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Millipore lysis buffer
Lysis Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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lysis buffer - by Bioz Stars, 2021-06
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Immunoprecipitation:

Article Title: Physiological changes during cellular ageing in fission yeast drive non-random patterns of genome rearrangements
Article Snippet: .. RIP-chip of Scw1-TAP was performed as described , except for the following modifications: immunoprecipitation was carried out using monoclonal antibodies against protein A (Sigma); the lysis buffer contained 10mg/ml heparin (sigma H7405), 1 mM PMSF and 1:100 protease inhibitor cocktail (Sigma P8340); and magnetic beads containing the immunoprecipitate were resuspended in 50 μl of wash buffer containing 1 mM DTT, 1 unit/ml of SuperaseIN (Ambion 2696) and 30 units/ml of AcTev protease (Life Technologies 12575015). .. The solution with the beads was incubated for 1.5 h at 18°C and the supernatant recovered and RNA extracted using PureLink RNA micro columns (Life Technologies), according to the manufacturer’s instructions.

Lysis:

Article Title: Physiological changes during cellular ageing in fission yeast drive non-random patterns of genome rearrangements
Article Snippet: .. RIP-chip of Scw1-TAP was performed as described , except for the following modifications: immunoprecipitation was carried out using monoclonal antibodies against protein A (Sigma); the lysis buffer contained 10mg/ml heparin (sigma H7405), 1 mM PMSF and 1:100 protease inhibitor cocktail (Sigma P8340); and magnetic beads containing the immunoprecipitate were resuspended in 50 μl of wash buffer containing 1 mM DTT, 1 unit/ml of SuperaseIN (Ambion 2696) and 30 units/ml of AcTev protease (Life Technologies 12575015). .. The solution with the beads was incubated for 1.5 h at 18°C and the supernatant recovered and RNA extracted using PureLink RNA micro columns (Life Technologies), according to the manufacturer’s instructions.

Article Title: Dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin mediates HIV-1 infection of and transmission by M2a-polarized macrophages in vitro
Article Snippet: An aliquot of cells were immediately washed five times and nucleic acid carry over with the viral stock was verified and excluded by PCR. .. After 48 h incubation in complete medium [ ], MDMs (1 ×106 cells) were harvested, washed, resuspended in lysis buffer containing polyoxyethylene (0.1%), lauryl ether 10 mol/l (Sigma) along with proteinase K (0.1 mg/ml) and digested for 2 h at 65°C; proteinase K was heat-inactivated for 15 min at 95°C [ ]. .. Quantitative real-time PCR amplification of HIV-1 gag DNA was performed using a primer/probe combination that detects all viral DNA synthesized after second-strand transfer including both unintegrated and integrated DNA species [ ].

Article Title: G-quadruplex ligands mediate downregulation of DUX4 expression
Article Snippet: The number of fibres having internal nuclei was counted manually using ImageJ software (National Institutes of Health, Maryland, USA) and displayed as per mm2 of the muscle section. .. Protein extraction and western blot quantification of DUX4 expression Frozen TA sections collected from cryosectioning were homogenized in lysis buffer (0.15 M NaCl, 0.05 M HEPES, 1% (v/v) NP-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 0.01 M EDTA; (reagents were purchased from Sigma, UK) containing 1× protease inhibitors (Roche, UK) at 25 Hz for 1–2 min on a TissueLyser II (QIAgen, UK). .. Following centrifugation at 14 000 × g , 10 min, 4°C, the supernatant was transferred to fresh pre-chilled 1.5 ml tubes.

Article Title: Hormonal therapy with estradiol and drospirenone improves endothelium-dependent vasodilation in the coronary bed of ovariectomized spontaneously hypertensive rats
Article Snippet: Western blotting The coronary arteries were pooled with frozen tissue from 3 rats (considered as n=1), and the total number of pooled samples per group was n=4. .. The samples were homogenized in lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl, 5 mM EDTA, 2 mM Na, and 1 mM MgCl2 plus protease inhibitors (Sigma Fast, USA). .. The protein concentration was determined by the Lowry method ( ) and bovine serum albumin was used as the standard.

Article Title: Rescue of an hTERT Mutant Defective in Telomere Elongation by Fusion with hPot1
Article Snippet: .. A Branson sonifier microtip (Branson Ultrasonics) was used for sonication (output 3; duty cycle, 30% for five 10-s bursts), after which insoluble material was pelleted by microcentrifugation (13,000 × g for 5 min at 4°C), the remaining lysate was diluted in lysis buffer (1:2), and 40 μl of 50% slurry-precoupled anti-Flag M2 agarose affinity gel (Sigma) was added. ..

Article Title: Protein-specific analysis of invertebrate glycoproteins
Article Snippet: .. For small amounts of biological samples, also a lysis buffer supplemented with protease inhibitor cocktail (Sigma) can be used prior to SDS-PAGE. .. The methanol precipitation step after cell lysis helps to desalt the sample and so avoid smearing upon electrophoresis.

Article Title: Characterization and Heterologous Expression of the Genes Encoding Enterocin A Production, Immunity, and Regulation in Enterococcus faecium DPC1146
Article Snippet: Plasmid DNA from E. coli (3 ml of fresh overnight culture) was isolated by using a QIAprep Spin Miniprep Kit (Qiagen Gmbh, Hilden, Germany) and resuspended in sterile distilled water. .. Plasmid DNA was extracted from lactococci and enterococci (3 ml of fresh overnight culture) by the rapid lysis method of Anderson and McKay ( ) and dialyzed on filters with a 0.025-μm pore size (Millipore Corp., Bedford, Mass.) before subsequent manipulations. ..

Article Title: G-actin Participates in RNA Polymerase II-dependent Transcription Elongation by Recruiting Positive Transcription Elongation Factor b (P-TEFb) *
Article Snippet: Immunoprecipitation was carried out using HeLa NEs and the indicated antibodies at 4 °C for 3 h, followed by incubation for another 3 h with Protein A/G-Sepharose. .. After washing with lysis buffer (20 m m Tris/HCl (pH 8.0), 137 m m NaCl, 1% Nonidet P40, 10% glycerol, 1 m m Na3 VO4 , 1 m m PMSF, 10 mg/ml aprotinin, and 20 mg/ml leupeptin), the immunoprecipitates were resolved on SDS-PAGE, electrotransferred onto nitrocellulose membranes (Millipore), and probed with the indicated antibodies. .. Chemiluminescent detection was performed using ECL (GE Healthcare) plus Western blot reagents.

Protease Inhibitor:

Article Title: Physiological changes during cellular ageing in fission yeast drive non-random patterns of genome rearrangements
Article Snippet: .. RIP-chip of Scw1-TAP was performed as described , except for the following modifications: immunoprecipitation was carried out using monoclonal antibodies against protein A (Sigma); the lysis buffer contained 10mg/ml heparin (sigma H7405), 1 mM PMSF and 1:100 protease inhibitor cocktail (Sigma P8340); and magnetic beads containing the immunoprecipitate were resuspended in 50 μl of wash buffer containing 1 mM DTT, 1 unit/ml of SuperaseIN (Ambion 2696) and 30 units/ml of AcTev protease (Life Technologies 12575015). .. The solution with the beads was incubated for 1.5 h at 18°C and the supernatant recovered and RNA extracted using PureLink RNA micro columns (Life Technologies), according to the manufacturer’s instructions.

Article Title: Protein-specific analysis of invertebrate glycoproteins
Article Snippet: .. For small amounts of biological samples, also a lysis buffer supplemented with protease inhibitor cocktail (Sigma) can be used prior to SDS-PAGE. .. The methanol precipitation step after cell lysis helps to desalt the sample and so avoid smearing upon electrophoresis.

Magnetic Beads:

Article Title: Physiological changes during cellular ageing in fission yeast drive non-random patterns of genome rearrangements
Article Snippet: .. RIP-chip of Scw1-TAP was performed as described , except for the following modifications: immunoprecipitation was carried out using monoclonal antibodies against protein A (Sigma); the lysis buffer contained 10mg/ml heparin (sigma H7405), 1 mM PMSF and 1:100 protease inhibitor cocktail (Sigma P8340); and magnetic beads containing the immunoprecipitate were resuspended in 50 μl of wash buffer containing 1 mM DTT, 1 unit/ml of SuperaseIN (Ambion 2696) and 30 units/ml of AcTev protease (Life Technologies 12575015). .. The solution with the beads was incubated for 1.5 h at 18°C and the supernatant recovered and RNA extracted using PureLink RNA micro columns (Life Technologies), according to the manufacturer’s instructions.

Incubation:

Article Title: Dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin mediates HIV-1 infection of and transmission by M2a-polarized macrophages in vitro
Article Snippet: An aliquot of cells were immediately washed five times and nucleic acid carry over with the viral stock was verified and excluded by PCR. .. After 48 h incubation in complete medium [ ], MDMs (1 ×106 cells) were harvested, washed, resuspended in lysis buffer containing polyoxyethylene (0.1%), lauryl ether 10 mol/l (Sigma) along with proteinase K (0.1 mg/ml) and digested for 2 h at 65°C; proteinase K was heat-inactivated for 15 min at 95°C [ ]. .. Quantitative real-time PCR amplification of HIV-1 gag DNA was performed using a primer/probe combination that detects all viral DNA synthesized after second-strand transfer including both unintegrated and integrated DNA species [ ].

Protein Extraction:

Article Title: G-quadruplex ligands mediate downregulation of DUX4 expression
Article Snippet: The number of fibres having internal nuclei was counted manually using ImageJ software (National Institutes of Health, Maryland, USA) and displayed as per mm2 of the muscle section. .. Protein extraction and western blot quantification of DUX4 expression Frozen TA sections collected from cryosectioning were homogenized in lysis buffer (0.15 M NaCl, 0.05 M HEPES, 1% (v/v) NP-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 0.01 M EDTA; (reagents were purchased from Sigma, UK) containing 1× protease inhibitors (Roche, UK) at 25 Hz for 1–2 min on a TissueLyser II (QIAgen, UK). .. Following centrifugation at 14 000 × g , 10 min, 4°C, the supernatant was transferred to fresh pre-chilled 1.5 ml tubes.

Western Blot:

Article Title: G-quadruplex ligands mediate downregulation of DUX4 expression
Article Snippet: The number of fibres having internal nuclei was counted manually using ImageJ software (National Institutes of Health, Maryland, USA) and displayed as per mm2 of the muscle section. .. Protein extraction and western blot quantification of DUX4 expression Frozen TA sections collected from cryosectioning were homogenized in lysis buffer (0.15 M NaCl, 0.05 M HEPES, 1% (v/v) NP-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 0.01 M EDTA; (reagents were purchased from Sigma, UK) containing 1× protease inhibitors (Roche, UK) at 25 Hz for 1–2 min on a TissueLyser II (QIAgen, UK). .. Following centrifugation at 14 000 × g , 10 min, 4°C, the supernatant was transferred to fresh pre-chilled 1.5 ml tubes.

Expressing:

Article Title: G-quadruplex ligands mediate downregulation of DUX4 expression
Article Snippet: The number of fibres having internal nuclei was counted manually using ImageJ software (National Institutes of Health, Maryland, USA) and displayed as per mm2 of the muscle section. .. Protein extraction and western blot quantification of DUX4 expression Frozen TA sections collected from cryosectioning were homogenized in lysis buffer (0.15 M NaCl, 0.05 M HEPES, 1% (v/v) NP-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 0.01 M EDTA; (reagents were purchased from Sigma, UK) containing 1× protease inhibitors (Roche, UK) at 25 Hz for 1–2 min on a TissueLyser II (QIAgen, UK). .. Following centrifugation at 14 000 × g , 10 min, 4°C, the supernatant was transferred to fresh pre-chilled 1.5 ml tubes.

Sonication:

Article Title: Rescue of an hTERT Mutant Defective in Telomere Elongation by Fusion with hPot1
Article Snippet: .. A Branson sonifier microtip (Branson Ultrasonics) was used for sonication (output 3; duty cycle, 30% for five 10-s bursts), after which insoluble material was pelleted by microcentrifugation (13,000 × g for 5 min at 4°C), the remaining lysate was diluted in lysis buffer (1:2), and 40 μl of 50% slurry-precoupled anti-Flag M2 agarose affinity gel (Sigma) was added. ..

SDS Page:

Article Title: Protein-specific analysis of invertebrate glycoproteins
Article Snippet: .. For small amounts of biological samples, also a lysis buffer supplemented with protease inhibitor cocktail (Sigma) can be used prior to SDS-PAGE. .. The methanol precipitation step after cell lysis helps to desalt the sample and so avoid smearing upon electrophoresis.

Article Title: G-actin Participates in RNA Polymerase II-dependent Transcription Elongation by Recruiting Positive Transcription Elongation Factor b (P-TEFb) *
Article Snippet: Immunoprecipitation was carried out using HeLa NEs and the indicated antibodies at 4 °C for 3 h, followed by incubation for another 3 h with Protein A/G-Sepharose. .. After washing with lysis buffer (20 m m Tris/HCl (pH 8.0), 137 m m NaCl, 1% Nonidet P40, 10% glycerol, 1 m m Na3 VO4 , 1 m m PMSF, 10 mg/ml aprotinin, and 20 mg/ml leupeptin), the immunoprecipitates were resolved on SDS-PAGE, electrotransferred onto nitrocellulose membranes (Millipore), and probed with the indicated antibodies. .. Chemiluminescent detection was performed using ECL (GE Healthcare) plus Western blot reagents.

Plasmid Preparation:

Article Title: Characterization and Heterologous Expression of the Genes Encoding Enterocin A Production, Immunity, and Regulation in Enterococcus faecium DPC1146
Article Snippet: Plasmid DNA from E. coli (3 ml of fresh overnight culture) was isolated by using a QIAprep Spin Miniprep Kit (Qiagen Gmbh, Hilden, Germany) and resuspended in sterile distilled water. .. Plasmid DNA was extracted from lactococci and enterococci (3 ml of fresh overnight culture) by the rapid lysis method of Anderson and McKay ( ) and dialyzed on filters with a 0.025-μm pore size (Millipore Corp., Bedford, Mass.) before subsequent manipulations. ..

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    Millipore gst lysis buffer
    (A) Endogenous MYCN and <t>p53</t> co-IP. Nuclear extracts from the neuroblastoma cell line IMR-32 treated with Nutlin-3a were co-immunoprecipitated using anti-p53 (Ab-7) antibody or IgG (negative control). Western blots of immunoprecipitated proteins were performed using anti-p53 (DO-1), anti- MYCN (B8.4.B), or anti-Max (C-17) antibodies. (B) Endogenous MYC and p53 co-IP. HeLa cells treated with Nutlin-3a were co-immunoprecipitated using anti-p53 antibody or negative control IgG. Immunoprecipitated proteins were analyzed by Western blotting, using with anti-p53 (DO-1), anti- MYC (N262), and anti-MAX (C-17) antibodies. (C) in vitro <t>GST-C-MYC</t> pull-down. Crude nuclear protein extract from transient p53 over-expressing HEK-293T cells was incubated overnight with full-length GST-MYC or GST control proteins immobilized on glutathione-agarose beads. Pull-down samples were immunoblotted with the anti-p53 antibody. Membrane Ponceau S staining is shown as a loading control. (D) MYCN and p53 in vitro pull-down. Purified recombinant MYCN-6×His, GST-p53 (full length), and GST-control proteins were loaded as input samples. Recombinant MYCN- 6×His protein was incubated with GST-p53 or GST-control proteins immobilized on glutathione-agarose beads. GST proteins were pulled down and associated MYCN was detected by Western Blotting. Stain-Free total protein staining was used as a loading control. (E) Recombinant p53 and MYCN co-immunoprecipitation. The p53-null, non-small cell lung carcinoma cell line H-1299 was transiently transfected with plasmids overexpressing p53-GFP and MYCN-3×Flag. Crude nuclear protein extract collected from cells cultured under different transfection conditions were immunoprecipitated (IP) with either anti-p53 (Ab-7) or anti-FLAG (M2) antibody, and Western blots were performed using either anti-FLAG (M2) or anti-p53 (DO-1) antibody. (F) MYCN interacts with tetrameric form of p53. Crude nuclear protein extracts from MYCN-amplified SK-N-BE2-(C) cells were incubated with GST alone and a series GST-p53 purified proteins: p53-WT (dimeric-tetrameric), p53-L344A (dimeric only) and p53-L344P (monomeric only). Input and pull-down samples were immunoblotted using anti-MYCN antibody and Ponceau staining was used as loading control.
    Gst Lysis Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gst lysis buffer/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    gst lysis buffer - by Bioz Stars, 2021-06
    99/100 stars
      Buy from Supplier

    99
    Millipore triton x 100
    Downregulation of  ATGs  blocks autophagosome formation and leads to the persistence of cytoplasm in the inside aperture. (A–C) Immuno-fluorescence of germination aperture of pollen from WT and  ATG -silencing lines using the ATG8 (red) antibody (bar: 5 μm). (D) Number of autophagosomes in the germination aperture labeled by immuno-fluorescence. Data are from three independent experiments, with 40 pollen grains analyzed in each experiment (n = 120). (E) Lipidation of ATG8 was confirmed by Phospholipase D treatment. Mem, membrane fraction collected after centrifugation and solubilized in 0.5% Triton X-100; PLD, phospholipase D. (F) ATG8–PE adduct was decreased in pollen from  ATG -silencing plants. Membrane fractions from WT and  ATG -silencing plants were used for immunoblot analysis. Ponceau staining was used to determine the loading control. (G) Relative ATG8–PE levels in WT,  ATG2  and  ATG5  RNAi pollen. Each data bar represents the mean ± SD from five independent experiments. (H) A layer of undegraded material (arrows) was observed in the aperture of germination-aborted pollen from  ATG -silencing plants (bar: 2 μm, n = 16–19). Arrowheads, autophagosomes; in, intine; ex, exine. (I) Area of undegraded layer at the germination aperture in  ATGs  RNAi pollen (n = 16–19). ** indicate statistical difference compared to the WT ( t -test,  p
    Triton X 100, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/triton x 100/product/Millipore
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    99
    Millipore rbc
    Mutants in the hTIM-4 CC′FG cavity do not phagocytose apoptotic cells ( A ) Binding of anti-hTIM-4 antibodies to mutant hTIM-4 transfected 3T3 cell lines. WT hTIM-4 and mutant hTIM-4 (W119A, F120A, N121A and D122A) were transfected into NIH-3T3 cells. WT or mutant hTIM-4 3T3 were stained with 9F4, 4E11 or polyclonal anti-hTIM-4 antibody and analyzed by flow cytometry. Open curves represent staining with anti-hTIM-4 antibody, and the filled gray curves represent staining with control mouse IgG1 for 9F4 and 4E11, or goat IgG for polyclonal anti-hTIM-4 antibody. Numbers in the panels are mean fluorescence intensity of the TIM-4 stained cells. (B) 9F4 but not 4E11 blocked phagocytosis of eryptotic <t>RBC</t> by hTIM-4 3T3. hTIM-4 3T3 were pre-incubated with 9F4, 4E11 or isotype control mouse IgG1 for 30 min, co-cultured with <t>PKH67-labeled</t> eryptotic RBC, non-adherent cells washed off, and remaining cells detached and analyzed for PKH67 positive RBC in hTIM-4 3T3 by flow cytometry. Assays were done in duplicate and average values are plotted with S.D. (C, D) Phagocytosis of PKH67-labeled eryptotic RBC by mutant TIM-4 3T3. The indicated TIM-4 3T3 transfectants were co-cultured with PKH67-labeled eryptotic RBC, non-adherent cells washed off, and remaining cells detached and analyzed for PKH67 positive RBC in hTIM-4 3T3 by flow cytometry. FACS profiles of 90 min culture are shown in C . Filled gray curves represent untransfected 3T3 with eryptotic RBC, and open curves represent the indicated TIM-4 transfectant with eryptotic RBC. Results at 45 and 90 minutes are presented graphically in D . Assays were done in duplicate and average values are plotted with S.D.
    Rbc, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore rna lysis buffer
    circAGFG1 functions as a sponge for miR-195-5p. a The miR-195-5p binding site on circAGFG1 predicted by targetScan and miRanda. b and c FISH was performed to observe the cellular location of circAGFG1 (red) and miR-195-5p (green) in cells (magnification, × 200, scale bar, 50 μm) and tissues (magnification, × 100, scale bar, 100 μm). d Relative expression of miR-195-5p in TNBC tissues (Tumor) and adjacent non-tumor tissues (Normal) was determined by qRT-PCR ( n = 40). e Relative expression of miR-195-5p in TNBC tissues (Tumor) compared with normal tissue (normal) was analyzed using TCGA data. f Kaplan-Meier survival analysis of overall survival based on TCGA data ( n = 100). g Schematic illustration of circAGFG1-WT and circAGFG1-Mut luciferase reporter vectors. h The relative luciferase activities were detected in 293 T cells after <t>transfection</t> with circAGFG1-WT or circAGFG1-Mut and miR-195-5p mimics or miR-NC, respectively. i and j Anti-AGO2 RIP was executed in MDA-MB-231 cells after transfection with miR-195-5p mimic or miR-NC, followed by western blot and qRT-PCR to detect AGO2 protein, circAGFG1 and miR-195-5p, respectively. k <t>RNA</t> pull-down was executed in MDA-MB-231 cells, followed by qRT-PCR to detect the enrichment of circAGFG1 and miR-195-5p. l The relative expression of miR-195-5p was detected by qRT-PCR after transfection with indicated vectors. m Pearson correlation analysis of circAGFG1 and miR-195-5p expression in 20 TNBC tissues. Data were indicated as mean ± SD, * P
    Rna Lysis Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    (A) Endogenous MYCN and p53 co-IP. Nuclear extracts from the neuroblastoma cell line IMR-32 treated with Nutlin-3a were co-immunoprecipitated using anti-p53 (Ab-7) antibody or IgG (negative control). Western blots of immunoprecipitated proteins were performed using anti-p53 (DO-1), anti- MYCN (B8.4.B), or anti-Max (C-17) antibodies. (B) Endogenous MYC and p53 co-IP. HeLa cells treated with Nutlin-3a were co-immunoprecipitated using anti-p53 antibody or negative control IgG. Immunoprecipitated proteins were analyzed by Western blotting, using with anti-p53 (DO-1), anti- MYC (N262), and anti-MAX (C-17) antibodies. (C) in vitro GST-C-MYC pull-down. Crude nuclear protein extract from transient p53 over-expressing HEK-293T cells was incubated overnight with full-length GST-MYC or GST control proteins immobilized on glutathione-agarose beads. Pull-down samples were immunoblotted with the anti-p53 antibody. Membrane Ponceau S staining is shown as a loading control. (D) MYCN and p53 in vitro pull-down. Purified recombinant MYCN-6×His, GST-p53 (full length), and GST-control proteins were loaded as input samples. Recombinant MYCN- 6×His protein was incubated with GST-p53 or GST-control proteins immobilized on glutathione-agarose beads. GST proteins were pulled down and associated MYCN was detected by Western Blotting. Stain-Free total protein staining was used as a loading control. (E) Recombinant p53 and MYCN co-immunoprecipitation. The p53-null, non-small cell lung carcinoma cell line H-1299 was transiently transfected with plasmids overexpressing p53-GFP and MYCN-3×Flag. Crude nuclear protein extract collected from cells cultured under different transfection conditions were immunoprecipitated (IP) with either anti-p53 (Ab-7) or anti-FLAG (M2) antibody, and Western blots were performed using either anti-FLAG (M2) or anti-p53 (DO-1) antibody. (F) MYCN interacts with tetrameric form of p53. Crude nuclear protein extracts from MYCN-amplified SK-N-BE2-(C) cells were incubated with GST alone and a series GST-p53 purified proteins: p53-WT (dimeric-tetrameric), p53-L344A (dimeric only) and p53-L344P (monomeric only). Input and pull-down samples were immunoblotted using anti-MYCN antibody and Ponceau staining was used as loading control.

    Journal: Oncotarget

    Article Title: MYCN acts as a direct co-regulator of p53 in MYCN amplified neuroblastoma

    doi: 10.18632/oncotarget.24859

    Figure Lengend Snippet: (A) Endogenous MYCN and p53 co-IP. Nuclear extracts from the neuroblastoma cell line IMR-32 treated with Nutlin-3a were co-immunoprecipitated using anti-p53 (Ab-7) antibody or IgG (negative control). Western blots of immunoprecipitated proteins were performed using anti-p53 (DO-1), anti- MYCN (B8.4.B), or anti-Max (C-17) antibodies. (B) Endogenous MYC and p53 co-IP. HeLa cells treated with Nutlin-3a were co-immunoprecipitated using anti-p53 antibody or negative control IgG. Immunoprecipitated proteins were analyzed by Western blotting, using with anti-p53 (DO-1), anti- MYC (N262), and anti-MAX (C-17) antibodies. (C) in vitro GST-C-MYC pull-down. Crude nuclear protein extract from transient p53 over-expressing HEK-293T cells was incubated overnight with full-length GST-MYC or GST control proteins immobilized on glutathione-agarose beads. Pull-down samples were immunoblotted with the anti-p53 antibody. Membrane Ponceau S staining is shown as a loading control. (D) MYCN and p53 in vitro pull-down. Purified recombinant MYCN-6×His, GST-p53 (full length), and GST-control proteins were loaded as input samples. Recombinant MYCN- 6×His protein was incubated with GST-p53 or GST-control proteins immobilized on glutathione-agarose beads. GST proteins were pulled down and associated MYCN was detected by Western Blotting. Stain-Free total protein staining was used as a loading control. (E) Recombinant p53 and MYCN co-immunoprecipitation. The p53-null, non-small cell lung carcinoma cell line H-1299 was transiently transfected with plasmids overexpressing p53-GFP and MYCN-3×Flag. Crude nuclear protein extract collected from cells cultured under different transfection conditions were immunoprecipitated (IP) with either anti-p53 (Ab-7) or anti-FLAG (M2) antibody, and Western blots were performed using either anti-FLAG (M2) or anti-p53 (DO-1) antibody. (F) MYCN interacts with tetrameric form of p53. Crude nuclear protein extracts from MYCN-amplified SK-N-BE2-(C) cells were incubated with GST alone and a series GST-p53 purified proteins: p53-WT (dimeric-tetrameric), p53-L344A (dimeric only) and p53-L344P (monomeric only). Input and pull-down samples were immunoblotted using anti-MYCN antibody and Ponceau staining was used as loading control.

    Article Snippet: GST-p53 cells were lysed in GST lysis buffer (1% Triton, 1 μg/μl lysozyme, 0.5 mM EDTA, and 1 mM PMSF in phosphate buffered saline), purified and immobilized on glutathione-agarose beads (Sigma Aldrich).

    Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Negative Control, Western Blot, In Vitro, Expressing, Incubation, Staining, Purification, Recombinant, Transfection, Cell Culture, Amplification

    (A) Graphical representations of p53 and MYCN proteins. p53 (upper panel) and MYCN (lower panel) protein domains and truncation constructs. p53 protein domains: Trans Activation Domain (TAD), SRC Homology 3 domain (SH3), DNA binding domain, Nuclear Localization Signal (NLS), Tetramerization domain (TET), Regulatory domain (REG). MYCN protein domains: MYC boxes (MB), the basic region helix loop helix (BR-HLH), and the leucine zipper. The GST protein fragments are indicated with bars, and numbers refer to amino-acid positions. p53 and MYCN protein fragments were cloned in frame with the N-terminal GST in a pGEX-2T vector. GST-p53 and GST-MYCN fragments were cloned, expressed in BL-21 E.Coli strain and purified using gluthatione-agarose beads. (B) MYCN interacts with the C-terminus of p53. Crude nuclear protein extracts from MYCN-amplified SK-N-BE-(2)-c cells were incubated with the different p53 truncations or GST alone (negative control) immobilized onto glutathione-agarose beads. Input and pull-down samples were immunoblotted using anti-MYCN and anti-MAX antibodies. Stain-Free total protein staining was used as the loading control. (C) GST pull-down assay of MYCN truncations. Crude nuclear protein extract from transiently transfected p53-overexpressing HEK-293T cells was incubated with different MYCN-GST fragments immobilized on glutathione-agarose beads. GST alone was used as a negative control. Input and pull-down samples were immunoblotted using anti-p53 (DO-1) antibody. Ponceau staining was used as a loading control.

    Journal: Oncotarget

    Article Title: MYCN acts as a direct co-regulator of p53 in MYCN amplified neuroblastoma

    doi: 10.18632/oncotarget.24859

    Figure Lengend Snippet: (A) Graphical representations of p53 and MYCN proteins. p53 (upper panel) and MYCN (lower panel) protein domains and truncation constructs. p53 protein domains: Trans Activation Domain (TAD), SRC Homology 3 domain (SH3), DNA binding domain, Nuclear Localization Signal (NLS), Tetramerization domain (TET), Regulatory domain (REG). MYCN protein domains: MYC boxes (MB), the basic region helix loop helix (BR-HLH), and the leucine zipper. The GST protein fragments are indicated with bars, and numbers refer to amino-acid positions. p53 and MYCN protein fragments were cloned in frame with the N-terminal GST in a pGEX-2T vector. GST-p53 and GST-MYCN fragments were cloned, expressed in BL-21 E.Coli strain and purified using gluthatione-agarose beads. (B) MYCN interacts with the C-terminus of p53. Crude nuclear protein extracts from MYCN-amplified SK-N-BE-(2)-c cells were incubated with the different p53 truncations or GST alone (negative control) immobilized onto glutathione-agarose beads. Input and pull-down samples were immunoblotted using anti-MYCN and anti-MAX antibodies. Stain-Free total protein staining was used as the loading control. (C) GST pull-down assay of MYCN truncations. Crude nuclear protein extract from transiently transfected p53-overexpressing HEK-293T cells was incubated with different MYCN-GST fragments immobilized on glutathione-agarose beads. GST alone was used as a negative control. Input and pull-down samples were immunoblotted using anti-p53 (DO-1) antibody. Ponceau staining was used as a loading control.

    Article Snippet: GST-p53 cells were lysed in GST lysis buffer (1% Triton, 1 μg/μl lysozyme, 0.5 mM EDTA, and 1 mM PMSF in phosphate buffered saline), purified and immobilized on glutathione-agarose beads (Sigma Aldrich).

    Techniques: Construct, Activation Assay, Binding Assay, Clone Assay, Plasmid Preparation, Purification, Amplification, Incubation, Negative Control, Staining, Pull Down Assay, Transfection

    Downregulation of  ATGs  blocks autophagosome formation and leads to the persistence of cytoplasm in the inside aperture. (A–C) Immuno-fluorescence of germination aperture of pollen from WT and  ATG -silencing lines using the ATG8 (red) antibody (bar: 5 μm). (D) Number of autophagosomes in the germination aperture labeled by immuno-fluorescence. Data are from three independent experiments, with 40 pollen grains analyzed in each experiment (n = 120). (E) Lipidation of ATG8 was confirmed by Phospholipase D treatment. Mem, membrane fraction collected after centrifugation and solubilized in 0.5% Triton X-100; PLD, phospholipase D. (F) ATG8–PE adduct was decreased in pollen from  ATG -silencing plants. Membrane fractions from WT and  ATG -silencing plants were used for immunoblot analysis. Ponceau staining was used to determine the loading control. (G) Relative ATG8–PE levels in WT,  ATG2  and  ATG5  RNAi pollen. Each data bar represents the mean ± SD from five independent experiments. (H) A layer of undegraded material (arrows) was observed in the aperture of germination-aborted pollen from  ATG -silencing plants (bar: 2 μm, n = 16–19). Arrowheads, autophagosomes; in, intine; ex, exine. (I) Area of undegraded layer at the germination aperture in  ATGs  RNAi pollen (n = 16–19). ** indicate statistical difference compared to the WT ( t -test,  p

    Journal: Autophagy

    Article Title: Autophagy-mediated compartmental cytoplasmic deletion is essential for tobacco pollen germination and male fertility

    doi: 10.1080/15548627.2020.1719722

    Figure Lengend Snippet: Downregulation of ATGs blocks autophagosome formation and leads to the persistence of cytoplasm in the inside aperture. (A–C) Immuno-fluorescence of germination aperture of pollen from WT and ATG -silencing lines using the ATG8 (red) antibody (bar: 5 μm). (D) Number of autophagosomes in the germination aperture labeled by immuno-fluorescence. Data are from three independent experiments, with 40 pollen grains analyzed in each experiment (n = 120). (E) Lipidation of ATG8 was confirmed by Phospholipase D treatment. Mem, membrane fraction collected after centrifugation and solubilized in 0.5% Triton X-100; PLD, phospholipase D. (F) ATG8–PE adduct was decreased in pollen from ATG -silencing plants. Membrane fractions from WT and ATG -silencing plants were used for immunoblot analysis. Ponceau staining was used to determine the loading control. (G) Relative ATG8–PE levels in WT, ATG2 and ATG5 RNAi pollen. Each data bar represents the mean ± SD from five independent experiments. (H) A layer of undegraded material (arrows) was observed in the aperture of germination-aborted pollen from ATG -silencing plants (bar: 2 μm, n = 16–19). Arrowheads, autophagosomes; in, intine; ex, exine. (I) Area of undegraded layer at the germination aperture in ATGs RNAi pollen (n = 16–19). ** indicate statistical difference compared to the WT ( t -test, p

    Article Snippet: Four volumes of lysis buffer (8 M urea, 1% Triton X-100 [Sigma-Aldrich, T8787], 10 mM dithiothreitol, and 1% protease inhibitor cocktail [Calbiochem, 535141]) were added to the powder, followed by sonication three times on ice using a high-intensity ultrasonic processor (Scientz, USA).

    Techniques: Fluorescence, Labeling, Centrifugation, Staining

    Mutants in the hTIM-4 CC′FG cavity do not phagocytose apoptotic cells ( A ) Binding of anti-hTIM-4 antibodies to mutant hTIM-4 transfected 3T3 cell lines. WT hTIM-4 and mutant hTIM-4 (W119A, F120A, N121A and D122A) were transfected into NIH-3T3 cells. WT or mutant hTIM-4 3T3 were stained with 9F4, 4E11 or polyclonal anti-hTIM-4 antibody and analyzed by flow cytometry. Open curves represent staining with anti-hTIM-4 antibody, and the filled gray curves represent staining with control mouse IgG1 for 9F4 and 4E11, or goat IgG for polyclonal anti-hTIM-4 antibody. Numbers in the panels are mean fluorescence intensity of the TIM-4 stained cells. (B) 9F4 but not 4E11 blocked phagocytosis of eryptotic RBC by hTIM-4 3T3. hTIM-4 3T3 were pre-incubated with 9F4, 4E11 or isotype control mouse IgG1 for 30 min, co-cultured with PKH67-labeled eryptotic RBC, non-adherent cells washed off, and remaining cells detached and analyzed for PKH67 positive RBC in hTIM-4 3T3 by flow cytometry. Assays were done in duplicate and average values are plotted with S.D. (C, D) Phagocytosis of PKH67-labeled eryptotic RBC by mutant TIM-4 3T3. The indicated TIM-4 3T3 transfectants were co-cultured with PKH67-labeled eryptotic RBC, non-adherent cells washed off, and remaining cells detached and analyzed for PKH67 positive RBC in hTIM-4 3T3 by flow cytometry. FACS profiles of 90 min culture are shown in C . Filled gray curves represent untransfected 3T3 with eryptotic RBC, and open curves represent the indicated TIM-4 transfectant with eryptotic RBC. Results at 45 and 90 minutes are presented graphically in D . Assays were done in duplicate and average values are plotted with S.D.

    Journal: Immunity

    Article Title: T cell Immunoglobulin Mucin Protein (TIM)-4 binds phosphatidylserine and mediates uptake of apoptotic cells

    doi: 10.1016/j.immuni.2007.11.011

    Figure Lengend Snippet: Mutants in the hTIM-4 CC′FG cavity do not phagocytose apoptotic cells ( A ) Binding of anti-hTIM-4 antibodies to mutant hTIM-4 transfected 3T3 cell lines. WT hTIM-4 and mutant hTIM-4 (W119A, F120A, N121A and D122A) were transfected into NIH-3T3 cells. WT or mutant hTIM-4 3T3 were stained with 9F4, 4E11 or polyclonal anti-hTIM-4 antibody and analyzed by flow cytometry. Open curves represent staining with anti-hTIM-4 antibody, and the filled gray curves represent staining with control mouse IgG1 for 9F4 and 4E11, or goat IgG for polyclonal anti-hTIM-4 antibody. Numbers in the panels are mean fluorescence intensity of the TIM-4 stained cells. (B) 9F4 but not 4E11 blocked phagocytosis of eryptotic RBC by hTIM-4 3T3. hTIM-4 3T3 were pre-incubated with 9F4, 4E11 or isotype control mouse IgG1 for 30 min, co-cultured with PKH67-labeled eryptotic RBC, non-adherent cells washed off, and remaining cells detached and analyzed for PKH67 positive RBC in hTIM-4 3T3 by flow cytometry. Assays were done in duplicate and average values are plotted with S.D. (C, D) Phagocytosis of PKH67-labeled eryptotic RBC by mutant TIM-4 3T3. The indicated TIM-4 3T3 transfectants were co-cultured with PKH67-labeled eryptotic RBC, non-adherent cells washed off, and remaining cells detached and analyzed for PKH67 positive RBC in hTIM-4 3T3 by flow cytometry. FACS profiles of 90 min culture are shown in C . Filled gray curves represent untransfected 3T3 with eryptotic RBC, and open curves represent the indicated TIM-4 transfectant with eryptotic RBC. Results at 45 and 90 minutes are presented graphically in D . Assays were done in duplicate and average values are plotted with S.D.

    Article Snippet: After treatment, U937 were washed 3 times and labeled with 2–5 μM of CellTracker Orange (CMTMR, Molecular Probes, FL2 channel) and thymocytes or RBC with 1–2 μM PKH67 (Sigma, FL1 channel) according to the manufacturer’s instructions.

    Techniques: Binding Assay, Mutagenesis, Transfection, Staining, Flow Cytometry, Cytometry, Fluorescence, Incubation, Cell Culture, Labeling, FACS

    hTIM-1 or hTIM-4 transfected NIH-3T3 cells phagocytose apoptotic U937 cells or eryptotic RBC but not live cells. Liposomes containing PS blocked phagocytosis ( A–D) CMTMR-labeled live or apoptotic U937 or PKH67 labeled live or eryptotic RBC were co-cultured with 3T3, hTIM-1 3T3 or hTIM-4 3T3 for 45 or 90 min., non-adherent cells washed off, and adherent cells detached and analyzed by flow cytometry or electron microscopy for phagocytosis of labeled cells. ( A ) FACS profiles for phagocytosis of live or apoptotic U937 by 3T3 (black line), hTIM-1 3T3 (blue line) or hTIM-4 3T3 (red line) after 90 min co-incubation. Phagocytosis assays with ( B ) U937 cells or ( E ) RBC were done in duplicate, and average values are plotted with S.D. (C) Electron micrograph of apoptotic U937 phagocytosed by hTIM-4 3T3. Original magnification 5000X. (D) Photomicrograph of CypHer5E-labeled apoptotic U937 compartmentalized to an acidic compartment of hTIM-4 3T3. (F) 3T3 cells or ( G–I ) hTIM-1 3T3 were labeled with CMFDA (green). U937 were labeled with CMTMR (red) and made apoptotic by 5 hr treatment with etoposide. 3T3 cells and apoptotic U937 cells were co-cultured for 2 hours, washed, and visualized by confocal microscopy as described in experimental procedures. An enlarged image of the boxed region in G is shown in H and a side image in I . ( J ) Binding of eryptotic RBC to hTIM-1 3T3 or hTIM-4 3T3 was inhibited by liposomes containing a 50:50 mix of PS and PC but not by PC alone. hTIM-1 or TIM-4 3T3 were pre-incubated with liposomes for 15 min. and co-cultured with PKH67 labeled eryptotic RBC for 45 min. After washing out non-attached cells, 3T3 cells were detached and analyzed by flow cytometry for phagocytosis of eryptotic RBC. Assays were done in duplicate and values of 100% represent phagocytosis of eryptotic RBC in the absence of liposomes. (K) Phagocytosis of eryptotic RBC by hTIM-1 or hTIM-4 3T3 was inhibited by liposomes containing PS but not PC, PE or PI. 1 or 10 μM of PS, PC, PE or PI liposomes were pre-incubated with hTIM-1 or TIM-4 3T3 cells. Assays were done in duplicate as described in H.

    Journal: Immunity

    Article Title: T cell Immunoglobulin Mucin Protein (TIM)-4 binds phosphatidylserine and mediates uptake of apoptotic cells

    doi: 10.1016/j.immuni.2007.11.011

    Figure Lengend Snippet: hTIM-1 or hTIM-4 transfected NIH-3T3 cells phagocytose apoptotic U937 cells or eryptotic RBC but not live cells. Liposomes containing PS blocked phagocytosis ( A–D) CMTMR-labeled live or apoptotic U937 or PKH67 labeled live or eryptotic RBC were co-cultured with 3T3, hTIM-1 3T3 or hTIM-4 3T3 for 45 or 90 min., non-adherent cells washed off, and adherent cells detached and analyzed by flow cytometry or electron microscopy for phagocytosis of labeled cells. ( A ) FACS profiles for phagocytosis of live or apoptotic U937 by 3T3 (black line), hTIM-1 3T3 (blue line) or hTIM-4 3T3 (red line) after 90 min co-incubation. Phagocytosis assays with ( B ) U937 cells or ( E ) RBC were done in duplicate, and average values are plotted with S.D. (C) Electron micrograph of apoptotic U937 phagocytosed by hTIM-4 3T3. Original magnification 5000X. (D) Photomicrograph of CypHer5E-labeled apoptotic U937 compartmentalized to an acidic compartment of hTIM-4 3T3. (F) 3T3 cells or ( G–I ) hTIM-1 3T3 were labeled with CMFDA (green). U937 were labeled with CMTMR (red) and made apoptotic by 5 hr treatment with etoposide. 3T3 cells and apoptotic U937 cells were co-cultured for 2 hours, washed, and visualized by confocal microscopy as described in experimental procedures. An enlarged image of the boxed region in G is shown in H and a side image in I . ( J ) Binding of eryptotic RBC to hTIM-1 3T3 or hTIM-4 3T3 was inhibited by liposomes containing a 50:50 mix of PS and PC but not by PC alone. hTIM-1 or TIM-4 3T3 were pre-incubated with liposomes for 15 min. and co-cultured with PKH67 labeled eryptotic RBC for 45 min. After washing out non-attached cells, 3T3 cells were detached and analyzed by flow cytometry for phagocytosis of eryptotic RBC. Assays were done in duplicate and values of 100% represent phagocytosis of eryptotic RBC in the absence of liposomes. (K) Phagocytosis of eryptotic RBC by hTIM-1 or hTIM-4 3T3 was inhibited by liposomes containing PS but not PC, PE or PI. 1 or 10 μM of PS, PC, PE or PI liposomes were pre-incubated with hTIM-1 or TIM-4 3T3 cells. Assays were done in duplicate as described in H.

    Article Snippet: After treatment, U937 were washed 3 times and labeled with 2–5 μM of CellTracker Orange (CMTMR, Molecular Probes, FL2 channel) and thymocytes or RBC with 1–2 μM PKH67 (Sigma, FL1 channel) according to the manufacturer’s instructions.

    Techniques: Transfection, Labeling, Cell Culture, Flow Cytometry, Cytometry, Electron Microscopy, FACS, Incubation, Confocal Microscopy, Binding Assay

    Human kidney cell line 769P expresses hTIM-1 and phagocytoses apoptotic U937 cells and eryptotic RBC through hTIM-1 (A) 769P cells were stained with PE-conjugated anti-hTIM-1 mAb (3D1) or anti-hTIM-4 mAb (9F4) and analyzed by flow cytometry. (B) CMFDA-labeled 769P cells (green) and CMTMR-labeled apoptotic U937 cells (red) were co-cultured for 2 hours, non-adherent cells washed off, and remaining cells fixed and visualized by confocal microscopy. (C) PKH67 labeled fresh and eryptotic RBC were co-cultured with 769P cells for 45 min or 90 min, non-adherent cells washed off, and remaining cells detached and analyzed for PKH67 positive RBC in 769Pcells by flow cytometry. The FACS profiles for PKH67 positive RBC in 769P cells (open curves) and 769P cells without RBC (filled gray curves) are shown in the upper panel and presented graphically in the lower panel. Assays were done in duplicate and average values are plotted with S.D. (D) Phagocytosis of eryptotic RBC was blocked by anti-hTIM-1 mAb. 769P cells were pre-incubated with 3D1, 1D12, or isotype control for 30 min., co-cultured with PKH67 labeled eryptotic RBC for 90 min, non-adherent cells washed off, and remaining cells detached and analyzed for PKH67 positive RBC in 769P by flow cytometry. FACS profiles for PKH67 positive RBC in 769P incubated with 2 μg/ml 3D1 (red line), 1D12 (blue line) or isotype control (black line) are shown in the upper panel and presented graphically in the lower panel. Assays were done in duplicate and average values are plotted with S.D.

    Journal: Immunity

    Article Title: T cell Immunoglobulin Mucin Protein (TIM)-4 binds phosphatidylserine and mediates uptake of apoptotic cells

    doi: 10.1016/j.immuni.2007.11.011

    Figure Lengend Snippet: Human kidney cell line 769P expresses hTIM-1 and phagocytoses apoptotic U937 cells and eryptotic RBC through hTIM-1 (A) 769P cells were stained with PE-conjugated anti-hTIM-1 mAb (3D1) or anti-hTIM-4 mAb (9F4) and analyzed by flow cytometry. (B) CMFDA-labeled 769P cells (green) and CMTMR-labeled apoptotic U937 cells (red) were co-cultured for 2 hours, non-adherent cells washed off, and remaining cells fixed and visualized by confocal microscopy. (C) PKH67 labeled fresh and eryptotic RBC were co-cultured with 769P cells for 45 min or 90 min, non-adherent cells washed off, and remaining cells detached and analyzed for PKH67 positive RBC in 769Pcells by flow cytometry. The FACS profiles for PKH67 positive RBC in 769P cells (open curves) and 769P cells without RBC (filled gray curves) are shown in the upper panel and presented graphically in the lower panel. Assays were done in duplicate and average values are plotted with S.D. (D) Phagocytosis of eryptotic RBC was blocked by anti-hTIM-1 mAb. 769P cells were pre-incubated with 3D1, 1D12, or isotype control for 30 min., co-cultured with PKH67 labeled eryptotic RBC for 90 min, non-adherent cells washed off, and remaining cells detached and analyzed for PKH67 positive RBC in 769P by flow cytometry. FACS profiles for PKH67 positive RBC in 769P incubated with 2 μg/ml 3D1 (red line), 1D12 (blue line) or isotype control (black line) are shown in the upper panel and presented graphically in the lower panel. Assays were done in duplicate and average values are plotted with S.D.

    Article Snippet: After treatment, U937 were washed 3 times and labeled with 2–5 μM of CellTracker Orange (CMTMR, Molecular Probes, FL2 channel) and thymocytes or RBC with 1–2 μM PKH67 (Sigma, FL1 channel) according to the manufacturer’s instructions.

    Techniques: Staining, Flow Cytometry, Cytometry, Labeling, Cell Culture, Confocal Microscopy, FACS, Incubation

    circAGFG1 functions as a sponge for miR-195-5p. a The miR-195-5p binding site on circAGFG1 predicted by targetScan and miRanda. b and c FISH was performed to observe the cellular location of circAGFG1 (red) and miR-195-5p (green) in cells (magnification, × 200, scale bar, 50 μm) and tissues (magnification, × 100, scale bar, 100 μm). d Relative expression of miR-195-5p in TNBC tissues (Tumor) and adjacent non-tumor tissues (Normal) was determined by qRT-PCR ( n = 40). e Relative expression of miR-195-5p in TNBC tissues (Tumor) compared with normal tissue (normal) was analyzed using TCGA data. f Kaplan-Meier survival analysis of overall survival based on TCGA data ( n = 100). g Schematic illustration of circAGFG1-WT and circAGFG1-Mut luciferase reporter vectors. h The relative luciferase activities were detected in 293 T cells after transfection with circAGFG1-WT or circAGFG1-Mut and miR-195-5p mimics or miR-NC, respectively. i and j Anti-AGO2 RIP was executed in MDA-MB-231 cells after transfection with miR-195-5p mimic or miR-NC, followed by western blot and qRT-PCR to detect AGO2 protein, circAGFG1 and miR-195-5p, respectively. k RNA pull-down was executed in MDA-MB-231 cells, followed by qRT-PCR to detect the enrichment of circAGFG1 and miR-195-5p. l The relative expression of miR-195-5p was detected by qRT-PCR after transfection with indicated vectors. m Pearson correlation analysis of circAGFG1 and miR-195-5p expression in 20 TNBC tissues. Data were indicated as mean ± SD, * P

    Journal: Molecular Cancer

    Article Title: The circRNA circAGFG1 acts as a sponge of miR-195-5p to promote triple-negative breast cancer progression through regulating CCNE1 expression

    doi: 10.1186/s12943-018-0933-7

    Figure Lengend Snippet: circAGFG1 functions as a sponge for miR-195-5p. a The miR-195-5p binding site on circAGFG1 predicted by targetScan and miRanda. b and c FISH was performed to observe the cellular location of circAGFG1 (red) and miR-195-5p (green) in cells (magnification, × 200, scale bar, 50 μm) and tissues (magnification, × 100, scale bar, 100 μm). d Relative expression of miR-195-5p in TNBC tissues (Tumor) and adjacent non-tumor tissues (Normal) was determined by qRT-PCR ( n = 40). e Relative expression of miR-195-5p in TNBC tissues (Tumor) compared with normal tissue (normal) was analyzed using TCGA data. f Kaplan-Meier survival analysis of overall survival based on TCGA data ( n = 100). g Schematic illustration of circAGFG1-WT and circAGFG1-Mut luciferase reporter vectors. h The relative luciferase activities were detected in 293 T cells after transfection with circAGFG1-WT or circAGFG1-Mut and miR-195-5p mimics or miR-NC, respectively. i and j Anti-AGO2 RIP was executed in MDA-MB-231 cells after transfection with miR-195-5p mimic or miR-NC, followed by western blot and qRT-PCR to detect AGO2 protein, circAGFG1 and miR-195-5p, respectively. k RNA pull-down was executed in MDA-MB-231 cells, followed by qRT-PCR to detect the enrichment of circAGFG1 and miR-195-5p. l The relative expression of miR-195-5p was detected by qRT-PCR after transfection with indicated vectors. m Pearson correlation analysis of circAGFG1 and miR-195-5p expression in 20 TNBC tissues. Data were indicated as mean ± SD, * P

    Article Snippet: MDA-MB-231 cells were harvested 48 h after transfection of miR-195-5p mimics or miR-NC, and lysed in complete RNA lysis buffer, then cell lysates were incubated with magnetic beads which were conjugated with anti-Argonaute2 (AGO2) (Millipore, Billerica, MA, USA) or negative control IgG antibody (Millipore, Billerica, MA, USA) at 4 °C for 4 h. The beads were washed using washing buffer.

    Techniques: Binding Assay, Fluorescence In Situ Hybridization, Expressing, Quantitative RT-PCR, Luciferase, Transfection, Multiple Displacement Amplification, Western Blot

    circAGFG1 promotes TNBC cell proliferation. a The schematic illustration of circAGFG1 expression vector and shRNAs. b and c qRT-PCR analysis of circAGFG1 and AGFG1 RNA expression in TNBC cells transfected with circAGFG1 expression vector, mock, sh-circ or sh-NC. d The growth curves of cells transfected with indicated vectors were evaluated by CCK8 assays. e and f EdU assays were conducted in cells after transfection with indicated plasmids (magnification, × 100). Scale bar, 100 μm. g and h Colony formation assays were executed to detect the proliferation of cells transfected with indicated vectors. Data were showed as mean ± SD, * P

    Journal: Molecular Cancer

    Article Title: The circRNA circAGFG1 acts as a sponge of miR-195-5p to promote triple-negative breast cancer progression through regulating CCNE1 expression

    doi: 10.1186/s12943-018-0933-7

    Figure Lengend Snippet: circAGFG1 promotes TNBC cell proliferation. a The schematic illustration of circAGFG1 expression vector and shRNAs. b and c qRT-PCR analysis of circAGFG1 and AGFG1 RNA expression in TNBC cells transfected with circAGFG1 expression vector, mock, sh-circ or sh-NC. d The growth curves of cells transfected with indicated vectors were evaluated by CCK8 assays. e and f EdU assays were conducted in cells after transfection with indicated plasmids (magnification, × 100). Scale bar, 100 μm. g and h Colony formation assays were executed to detect the proliferation of cells transfected with indicated vectors. Data were showed as mean ± SD, * P

    Article Snippet: MDA-MB-231 cells were harvested 48 h after transfection of miR-195-5p mimics or miR-NC, and lysed in complete RNA lysis buffer, then cell lysates were incubated with magnetic beads which were conjugated with anti-Argonaute2 (AGO2) (Millipore, Billerica, MA, USA) or negative control IgG antibody (Millipore, Billerica, MA, USA) at 4 °C for 4 h. The beads were washed using washing buffer.

    Techniques: Expressing, Plasmid Preparation, Quantitative RT-PCR, RNA Expression, Transfection