity purification, HEK293T cells stably expressing tagged proteins were lysed with NETN buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, and 0.5% Nonidet P-40) containing Benzonase (EMD Millipore) for 20 min. .. The supernatants were cleared at 14,000 rpm to remove debris and then incubated with streptavidin-conjugated beads (GE Healthcare) for 2 h at 4°C.

    Isolation:

    Article Title: Novel Filaments 5 nm in Diameter Constitute the Cytosolic Ring of the Plastid Division Apparatus
    Article Snippet: .. Dividing and nondividing chloroplasts were lysed, and protein concentrations were normalized (1 mg/mL) in sucrose-free isolation medium containing 0.5% Nonidet P-40 and 100 μg/mL DNase I (DN-25; Sigma) for 1 hr on ice. ..

    Lysis:

    Article Title: The Modulation of CD40 Ligand Signaling by Transmembrane CD28 Splice Variant in Human T Cells
    Article Snippet: .. Cell extracts were prepared using lysis buffer containing 1% Nonidet P-40 (BDH Chemicals) or 1% digitonin (Sigma-Aldrich; reference ). .. To immunoprecipitate CD28i-HA with anti-HA Ab (rat IgG1), protein A/G agarose and protein L agarose bead cocktail (sc-2336; Santa Cruz Biotechnology, Inc.) were used.

    Affinity Purification:

    Article Title: SIVA1 directs the E3 ubiquitin ligase RAD18 for PCNA monoubiquitination
    Article Snippet: .. For affinity purification, HEK293T cells stably expressing tagged proteins were lysed with NETN buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, and 0.5% Nonidet P-40) containing Benzonase (EMD Millipore) for 20 min. .. The supernatants were cleared at 14,000 rpm to remove debris and then incubated with streptavidin-conjugated beads (GE Healthcare) for 2 h at 4°C.

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  • 92
    Millipore triton x 114
    Assembly of transgenic  HA Gγ 1  and  HA Gγ 1 C71S with the endogenous Gβ 1  in retinal photoreceptors.  (A)  Transgenic construct used for generation of  HA Gγ 1  mice and amino acid sequence of the encoded protein with HA-tag (underlined) and C71 (asterisk).  (B)  SDS polyacrylamide gel stained with Coomassie blue showing anti-HA pull downs from 10 retinas of the transgene-negative (1) and transgene-positive (2)  HA Gγ 1  mice.  (B)  Western blot analysis of anti-HA pull downs from the retinas of  HA Gγ 1  and  HA Gγ 1 C71S mice with antibodies against Gβ 1  and HA.  (C)  Representative Western blot showing co-precipitation of phosducin (Pdc) with  HA Gγ 1  or  HA Gγ 1 C71S (HA). Tg(-) indicates transgene-negative littermates. The break separates two blots that were adjusted differently. Identical amounts of phosducin co-precipitated with  HA Gγ 1  and  HA Gγ 1  C71S ( n  = 4).  (E)  Representative experiments illustrating partitioning of  HA Gγ 1 C71S,  HA Gγ 1 , and endogenous Gγ 1  between the detergent (Triton X-114) and aqueous phases. Specific bands were visualized by Western blotting with anti-HA ( HA Gγ 1 C71S,  HA Gγ 1 ) and anti-Gγ 1  (Gγ 1 ).  (D)  Average distribution of each protein in the aqueous and detergent phases ( HA Gγ 1 C71S: 87/13 ± 4%;  HA Gγ 1 : 36/64 ± 9%, Gγ 1 : 41/59 ± 8%, error bars are SEM,  n  = 4).
    Triton X 114, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/triton x 114/product/Millipore
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    triton x 114 - by Bioz Stars, 2020-11
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    99
    Millipore nonidet p 40
    In vitro  phosphorylation of recombinant GST-BAD by kinases overexpressed in HEK293 cells.  HEK293 cells were transiently transfected with the indicated plasmids. Inactive mutants of PAK1 and Akt/PKB (K299R and K179A, respectively) were used as negative controls. 16 h post-transfection, cells were cultivated for an additional 30 h in medium supplemented with 0.3% serum. Afterward, cells were washed once in PBS and lysed by the direct addition of Nonidet P-40 buffer. For the kinase assay, 35 μg of the protein lysates containing the desired kinases were mixed with 1 μg of recombinant GST-BAD (purified from  E. coli ) in kinase buffer. Proteins were separated on a 12% SDS-polyacrylamide gel and blotted. Phosphorylation of BAD was visualized with phosphospecific BAD antibodies. Expression levels of RAF kinases, PAK1, and Akt/PKB are shown in the  lower panels . This experiment was repeated three times with the same results.
    Nonidet P 40, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 205 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Assembly of transgenic  HA Gγ 1  and  HA Gγ 1 C71S with the endogenous Gβ 1  in retinal photoreceptors.  (A)  Transgenic construct used for generation of  HA Gγ 1  mice and amino acid sequence of the encoded protein with HA-tag (underlined) and C71 (asterisk).  (B)  SDS polyacrylamide gel stained with Coomassie blue showing anti-HA pull downs from 10 retinas of the transgene-negative (1) and transgene-positive (2)  HA Gγ 1  mice.  (B)  Western blot analysis of anti-HA pull downs from the retinas of  HA Gγ 1  and  HA Gγ 1 C71S mice with antibodies against Gβ 1  and HA.  (C)  Representative Western blot showing co-precipitation of phosducin (Pdc) with  HA Gγ 1  or  HA Gγ 1 C71S (HA). Tg(-) indicates transgene-negative littermates. The break separates two blots that were adjusted differently. Identical amounts of phosducin co-precipitated with  HA Gγ 1  and  HA Gγ 1  C71S ( n  = 4).  (E)  Representative experiments illustrating partitioning of  HA Gγ 1 C71S,  HA Gγ 1 , and endogenous Gγ 1  between the detergent (Triton X-114) and aqueous phases. Specific bands were visualized by Western blotting with anti-HA ( HA Gγ 1 C71S,  HA Gγ 1 ) and anti-Gγ 1  (Gγ 1 ).  (D)  Average distribution of each protein in the aqueous and detergent phases ( HA Gγ 1 C71S: 87/13 ± 4%;  HA Gγ 1 : 36/64 ± 9%, Gγ 1 : 41/59 ± 8%, error bars are SEM,  n  = 4).

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Farnesylation of the Transducin G Protein Gamma Subunit Is a Prerequisite for Its Ciliary Targeting in Rod Photoreceptors

    doi: 10.3389/fnmol.2018.00016

    Figure Lengend Snippet: Assembly of transgenic HA Gγ 1 and HA Gγ 1 C71S with the endogenous Gβ 1 in retinal photoreceptors. (A) Transgenic construct used for generation of HA Gγ 1 mice and amino acid sequence of the encoded protein with HA-tag (underlined) and C71 (asterisk). (B) SDS polyacrylamide gel stained with Coomassie blue showing anti-HA pull downs from 10 retinas of the transgene-negative (1) and transgene-positive (2) HA Gγ 1 mice. (B) Western blot analysis of anti-HA pull downs from the retinas of HA Gγ 1 and HA Gγ 1 C71S mice with antibodies against Gβ 1 and HA. (C) Representative Western blot showing co-precipitation of phosducin (Pdc) with HA Gγ 1 or HA Gγ 1 C71S (HA). Tg(-) indicates transgene-negative littermates. The break separates two blots that were adjusted differently. Identical amounts of phosducin co-precipitated with HA Gγ 1 and HA Gγ 1 C71S ( n = 4). (E) Representative experiments illustrating partitioning of HA Gγ 1 C71S, HA Gγ 1 , and endogenous Gγ 1 between the detergent (Triton X-114) and aqueous phases. Specific bands were visualized by Western blotting with anti-HA ( HA Gγ 1 C71S, HA Gγ 1 ) and anti-Gγ 1 (Gγ 1 ). (D) Average distribution of each protein in the aqueous and detergent phases ( HA Gγ 1 C71S: 87/13 ± 4%; HA Gγ 1 : 36/64 ± 9%, Gγ 1 : 41/59 ± 8%, error bars are SEM, n = 4).

    Article Snippet: 20 μl of 10% Triton X-114 (648468, Calbiochem) was added to 180 μl of the retinal extract, mixed by gentle inversion and pre-warmed to 37°C for 5 min.

    Techniques: Transgenic Assay, Construct, Mouse Assay, Sequencing, Staining, Western Blot

    In vitro  phosphorylation of recombinant GST-BAD by kinases overexpressed in HEK293 cells.  HEK293 cells were transiently transfected with the indicated plasmids. Inactive mutants of PAK1 and Akt/PKB (K299R and K179A, respectively) were used as negative controls. 16 h post-transfection, cells were cultivated for an additional 30 h in medium supplemented with 0.3% serum. Afterward, cells were washed once in PBS and lysed by the direct addition of Nonidet P-40 buffer. For the kinase assay, 35 μg of the protein lysates containing the desired kinases were mixed with 1 μg of recombinant GST-BAD (purified from  E. coli ) in kinase buffer. Proteins were separated on a 12% SDS-polyacrylamide gel and blotted. Phosphorylation of BAD was visualized with phosphospecific BAD antibodies. Expression levels of RAF kinases, PAK1, and Akt/PKB are shown in the  lower panels . This experiment was repeated three times with the same results.

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of Novel in Vivo Phosphorylation Sites of the Human Proapoptotic Protein BAD

    doi: 10.1074/jbc.M109.010702

    Figure Lengend Snippet: In vitro phosphorylation of recombinant GST-BAD by kinases overexpressed in HEK293 cells. HEK293 cells were transiently transfected with the indicated plasmids. Inactive mutants of PAK1 and Akt/PKB (K299R and K179A, respectively) were used as negative controls. 16 h post-transfection, cells were cultivated for an additional 30 h in medium supplemented with 0.3% serum. Afterward, cells were washed once in PBS and lysed by the direct addition of Nonidet P-40 buffer. For the kinase assay, 35 μg of the protein lysates containing the desired kinases were mixed with 1 μg of recombinant GST-BAD (purified from E. coli ) in kinase buffer. Proteins were separated on a 12% SDS-polyacrylamide gel and blotted. Phosphorylation of BAD was visualized with phosphospecific BAD antibodies. Expression levels of RAF kinases, PAK1, and Akt/PKB are shown in the lower panels . This experiment was repeated three times with the same results.

    Article Snippet: After excessive washing with 10 m m Hepes, pH 7.4, 150 m m NaCl, and 0.01% Nonidet P-40, the phosphorylated hBAD fraction was released from the complex by 1% Empigen (Calbiochem).

    Techniques: In Vitro, Recombinant, Transfection, Kinase Assay, Purification, Expressing

    Immunoblot analysis of Nonidet P-40 extracted and insoluble fractions. Equal volumes of total cell lysate, extracted and insoluble fractions were analyzed by immunoblot (left) and densitometric analysis (right). Representative immunoblots are shown. The graph displays the means and SEM of three independent experiments. A large proportion of Krp1 remains in the insoluble pellet along with most of the cytoskeletal components (myosin, actin, N-RAP) and caveolin-3, although the majority of Krp1 is present in the extracted supernatant along with the cytosolic marker GAPDH and the integral membrane marker ß1-integrin.

    Journal: Experimental cell research

    Article Title: Krp1 (Sarcosin) Promotes Lateral Fusion of Myofibril Assembly Intermediates in Cultured Mouse Cardiomyocytes

    doi: 10.1016/j.yexcr.2007.12.009

    Figure Lengend Snippet: Immunoblot analysis of Nonidet P-40 extracted and insoluble fractions. Equal volumes of total cell lysate, extracted and insoluble fractions were analyzed by immunoblot (left) and densitometric analysis (right). Representative immunoblots are shown. The graph displays the means and SEM of three independent experiments. A large proportion of Krp1 remains in the insoluble pellet along with most of the cytoskeletal components (myosin, actin, N-RAP) and caveolin-3, although the majority of Krp1 is present in the extracted supernatant along with the cytosolic marker GAPDH and the integral membrane marker ß1-integrin.

    Article Snippet: Krp1 association with cellular compartments was further explored by separation of extracted and insoluble fractions following treatment with Nonidet P-40.

    Techniques: Western Blot, Marker

    Western blot analysis indicates Oda5p is a salt-extractable,  M r  76,000 axonemal protein that sediments at ∼5S in sucrose density gradients. (A) The Oda5 antibody recognizes an  M r  76,000 band in wild-type whole cells that is absent from the  oda5-2  whole cells, confirming the antibody recognizes the correct protein. This band is not detected in cell bodies lacking flagella (middle), yet it is readily detected in whole flagella (right). (B) Oda5p remains associated with the axoneme after Nonidet P-40 detergent extraction (demembranated axonemes) and is not detected in the Nonidet P-40 detergent-soluble membrane + matrix fraction (membrane + matrix). Extraction of demembranated axonemes with 0.6 M KCl releases Oda5p into the KCl extract; none remains in the KCl-extracted axonemes. (C) Sucrose gradient fractions were probed with antibodies to outer dynein arm components IC2 and γ-DHC, and with the Oda5p-antibody. The outer dynein arm/ODA-DC complex sediments at ∼23S as expected; however, Oda5p sediments at ∼5S.

    Journal: Molecular Biology of the Cell

    Article Title: Oda5p, a Novel Axonemal Protein Required for Assembly of the Outer Dynein Arm and an Associated Adenylate Kinase D⃞

    doi: 10.1091/mbc.E03-11-0820

    Figure Lengend Snippet: Western blot analysis indicates Oda5p is a salt-extractable, M r 76,000 axonemal protein that sediments at ∼5S in sucrose density gradients. (A) The Oda5 antibody recognizes an M r 76,000 band in wild-type whole cells that is absent from the oda5-2 whole cells, confirming the antibody recognizes the correct protein. This band is not detected in cell bodies lacking flagella (middle), yet it is readily detected in whole flagella (right). (B) Oda5p remains associated with the axoneme after Nonidet P-40 detergent extraction (demembranated axonemes) and is not detected in the Nonidet P-40 detergent-soluble membrane + matrix fraction (membrane + matrix). Extraction of demembranated axonemes with 0.6 M KCl releases Oda5p into the KCl extract; none remains in the KCl-extracted axonemes. (C) Sucrose gradient fractions were probed with antibodies to outer dynein arm components IC2 and γ-DHC, and with the Oda5p-antibody. The outer dynein arm/ODA-DC complex sediments at ∼23S as expected; however, Oda5p sediments at ∼5S.

    Article Snippet: Because the AK is not predicted to contain transmembrane domains, and because its presence in the flagellum is dependent upon Oda5p, which is an axonemal protein, it is probable that the AK also is an axonemal component and that it is associated with the axoneme via interactions that survive the Tergitol treatment but are disrupted by Nonidet P-40.

    Techniques: Western Blot