Journal: Journal of Histochemistry and Cytochemistry

Article Title: Refined Immunochemical Characterization in Healthy Dog Skin of the Epidermal Cornification Proteins, Filaggrin, and Corneodesmosin

doi: 10.1369/0022155418798807

Figure Lengend Snippet: Western blotting of dog epidermis extracts. Beagle (Bea) and golden (Gol) retriever dog epidermis was sequentially extracted in Tris-EDTA buffer containing either Nonidet-P40 (TE-NP40 extracts, NP) or 8 M urea (TE-U extracts, U). The extracted proteins were separated by SDS-PAGE, Coomassie blue stained, or immunodetected with AHF10 and G36-19 mAbs, as indicated. The immunoblot corresponding to AHF10 reactivity on the extracts of the beagle dog epidermis was exposed for a longer time (NP′ and U′) to highlight the absence of FLGa-corresponding band. The migration of molecular mass markers (m) is indicated on the left in kDa. ProFLG (Pro) and FLG monomers (FLGa and FLGb) are indicated by arrows, as well as the entire CDSN and its proteolytically derived fragments. For comparison, the Western blotting with AHF10 of TE-NP40 and TE-U extracts of human epidermis (Hum) is shown. Abbreviations: AHF, anti-human filaggrin; PAGE, polyacrylamide gel electrophoresis; FLG, Filaggrin; CDSN, corneodesmosin.

Article Snippet: In brief, human and dog epidermal proteins were sequentially extracted in Tris-HCl pH 7.4 buffer containing nonidet-P40 detergent (Sigma-Aldrich) and EDTA (TE-NP40 extracts), then in TE buffer containing 8 M urea (TE-U extracts), as previously reported.

Techniques: Western Blot, SDS Page, Staining, Migration, Derivative Assay, Polyacrylamide Gel Electrophoresis

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

Journal: The FASEB Journal

Article Title: Keratin 8 phosphorylation regulates its transamidation and hepatocyte Mallory-Denk body formation

doi: 10.1096/fj.11-198580

Figure Lengend Snippet: K8 S74A mutation inhibits K8 transamidation by TG2 in vitro . A ) HT29 cells were treated with vehicle or 1 μM OA for 30 or 60 min. Treated cells were solubilized in Nonidet P-40-containing buffer, then incubated with or without TG2 for 15 min (37°C). Extracts were analyzed by blotting, using antibodies to K8, K8 pS74, and TG2. TG2 blot (bottom panel) confirms the addition of exogenous TG2 to the designated extracts. Note that hyperphosphorylation promotes K8-containing cross-link formation. B ) BHK cells were transfected with WT K18 plus the indicated K8 constructs. After 48 h, cells were solubilized with Nonidet P-40, followed by TG2 reactions (15 min, 37°C) using the undiluted extracts or extracts diluted by 1/2 and 1/4. After quenching, samples were separated by SDS-PAGE, followed by immune blotting using anti-K8 antibodies to assess K8 cross-linking (indicated by bracket). Bottom panel shows a lower exposure of the gel in order to demonstrate near-equal loading. C ) BHK cells were transfected with WT K18 plus the indicated K8 constructs. After 48 h, OA (1 μM, 30 min) was added to the cultured cells, followed by detergent solubilization. Cell extracts were either left untreated or treated with TG2 for 15 min (37°C), followed by quenching, then analysis of K8 cross-links (indicated by bracket) by immunoblotting with an anti-K8 antibody. Bottom panel shows a lower exposure of the gel in order to demonstrate near-equal loading for each dilution set. D ) Primary hepatocytes were isolated from K8 S74A and K8 WT transgenic mice; after 8 h of culture, hepatocytes were treated with either DMSO alone or 1 μM OA (45 min, 37°C). Cultured hepatocytes were then lysed in Nonidet P-40 buffer, and extracts were subjected to in vitro transamidation. Middle panel shows human K8 expression (input) in the hepatocyte extracts. Bottom panel shows the induction of S74 phosphorylation in WT K8 transgenic liver after OA treatment. Top panel shows K8 cross-linking (indicated by bracket) before and after OA treatment.

Article Snippet: After 48 h, the transfected cells were lysed in Nonidet P-40 buffer [1% Nonidet P-40, 1× PBS (pH 7.4), 5 mM EDTA, and protease inhibitor cocktail from Sigma-Aldrich], and equal volumes of extracts were incubated with 3.5 μg/ml recombinant TG2 in the presence of 15 mM CaCl2 (37°C).

Techniques: Mutagenesis, In Vitro, Incubation, Transfection, Construct, SDS Page, Cell Culture, Isolation, Transgenic Assay, Mouse Assay, Expressing

Journal: Molecular Biology of the Cell

Article Title: The Interplay between Folding-facilitating Mechanisms in Trypanosoma cruzi Endoplasmic Reticulum

doi: 10.1091/mbc.E03-04-0228

Figure Lengend Snippet: CRT-CZP interaction in wild-type and GT null mutant cells. Cells were pulsed for 2 min with [ 35 S]Met plus [ 35 S]Cys, lysed with 1% Nonidet P-40 (A) or with indicated concentrations of the same detergent (NP-40) (B), and lysates were subjected to immunoprecipitation with CRT antiserum. Immunoprecipitates were run on 10% SDS-PAGE and subjected to autoradiography. Where indicated 6 mM DNJ was added 30 min before the pulse. CZP and CRT stand for cruzipain and calreticulin, respectively. For further details, see MATERIALS AND METHODS.

Article Snippet: For immunoprecipitation experiments suspensions were centrifuged and cells were lysed on addition of 350 μl of 50 mM HEPES buffer, pH 7.5 containing 0.2 M NaCl, the indicated Nonidet P-40 concentrations, 0.3 M iodoacetamide, 1 mM phenylmethylsufonylfluoride (PMSF, Sigma), and 100 μM trans -epoxysuccinyl-1-leucylamido(4-guanidino)butane (E64, Sigma; this compound irreversibly inhibits CZP activity).

Techniques: Mutagenesis, Immunoprecipitation, SDS Page, Autoradiography

Journal: The Plant Cell

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

doi:

Figure Lengend 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.

Article Snippet: Supernatants from Nonidet P-40 treatment (10,000 g for 15 min) were enriched 20-fold by ultrafiltration with Centricon-10 (10-kD cutoff; Millipore, Bedford, MA) and mixed with one-third volume of 4 × Laemmli sample buffer.

Techniques: SDS Page, Isolation

Journal: The Plant Cell

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

doi:

Figure Lengend 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.

Article Snippet: Supernatants from Nonidet P-40 treatment (10,000 g for 15 min) were enriched 20-fold by ultrafiltration with Centricon-10 (10-kD cutoff; Millipore, Bedford, MA) and mixed with one-third volume of 4 × Laemmli sample buffer.

Techniques: SDS Page, Recombinant, Purification, Isolation

Journal: The Plant Cell

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

doi:

Figure Lengend 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.

Article Snippet: Supernatants from Nonidet P-40 treatment (10,000 g for 15 min) were enriched 20-fold by ultrafiltration with Centricon-10 (10-kD cutoff; Millipore, Bedford, MA) and mixed with one-third volume of 4 × Laemmli sample buffer.

Techniques: Isolation

Journal: The Plant Cell

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

doi:

Figure Lengend 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.

Article Snippet: Supernatants from Nonidet P-40 treatment (10,000 g for 15 min) were enriched 20-fold by ultrafiltration with Centricon-10 (10-kD cutoff; Millipore, Bedford, MA) and mixed with one-third volume of 4 × Laemmli sample buffer.

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

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

Article Title: Sel1L is indispensable for mammalian endoplasmic reticulum-associated degradation, endoplasmic reticulum homeostasis, and survival

doi: 10.1073/pnas.1318114111

Figure Lengend Snippet: Sel1L is indispensable for the stability of Sel1L-Hrd1 complex in vivo. Pancreas was harvested at days 4, 8, and 13. ( A ) Western blot analysis of Sel1L and Hrd1 in WT and IKO pancreas at day 4 and 8, with quantitation shown in B upon being normalized to the loading control HSP90. ( C ) qPCR analysis of Sel1L and Hrd1 genes at day 13; n = 5. ( D ) Immunohistochemical staining of Sel1L and Hrd1 in pancreas at day 13; n = 3 each. ( E ) Western blot analysis of Sel1L and Hrd1 in the ileum and kidney of IKO mice. ( F ) Linear regression analysis between Hrd1 and Sel1L protein levels in various WT or Sel1L for details. Each dot represents one sample ( n = 25). The slope of the regression line and the square of the correlation coefficient ( R 2 ) are shown. ( G ) Western blot analysis of Sel1L-associated factors (EDEM1, OS9, and XTP3B) in WT and IKO pancreas at day 13 with quantitation shown in H . ( I ) qPCR analysis of Edem1 and Os9 in the pancreas at day 13. ( J ) Percentage of Nonidet P-40 insoluble pellet weight in total tissue weight at the indicated times following tamoxifen injection; n = 2–6. WT samples were pooled from two samples of day 8 and four samples of day 13. ( K ) Western blot analysis of various proteins in the NP40P and NP40S fractions of pancreas at day 13 using lysis buffer containing 0.5% Nonidet P-40. The distribution of Bag6 and H2A marks the soluble (S) and insoluble (P) fractions, respectively. Representative data of three samples each shown. Data are mean ± SEM. * P

Article Snippet: Frozen pancreas tissue from WT and Sel1L IKO mice at day 13 (∼15 mg) was weighed and homogenized in Nonidet P-40 lysis buffer (50 mM Tris⋅HCl pH 8.0, 0.5% Nonidet P-40, 150 mM NaCl, 5 mM MgCl2 ) supplied with protease inhibitor (Sigma).

Techniques: In Vivo, Western Blot, Quantitation Assay, Real-time Polymerase Chain Reaction, Immunohistochemistry, Staining, Mouse Assay, Injection, Lysis

Journal: PLoS Pathogens

Article Title: Mechanisms of Assembly and Cellular Interactions for the Bacterial Genotoxin CDT

doi: 10.1371/journal.ppat.0010028

Figure Lengend Snippet: Immunoprecipitation of CDT Complexes Containing Deletion Mutants of CdtA (A) The complexes (10 μg each) were immunoprecipitated with 10 μg anti-myc antibody (myc tag was fused to C-terminus of CdtC) in 150 mM NaCl, 20 mM HEPES (pH 7.5), 0.1% Nonidet P40 substitute, followed by exposure to protein G Sepharose beads, and subjected to nondenaturing SDS-PAGE. (B) Nonspecific binding of CDT complexes to protein G Sepharose was not detected under the same experimental conditions. A, CdtA; B, CdtB; C, CdtC; A*, truncated CdtA; A1, holotoxin with CdtA Δ18−56; A2, holotoxin with CdtA Δ18−67; A3, holotoxin with CdtA Δ18−75; WT, wild-type; Ab, only anti- myc antibody, no CDT; IgG (H+L), nonreduced immunoglobulin G; IgG H, immunoglobulin G heavy chain; IgG L, immunoglobulin G light chain; SN, supernatant.

Article Snippet: Then, 10 μg of protein complex was incubated with 10 μg of antibody in binding buffer (20 mM HEPES [pH 7.5], 150 mM NaCl, 0.1% Nonidet P40 Substitute [Sigma, St. Louis, Missouri, United States]) for 3 hr at 4 °C.

Techniques: Immunoprecipitation, SDS Page, Binding Assay

Journal: The Journal of Biological Chemistry

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

doi: 10.1074/jbc.M110.162305

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

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

Techniques: Expressing, Multiple Displacement Amplification

Journal: Journal of Cellular and Molecular Medicine

Article Title: Identification of pY19-caveolin-2 as a positive regulator of insulin-stimulated actin cytoskeleton-dependent mitogenesis

doi: 10.1111/j.1582-4934.2008.00391.x

Figure Lengend Snippet: Effect of nonionic, non-denaturing detergents on the caveolin-2 solubilization and interaction between caveolin-2 and ERK. Hirc-B cells were treated with 10 μM U0126 for 2 hrs before100 nM insulin treatment for 10 min. Cells were then lysed in either 1% Triton X-100 lysis buffer containing 0.5% Nonidet P-40 alone (buffer A) , 1% Triton X-100 lysis buffer containing 0.5% Nonidet P-40 + 60 mM OG (buffer B) , or 1% Triton X-100 lysis buffer containing 60 mM OG alone (buffer C) as described under ‘Materials and Methods’. WCL were immunoprecipitated with anti-caveolin-2 and anti-ERK antibodies and subjected to immunoblot analysis with anti-phospho-ERK, anti-ERK, anti-pY19-caveolin-2 and anti-caveolin-2 antibodies as indicated.

Article Snippet: Immunoprecipitation Serum-starved cells were incubated with or without 100 nM insulin for 10 min. after being preincubated with or without 10 μM U0126 for 2 hrs or 100 nM wortmannin for 1 hr, washed with ice-cold PBS, and lysed in buffer A (1% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA (pH 8.0), 0.2 mM sodium ortho-vanadate, 0.2 mM PMSF, 0.5% Nonidet P-40 (Igepal CA-630, octyl phenoxylpolyethoxylethanol, 198596: ICN Biomedicals) [ , ].

Techniques: Lysis, Immunoprecipitation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Identification of pY19-caveolin-2 as a positive regulator of insulin-stimulated actin cytoskeleton-dependent mitogenesis

doi: 10.1111/j.1582-4934.2008.00391.x

Figure Lengend Snippet: Effect of transient expression of mutant caveolin-2 (Y19A) on the insulin-induced association of caveolin-2 with phospho-ERK. Hirc-B cells were transiently transfected with pcDNA3 alone (pcDNA3), pcDNA3 + caveolin-2 (WT: wild type), or pcDNA3 + caveolin-2 (Y19A: phosphorylation-deficient mutant) using the Lipofectamin trans-fection reagent. Thirty-six hours after transfection, the cells were lysed in 1% Triton X-100 lysis buffer B containing 0.5% Nonidet P-40 + 60 mM OG. The WCL were processed for immunoprecipitation with anti-caveolin-2 and anti-ERK antibodies and subjected to immunoblot analysis with anti-phos-pho-ERK, anti-ERK, anti-pY19-cave-olin-2, and anti-caveolin-2 antibodies as indicated.

Article Snippet: Immunoprecipitation Serum-starved cells were incubated with or without 100 nM insulin for 10 min. after being preincubated with or without 10 μM U0126 for 2 hrs or 100 nM wortmannin for 1 hr, washed with ice-cold PBS, and lysed in buffer A (1% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA (pH 8.0), 0.2 mM sodium ortho-vanadate, 0.2 mM PMSF, 0.5% Nonidet P-40 (Igepal CA-630, octyl phenoxylpolyethoxylethanol, 198596: ICN Biomedicals) [ , ].

Techniques: Expressing, Mutagenesis, Transfection, Lysis, Immunoprecipitation

Journal: International Journal of Molecular Sciences

Article Title: Stimulation of Peritoneal Mesothelial Cells to Secrete Matrix Metalloproteinase-9 (MMP-9) by TNF-α: A Role in the Invasion of Gastric Carcinoma Cells

doi: 10.3390/ijms19123961

Figure Lengend Snippet: Invasion and adhesion of MKN1 cells and effects of the anti-α3 integrin antibody. A monolayer of mesothelial cells was stimulated with TNF-α (10 ng/mL) for 6 h. ( A ) MKN1 cells (1 × 10 5 cells/0.2 mL) in ASF104 serum-free medium were added to the inner chamber of the reconstituted mesothelium and incubated at 37 °C for 16 h. The cells that had migrated into the outer chamber through the membrane were counted under a microscope. ( B ) Fluorescently labeled MKN1 cells were added to the monolayer of mesothelial cells in a 96-well culture plate, and incubated at 37 °C for 40 min. After non-adherent cells were removed by washing, adherent cells were lysed with 1% Nonidet P-40 and measured with a fluorescence spectrophotometer (Ex = 490 nm, Em = 520 nm). For the inhibition experiments, MKN1 cells were treated with the anti-α3 integrin antibody (SM-T1, 10 μg/mL) at 0 °C for 30 min. Experiments were performed in triplicate, and the data are presented as the mean ± SEM. Statistical data analysis was conducted using the Student’s t -test. ** p

Article Snippet: Nonidet P-40 was from Nacalai Tesque (Kyoto, Japan).

Techniques: Incubation, Microscopy, Labeling, Fluorescence, Spectrophotometry, Inhibition

Journal: Biochimica et biophysica acta

Article Title: BAX insertion, oligomerization, and outer membrane permeabilization in brain mitochondria: role of permeability transition and SH-redox regulation

doi: 10.1016/j.bbabio.2010.07.006

Figure Lengend Snippet: CHAPS but not Nonidet P-40, Triton X-100, or octyl glucoside produced oligomerization of BAX in the solution without mitochondria. Analytical gel-filtration confirmed BAX oligomerization by CHAPS

Article Snippet: Alkali-resistant BAX insertion was assessed by non-reducing SDS-PAGE followed by western blotting with mitochondrial membranes solubilized in 1% Nonidet P-40. (21K, pdf)

Techniques: Produced, Filtration

Journal: Molecules and Cells

Article Title: Mass Spectrometry-Based Screening Platform Reveals Orco Interactome in Drosophila melanogaster

doi: 10.14348/molcells.2018.2305

Figure Lengend Snippet: Detergents and temperature affect Orco solubility (A) Various laboratory detergents solubilizing Orco from enriched tissues. All detergents are at 1% (v/v or w/v). T20, Tween 20; TX100, Triton X-100; NP40, Nonidet P-40; OG, Octyl-β-D-glucopyranoside; DDM, n-Dodecyl β-D-maltoside; Z3–16, Zwittergent 3–16; SDS, sodium dodecyl sulfate. (B) Temperature sensitivity of Orco samples prepared from a 1% DDM lysate in the presence of SDS and β-mercaptoethanol. Antibodies are against EGFP and native GAPDH.

Article Snippet: The detergents used were Tween 20 (Biosesang, T1027), Triton X-100 (Bio Basic, TB0198), Nonidet P-40 (Bio Basic, NDB0385), Octyl-β-D-glucopyranoside (Fluka, 75083), n-Dodecyl β-D-maltoside (DDM; ThermoFisher, 89903), CHAPS (Sigma-Aldrich, C5070), CHAPSO (Calbiochem, 220201), and Zwittergent 3–16 (Calbiochem, 693023).

Techniques: Solubility

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

Article Title: Covalent modification of the homeodomain-interacting protein kinase 2 (HIPK2) by the ubiquitin-like protein SUMO-1

doi:

Figure Lengend Snippet: Localization of SUMO-1-modified HIPK2 to nuclear speckles (dots). ( A ) Fractionation of SUMO-1-modified HIPK2. Cellular extracts from CV-1 cells cotransfected with GFP-SUMO-1 and Myc-HIPK2 expression vectors were fractionated. Proteins from each fraction were separated on SDS/polyacrylamide gradient (4–12%) gels and analyzed by Western blotting using a mouse anti-Myc Ab. HIPK2 and SUMO-1-modified HIPK2 are detected in the nuclear fraction (lane 3) and in the Nonidet P-40-insoluble fractions (lanes 7 and 8). SUMO-1-modified HIPK2 and unmodified HIPK2 are marked by bracket and arrowhead, respectively. ( B ) Confocal microscopic image of the nucleus showing colocalization of HIPK2 and SUMO-1 to nuclear speckles (dots). CV-1 cells overexpressing Myc-HIPK2 and GFP-SUMO-1 were fixed and subjected to immunofluorescence staining with a mouse anti-Myc mAb. The red signal (HIPK2) is obtained with an anti-mouse IgG rhodamine red-conjugated secondary Ab, whereas the green signal (SUMO-1) is obtained with GFP fluorescence. Superimposing two colors (MERGE) results in a yellow signal, indicating colocalization of the two proteins.

Article Snippet: For immunoprecipitation, cell extracts (500 μg) were diluted (1:10) in 1× NETN [50 mM Tris⋅HCl (pH 7.5)/5 mM EDTA/300 mM NaCl/1 mM DTT/1% Nonidet P-40] containing protease inhibitors and incubated with an anti-GFP rabbit polyclonal Ab (10 μg/ml) or anti-SUMO-1 goat Ab (Santa Cruz Biotechnology) for 1 hr at 4°C.

Techniques: Modification, Fractionation, Expressing, Western Blot, Immunofluorescence, Staining, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Cross-talk between Dopachrome Tautomerase and Caveolin-1 Is Melanoma Cell Phenotype-specific and Potentially Involved in Tumor Progression *

doi: 10.1074/jbc.M116.714733

Figure Lengend Snippet: DCT and CAV1 expressions are oppositely modulated by environmental and cellular factors in MJS phenotype. A and B, MJS cells were plated and cultured for 48, 72, or 96 h, and in one experiment the culture medium was replenished with a fresh one every 24 h ( MR +) or not changed during the indicated time periods ( MR −). C, MJS cells cultured for 24 h were incubated with fresh culture medium ( fresh ) or with medium resulting from a 96-h ( old ) culture for an additional 24 h ( left panel ); MJS cells were cultured for 72 h in culture medium with 10 or 1% FCS ( right panel ). D, MJS and MNT cells were cultured and treated for the indicated time periods as described for A and B . For all experiments, the Nonidet P-40- soluble and Nonidet P-40-insoluble fractions of the MJS cell lysates were analyzed by WB for the expression of CAV1 (with α-CAV1-CS), DCT (with D18 or α-hDCT), TYR (with α-Pep7h), and TRP-1 (with αPep1h). Actin was used as loading control. Each experiment is a representative one of three. E, DCT ( red ) and CAV1 ( green ) distribution in cell populations during sub-confluent (48 h) to semi-confluent (72 h) and confluent (96 h) transition assessed by immunofluorescence microscopy. Last columns represent enlarged details of separate images. The antibody combinations for DCT and CAV1 immunostaining are indicated in each panel. Images were acquired using ×40 objective. Scale bar represents 20 μm. Each experiment is a representative one of three. F, image cytometry analysis of triple labeled (DCT/CAV1/nuclei with the two antibody combinations and DAPI) samples performed using the TF system. MFI and MSI were determined for DCT and for CAV1 expression, respectively. For each staining, DCT expression scattergrams for 48 h ( upper left ), 72 h ( middle left ), and 96 h ( lower left ) are gated to select cells with high marker expression ( upper right quadrant represents cells positive for DCT, and the rectangle delineates the gated DCT high cell subpopulation, shown as red dots ). For the 96-h time point, CAV1 scattergrams of all cells ( bottom middle ) as compared with DCT high cell subpopulation ( bottom right ) were generated. Values representing mean expression for DCT and CAV1 as well as % of cells in DCT high subset were derived from Tissue Quest data statistics and are represented as graphs . One representative experiment of two performed is shown.

Article Snippet: Trypsin-EDTA (0.05%, 25300, Gibco) was from Thermo Fisher; phenylmethylsulfonyl fluoride (PMSF) (P7626), paraformaldehyde, Tricine (T0377), Tween 20 (P7949), Coomassie SERVA Blue R (35051) were from Sigma; β-glycerophosphate (35675) was from Calbiochem; NaF (sc-24988), orthovanadate (sc-3540), DTT (sc-29089), nonfat dry milk, blotto (sc2325), and nitrocellulose membrane (sc-3724) were from Santa Cruz Biotechnology; protease inhibitor mixture (Complete, 11 697 498 001) was from Roche Applied Science; Nonidet P-40 (RIST1315) was from MP Biomedicals; BCA protein assay reagent was from Pierce; protein A-Sepharose 4B (101041) was from Invitrogen; saponin (from quillaja bark, 47036) was from Fluka); TRIzol was from Life Technologies, Inc.; and acrylamide (acrylamide/bisacrylamide solution 30%, 1006391000) was from Merck.

Techniques: Cell Culture, Incubation, Western Blot, Expressing, Immunofluorescence, Microscopy, Immunostaining, Cytometry, Labeling, Staining, Marker, Generated, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Cross-talk between Dopachrome Tautomerase and Caveolin-1 Is Melanoma Cell Phenotype-specific and Potentially Involved in Tumor Progression *

doi: 10.1074/jbc.M116.714733

Figure Lengend Snippet: Characterization of anti-DCT and anti-CAV1 antibodies in Western blot and immunofluorescence microscopy. MJS (72 h) and SK28 (96 h) melanoma cell lines transfected with siRNA control ( si-ct ), si-DCT ( si-DCT ), or si-CAV1( si-CAV1 ) were assessed for DCT and CAV1 expression in Western blot ( WB ) and immunofluorescence microscopy ( IF ) with D18 or α-hDCT and α-CAV1-CS or α-CAV1-sc, respectively. WBs were assessed with ECL or SuperSignal West Femto chemiluminescent substrate ( Femto ) detection systems. Both anti-DCT and anti-CAV1 antibodies recognize specifically in the two cell lines the products encoded by the DCT and CAV1 gene, respectively, different CAV1 constituents (monomers, oligomers, in Nonidet P-40-soluble ( sol ) and Nonidet P-40-insoluble ( insol ) fractions). Importantly, in both cell lines, an insoluble oligomeric CAV1 pool detected with both anti-CAV1 antibodies was resistant to CAV1 down-regulation. Unlike in MJS, the soluble oligomeric CAV1 detected preponderantly with α-CAV1-sc in SK28 cells was also resistant to si-CAV1 indicating that CAV1 aggregation is different in the two cell lines. The nonspecific DCT and CAV1 bands are indicated by stars . Calnexin ( Clx ) was used as loading control for Nonidet P-40-soluble fractions. The IF images were acquired using ×40 objective. Scale bar represents 10 μm. Each experiment is a representative one of three.

Article Snippet: Trypsin-EDTA (0.05%, 25300, Gibco) was from Thermo Fisher; phenylmethylsulfonyl fluoride (PMSF) (P7626), paraformaldehyde, Tricine (T0377), Tween 20 (P7949), Coomassie SERVA Blue R (35051) were from Sigma; β-glycerophosphate (35675) was from Calbiochem; NaF (sc-24988), orthovanadate (sc-3540), DTT (sc-29089), nonfat dry milk, blotto (sc2325), and nitrocellulose membrane (sc-3724) were from Santa Cruz Biotechnology; protease inhibitor mixture (Complete, 11 697 498 001) was from Roche Applied Science; Nonidet P-40 (RIST1315) was from MP Biomedicals; BCA protein assay reagent was from Pierce; protein A-Sepharose 4B (101041) was from Invitrogen; saponin (from quillaja bark, 47036) was from Fluka); TRIzol was from Life Technologies, Inc.; and acrylamide (acrylamide/bisacrylamide solution 30%, 1006391000) was from Merck.

Techniques: Western Blot, Immunofluorescence, Microscopy, Transfection, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Cross-talk between Dopachrome Tautomerase and Caveolin-1 Is Melanoma Cell Phenotype-specific and Potentially Involved in Tumor Progression *

doi: 10.1074/jbc.M116.714733

Figure Lengend Snippet: DCT silencing affects CAV1 stability, assembly, and subcellular distribution in amelanotic melanoma cell phenotypes. A and B, CAV1 expression and assembly in si-DCT cells. MJS ( A ) and SK28 ( B ) sub-confluent (48 and 72 h), semi-confluent (72 and 96 h), and confluent (96 and 120 h) cultures in si-ct and si-DCT experiments analyzed for CAV1 MOs and OGs in Nonidet P-40-soluble and Nonidet P-40-insoluble fractions in thermally treated (+) or not (−) samples by WB. CAV1 was assessed with α-CAV1-CS and DCT with D18. Actin was used as loading control. C and D, impact of DCT gene silencing on CAV1 mRNA expression. DCT and CAV1 mRNA in si-ct and si-DCT MJS and SK28 cells were determined by real time RT-qPCR. Graphs show average of experiments ( n = 6 replicates for each cell line and time point); error bars represent S.E. E, subcellular distribution of DCT ( red ), CAV1 ( green ), and Cavin-1 ( blue ) in MJS (72 h) and SK28 (96 h) si-ct and si-DCT cells assessed by immunofluorescence confocal microscopy. Last columns represent enlarged details of merged images. Scale bar, 10 μm in merged images and 5 μm in enlarged details. Images were acquired using ×63 oil immersion objective. Each experiment is a representative one of three.

Article Snippet: Trypsin-EDTA (0.05%, 25300, Gibco) was from Thermo Fisher; phenylmethylsulfonyl fluoride (PMSF) (P7626), paraformaldehyde, Tricine (T0377), Tween 20 (P7949), Coomassie SERVA Blue R (35051) were from Sigma; β-glycerophosphate (35675) was from Calbiochem; NaF (sc-24988), orthovanadate (sc-3540), DTT (sc-29089), nonfat dry milk, blotto (sc2325), and nitrocellulose membrane (sc-3724) were from Santa Cruz Biotechnology; protease inhibitor mixture (Complete, 11 697 498 001) was from Roche Applied Science; Nonidet P-40 (RIST1315) was from MP Biomedicals; BCA protein assay reagent was from Pierce; protein A-Sepharose 4B (101041) was from Invitrogen; saponin (from quillaja bark, 47036) was from Fluka); TRIzol was from Life Technologies, Inc.; and acrylamide (acrylamide/bisacrylamide solution 30%, 1006391000) was from Merck.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Immunofluorescence, Confocal Microscopy