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    Millipore polyvinylidene fluoride pvdf membrane
    Detection and characterization of HcAPN3 expressed in H. cunea BBMV from different development larval stages and infected Sf9 cells. Protein from BBMV prepared from different developmental instars (lane 1–5), Sf9 cells containing control baculovirus alone (lane 6) and Sf9 cells infected with recombinant baculoviruse (lane 7) were separated by 10% <t>SDS-PAGE</t> and electrotransferred to <t>PVDF</t> membrane. ( A ) Western blot analysis of the expression of HcAPN3 with anti-HcAPN3 antibodies; ( B ) Binding detection of Cry1Ab35 toxin to HcAPN3 by ligand blot assay with anti-Cry1Ab35 antibodies. Lane M shows molecular weight markers and arrows indicate the position of the HcAPN3 protein in H. cunea BBMV.

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    1) Product Images from "Identification and Characterization of Hyphantria cunea Aminopeptidase N as a Binding Protein of Bacillus thuringiensis Cry1Ab35 Toxin"

    Article Title: Identification and Characterization of Hyphantria cunea Aminopeptidase N as a Binding Protein of Bacillus thuringiensis Cry1Ab35 Toxin

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms18122575

    Detection and characterization of HcAPN3 expressed in H. cunea BBMV from different development larval stages and infected Sf9 cells. Protein from BBMV prepared from different developmental instars (lane 1–5), Sf9 cells containing control baculovirus alone (lane 6) and Sf9 cells infected with recombinant baculoviruse (lane 7) were separated by 10% SDS-PAGE and electrotransferred to PVDF membrane. ( A ) Western blot analysis of the expression of HcAPN3 with anti-HcAPN3 antibodies; ( B ) Binding detection of Cry1Ab35 toxin to HcAPN3 by ligand blot assay with anti-Cry1Ab35 antibodies. Lane M shows molecular weight markers and arrows indicate the position of the HcAPN3 protein in H. cunea BBMV.
    Figure Legend Snippet: Detection and characterization of HcAPN3 expressed in H. cunea BBMV from different development larval stages and infected Sf9 cells. Protein from BBMV prepared from different developmental instars (lane 1–5), Sf9 cells containing control baculovirus alone (lane 6) and Sf9 cells infected with recombinant baculoviruse (lane 7) were separated by 10% SDS-PAGE and electrotransferred to PVDF membrane. ( A ) Western blot analysis of the expression of HcAPN3 with anti-HcAPN3 antibodies; ( B ) Binding detection of Cry1Ab35 toxin to HcAPN3 by ligand blot assay with anti-Cry1Ab35 antibodies. Lane M shows molecular weight markers and arrows indicate the position of the HcAPN3 protein in H. cunea BBMV.

    Techniques Used: Infection, Recombinant, SDS Page, Western Blot, Expressing, Binding Assay, Molecular Weight

    HcAPN3G and HcAPN3E fragment peptides expressed in E.coli BL21 (DE3) cells. HcAPN3G (lane 1) and HcAPN3E (lane 2) peptides expressed in E. coli BL21 (DE3) cells were separated by 10% SDS-PAGE, and either stained with Coomassie Brilliant Blue R-250 ( A ) or transferred to a PVDF membrane and probed by Cry1Ab35 toxin in ligand blot assay ( B ) with anti-Cry1Ab35 antibodies. Lane 3 and 4 was HcAPN3G and HcAPN3E control without Cry1Ab35 toxin ligand respectivily. Lane M shows molecular weight markers and arrows indicate the 58 kDa HcAPN3G and 49 kDa HcAPN3E protein bands.
    Figure Legend Snippet: HcAPN3G and HcAPN3E fragment peptides expressed in E.coli BL21 (DE3) cells. HcAPN3G (lane 1) and HcAPN3E (lane 2) peptides expressed in E. coli BL21 (DE3) cells were separated by 10% SDS-PAGE, and either stained with Coomassie Brilliant Blue R-250 ( A ) or transferred to a PVDF membrane and probed by Cry1Ab35 toxin in ligand blot assay ( B ) with anti-Cry1Ab35 antibodies. Lane 3 and 4 was HcAPN3G and HcAPN3E control without Cry1Ab35 toxin ligand respectivily. Lane M shows molecular weight markers and arrows indicate the 58 kDa HcAPN3G and 49 kDa HcAPN3E protein bands.

    Techniques Used: SDS Page, Staining, Molecular Weight

    2) Product Images from "5-Keto-d-Gluconate Production Is Catalyzed by a Quinoprotein Glycerol Dehydrogenase, Major Polyol Dehydrogenase, in Gluconobacter Species"

    Article Title: 5-Keto-d-Gluconate Production Is Catalyzed by a Quinoprotein Glycerol Dehydrogenase, Major Polyol Dehydrogenase, in Gluconobacter Species

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.69.4.1959-1966.2003

    Immunoblotting analysis of the membrane fractions of G. suboxydans IFO 3257 and 3255 strains and their SLDH disruptants and of the purified ARDH with anti-SLDH. Protein bands in the gel of SDS-PAGE were transferred electrophoretically onto PVDF membrane, and the enzyme band was visualized as described in Materials and Methods. Lane M: prestained marker proteins; lanes 1 to 4: membrane fractions (2 μg of protein each) of G. suboxydans strain 3255, 3255 sldA ::Km, 3275, and 3275 sldA ::Km, respectively; lane 5: ARDH (0.5 μg of protein) purified from G. suboxydans IFO 3257.
    Figure Legend Snippet: Immunoblotting analysis of the membrane fractions of G. suboxydans IFO 3257 and 3255 strains and their SLDH disruptants and of the purified ARDH with anti-SLDH. Protein bands in the gel of SDS-PAGE were transferred electrophoretically onto PVDF membrane, and the enzyme band was visualized as described in Materials and Methods. Lane M: prestained marker proteins; lanes 1 to 4: membrane fractions (2 μg of protein each) of G. suboxydans strain 3255, 3255 sldA ::Km, 3275, and 3275 sldA ::Km, respectively; lane 5: ARDH (0.5 μg of protein) purified from G. suboxydans IFO 3257.

    Techniques Used: Purification, SDS Page, Marker

    3) Product Images from "Chemical Characterization of N-Linked Oligosaccharide As the Antigen Epitope Recognized by an Anti-Sperm Auto-Monoclonal Antibody, Ts4"

    Article Title: Chemical Characterization of N-Linked Oligosaccharide As the Antigen Epitope Recognized by an Anti-Sperm Auto-Monoclonal Antibody, Ts4

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0133784

    Reactivity of lectins against proteins in the mouse testicular TS fraction co-immunoprecipitated with Ts4. The immunoprecipitated proteins from testicular TS fraction with either Ts4 or normal control IgM (n.c.) were separated by SDS-PAGE under reducing conditions. Control experiments were conducted under the same conditions except for the absence of the TS fraction (buf). The testicular TS fraction was used as a positive control (IP (-)). Proteins were electroblotted onto PVDF membranes and then probed with E-PHA (A), PSA (B), WGA (C), DSA (D), L-PHA (E), DBA (F), or SJA (G). Arrowheads indicate the lectin-reactive bands corresponding to TEX101. Mr, molecular mass.
    Figure Legend Snippet: Reactivity of lectins against proteins in the mouse testicular TS fraction co-immunoprecipitated with Ts4. The immunoprecipitated proteins from testicular TS fraction with either Ts4 or normal control IgM (n.c.) were separated by SDS-PAGE under reducing conditions. Control experiments were conducted under the same conditions except for the absence of the TS fraction (buf). The testicular TS fraction was used as a positive control (IP (-)). Proteins were electroblotted onto PVDF membranes and then probed with E-PHA (A), PSA (B), WGA (C), DSA (D), L-PHA (E), DBA (F), or SJA (G). Arrowheads indicate the lectin-reactive bands corresponding to TEX101. Mr, molecular mass.

    Techniques Used: Immunoprecipitation, SDS Page, Positive Control, Whole Genome Amplification

    Immunoreactivity of Ts4 against testicular proteins pretreated with periodic acid. The testicular TS fractions (each 5 μg protein) were loaded on a 10% gel, separated by SDS-PAGE under reducing conditions, and then blotted onto a PVDF membrane. The PVDF membrane was divided into individual lanes, which were treated with 0.075 M NaIO 4 and HIO 4 2H 2 O in PBS for various times. Specific bands were detected with Ts4 or 6035 (arrowheads). Mr, molecular mass.
    Figure Legend Snippet: Immunoreactivity of Ts4 against testicular proteins pretreated with periodic acid. The testicular TS fractions (each 5 μg protein) were loaded on a 10% gel, separated by SDS-PAGE under reducing conditions, and then blotted onto a PVDF membrane. The PVDF membrane was divided into individual lanes, which were treated with 0.075 M NaIO 4 and HIO 4 2H 2 O in PBS for various times. Specific bands were detected with Ts4 or 6035 (arrowheads). Mr, molecular mass.

    Techniques Used: SDS Page

    SDS-PAGE analyses of mouse testicular proteins immunoprecipitated with Ts4. Western blot analyses using Ts4 (A). Testicular TS proteins immunoprecipitated with either Ts4 or normal control mouse IgM (n.c.) were separated via SDS-PAGE on 10% gels under reducing conditions. Control experiments were conducted under the same conditions, but in the absence of the testicular extract (buf). Separated proteins were electroblotted onto PVDF membranes and then probed with Ts4. Arrowheads indicate molecular mass (Mr) of the specific immunoreactive bands. Visualized by CBB-staining (B). The same samples were applied to lanes of the 10% SDS-PAGE gel under reducing conditions, and then the gel was CBB-stained. Apparent positions of dominant bands obtained via immunoprecipitation with Ts4 are indicated with arrowheads.
    Figure Legend Snippet: SDS-PAGE analyses of mouse testicular proteins immunoprecipitated with Ts4. Western blot analyses using Ts4 (A). Testicular TS proteins immunoprecipitated with either Ts4 or normal control mouse IgM (n.c.) were separated via SDS-PAGE on 10% gels under reducing conditions. Control experiments were conducted under the same conditions, but in the absence of the testicular extract (buf). Separated proteins were electroblotted onto PVDF membranes and then probed with Ts4. Arrowheads indicate molecular mass (Mr) of the specific immunoreactive bands. Visualized by CBB-staining (B). The same samples were applied to lanes of the 10% SDS-PAGE gel under reducing conditions, and then the gel was CBB-stained. Apparent positions of dominant bands obtained via immunoprecipitation with Ts4 are indicated with arrowheads.

    Techniques Used: SDS Page, Immunoprecipitation, Western Blot, Staining

    4) Product Images from "Human replication protein A (RPA) binds a primer-template junction in the absence of its major ssDNA-binding domains"

    Article Title: Human replication protein A (RPA) binds a primer-template junction in the absence of its major ssDNA-binding domains

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkh346

    Identification of the low-weight crosslinking product. Western blot analysis was performed to identify the origin of the low molecular weight crosslinking product of RPA-ABCD. The reaction mixtures contained the photoreactive DNA-30 duplex structure with the ‘long’ FAP-dUMP photoreactive residue (1 µM) and RPA-ABCD (1 µM). Mixtures were preincubated for 20 min at 25°C and were either UV-irradiated (+) or not irradiated (–). Protein samples were then separated on 15% SDS–polyacrylamide gels and either silver stained (lanes 5 and 6 for non-irradiated and irradiated mixtures, respectively) or transferred to a PVDF membrane and visualized with antibodies. Lanes 1 and 3 contained non-irradiated probes, and lanes 2 and 4 contained irradiated probes preadsorbed with monoclonal antibodies specific to p32 and p14, respectively. The positions of protein markers are indicated in the left margin.
    Figure Legend Snippet: Identification of the low-weight crosslinking product. Western blot analysis was performed to identify the origin of the low molecular weight crosslinking product of RPA-ABCD. The reaction mixtures contained the photoreactive DNA-30 duplex structure with the ‘long’ FAP-dUMP photoreactive residue (1 µM) and RPA-ABCD (1 µM). Mixtures were preincubated for 20 min at 25°C and were either UV-irradiated (+) or not irradiated (–). Protein samples were then separated on 15% SDS–polyacrylamide gels and either silver stained (lanes 5 and 6 for non-irradiated and irradiated mixtures, respectively) or transferred to a PVDF membrane and visualized with antibodies. Lanes 1 and 3 contained non-irradiated probes, and lanes 2 and 4 contained irradiated probes preadsorbed with monoclonal antibodies specific to p32 and p14, respectively. The positions of protein markers are indicated in the left margin.

    Techniques Used: Western Blot, Molecular Weight, Recombinase Polymerase Amplification, Irradiation, Staining

    5) Product Images from "Sigma factor 1 in chloroplast gene transcription and photosynthetic light acclimation"

    Article Title: Sigma factor 1 in chloroplast gene transcription and photosynthetic light acclimation

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/erz464

    T-DNA insertional mutagenesis decreases SIG1 transcript and protein levels. (A) SIG1 transcript abundance in sig1-1 and sig1-2 mutants as quantified by qRT-PCR. The log2 fold change after normalization with the wild type is shown. Error bars represent ±SE of the mean of four biological replicates. (B) SIG1 protein level as estimated by immunoblotting. Representative SIG1 and actin blots are shown with the corresponding stained PVDF membrane. Both SIG1 and actin are detected on the same membrane. Numbers below each lane denote the ratio of SIG1 to actin band intensity. The percentage decreases in SIG1 relative to the wild-type control are also given. The full uncropped versions of SIG1 and actin immunoblots are given in Supplementary Fig. S5 . (C) An immunoblot of SIG1 with serial dilutions of the wild-type sample. The corresponding stained membrane is also shown. Molecular weight markers are indicated on the left. The mature SIG1 protein has a predicted mol. wt of 54 kDa. The SIG1 protein, however, runs on an 11.5% (w/v) SDS–6 M urea–PAGE gel with an apparent mol. wt of ~49 kDa.
    Figure Legend Snippet: T-DNA insertional mutagenesis decreases SIG1 transcript and protein levels. (A) SIG1 transcript abundance in sig1-1 and sig1-2 mutants as quantified by qRT-PCR. The log2 fold change after normalization with the wild type is shown. Error bars represent ±SE of the mean of four biological replicates. (B) SIG1 protein level as estimated by immunoblotting. Representative SIG1 and actin blots are shown with the corresponding stained PVDF membrane. Both SIG1 and actin are detected on the same membrane. Numbers below each lane denote the ratio of SIG1 to actin band intensity. The percentage decreases in SIG1 relative to the wild-type control are also given. The full uncropped versions of SIG1 and actin immunoblots are given in Supplementary Fig. S5 . (C) An immunoblot of SIG1 with serial dilutions of the wild-type sample. The corresponding stained membrane is also shown. Molecular weight markers are indicated on the left. The mature SIG1 protein has a predicted mol. wt of 54 kDa. The SIG1 protein, however, runs on an 11.5% (w/v) SDS–6 M urea–PAGE gel with an apparent mol. wt of ~49 kDa.

    Techniques Used: Mutagenesis, Quantitative RT-PCR, Staining, Western Blot, Molecular Weight, Polyacrylamide Gel Electrophoresis

    6) Product Images from "ADP-Ribosylation Factor 6 Expression and Activation Are Reduced in Myometrium in Complicated Pregnancies"

    Article Title: ADP-Ribosylation Factor 6 Expression and Activation Are Reduced in Myometrium in Complicated Pregnancies

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0037954

    Expression of ARF1, ARF6 and CYTH3 proteins in human myometrium. A. Immunoblot analysis of the expression of ARF6, ARF1, CYTH1-4 and tubulin proteins in human placenta and myometrium. Lanes 1, Placental lysate, and 2, NP myometrium lysate. Immunoblot analysis of ARF6 and ARF6 (B) and CYTH1-4. (C) protein expression in human myometrium of NP, NIL, SL, NP-SL and PT-NIL. The tissue lysate proteins (50 µg) were separated by SDS-PAGE, blotted onto PVDF membranes, and probed with the indicated antibody (i). The lysate of MDA-MB-231 cells was used as a positive control (+ve) where indicated. The intensity of the bands was quantified by densitometric scanning. The data were normalised to the amount of tubulin in each sample and shown as means ± SE of 4 samples in the bottom graph (ii).
    Figure Legend Snippet: Expression of ARF1, ARF6 and CYTH3 proteins in human myometrium. A. Immunoblot analysis of the expression of ARF6, ARF1, CYTH1-4 and tubulin proteins in human placenta and myometrium. Lanes 1, Placental lysate, and 2, NP myometrium lysate. Immunoblot analysis of ARF6 and ARF6 (B) and CYTH1-4. (C) protein expression in human myometrium of NP, NIL, SL, NP-SL and PT-NIL. The tissue lysate proteins (50 µg) were separated by SDS-PAGE, blotted onto PVDF membranes, and probed with the indicated antibody (i). The lysate of MDA-MB-231 cells was used as a positive control (+ve) where indicated. The intensity of the bands was quantified by densitometric scanning. The data were normalised to the amount of tubulin in each sample and shown as means ± SE of 4 samples in the bottom graph (ii).

    Techniques Used: Expressing, SDS Page, Multiple Displacement Amplification, Positive Control

    7) Product Images from "2,4-Dichlorophenoxyacetic acid promotes S-nitrosylation and oxidation of actin affecting cytoskeleton and peroxisomal dynamics"

    Article Title: 2,4-Dichlorophenoxyacetic acid promotes S-nitrosylation and oxidation of actin affecting cytoskeleton and peroxisomal dynamics

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/eru237

    Analysis of post-translational modifications of actin by carbonylation and S -nitrosylation. (A) Detection of carbonylated actin. Proteins from leaf extracts (500 μg) were derivatized with DNPH and immunoprecipitated with anti-DNP-IPA as indicated in the Materials and methods. Oxidized-purified proteins (10 μl) were subjected to SDS–PAGE, transferred onto PVDF membranes, and analysed with an anti-actin antibody. The figure is representative of four independent experiments. (B) Detection of S -nitrosylated actin. S -Nitrosylated proteins were labelled with biotin and immunopurified with anti-biotin–IPA, separated by SDS–PAGE, and the actin was identify by western blot analysis using an anti-actin antibody. The figure is representative of three independent experiments.
    Figure Legend Snippet: Analysis of post-translational modifications of actin by carbonylation and S -nitrosylation. (A) Detection of carbonylated actin. Proteins from leaf extracts (500 μg) were derivatized with DNPH and immunoprecipitated with anti-DNP-IPA as indicated in the Materials and methods. Oxidized-purified proteins (10 μl) were subjected to SDS–PAGE, transferred onto PVDF membranes, and analysed with an anti-actin antibody. The figure is representative of four independent experiments. (B) Detection of S -nitrosylated actin. S -Nitrosylated proteins were labelled with biotin and immunopurified with anti-biotin–IPA, separated by SDS–PAGE, and the actin was identify by western blot analysis using an anti-actin antibody. The figure is representative of three independent experiments.

    Techniques Used: Immunoprecipitation, Indirect Immunoperoxidase Assay, Purification, SDS Page, Western Blot

    8) Product Images from "Photoconductivity of acid exfoliated and flash-light-processed MoS2 films"

    Article Title: Photoconductivity of acid exfoliated and flash-light-processed MoS2 films

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-21688-0

    ( a ) Schematic drawing of experimental procedures. MoS 2 was first exfoliated in a pH 1.0 nitric acid solution and then collected on a PVDF membrane by filtration, followed by flash-light processing and polishing. Gold electrodes with a designed pattern were then deposited onto the film, and the photoresponse was measured under illumination. ( b ) TEM image of exfoliated MoS2 sheets. The insert is the electron diffraction pattern of the flake. ( c ) AFM image of MoS 2 sheets, ( d ) A photograph of exfoliated MoS 2 after two months of settling and the UV/Vis absorbance of exfoliated MoS 2 in water. Low ( e ) and high ( f ) magnification SEM images of MoS 2 film deposited onto a PVDF membrane after flash-light processing and polishing. ( g ) Cross-section SEM image of MoS 2 film.
    Figure Legend Snippet: ( a ) Schematic drawing of experimental procedures. MoS 2 was first exfoliated in a pH 1.0 nitric acid solution and then collected on a PVDF membrane by filtration, followed by flash-light processing and polishing. Gold electrodes with a designed pattern were then deposited onto the film, and the photoresponse was measured under illumination. ( b ) TEM image of exfoliated MoS2 sheets. The insert is the electron diffraction pattern of the flake. ( c ) AFM image of MoS 2 sheets, ( d ) A photograph of exfoliated MoS 2 after two months of settling and the UV/Vis absorbance of exfoliated MoS 2 in water. Low ( e ) and high ( f ) magnification SEM images of MoS 2 film deposited onto a PVDF membrane after flash-light processing and polishing. ( g ) Cross-section SEM image of MoS 2 film.

    Techniques Used: Filtration, Transmission Electron Microscopy

    9) Product Images from "Novel DNA Aptamers for Parkinson’s Disease Treatment Inhibit α-Synuclein Aggregation and Facilitate its Degradation"

    Article Title: Novel DNA Aptamers for Parkinson’s Disease Treatment Inhibit α-Synuclein Aggregation and Facilitate its Degradation

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2018.02.011

    The Aptamers of F5R1 and F5R2 Enhanced Lysosomal Degradation of α-syn and Rescued the Cell Defects (A) SK-N-SH cells pre-treated with F5R1, F5R2 or random DNA sequence were transfected the α-syn or vector control vectors and incubated for 24 hr. The extracts were separated by SDS-PAGE and blotted onto PVDF membrane. The membrane was blocked and probed with the α-syn specific polyclonal antibody. β-actin served as the loading control. (B) Quantitative analysis of the total protein level of α-syn from (A). (C) SK-N-SH cells were similarly treated as in (A) except for incubation time (48 hr). The cell extracts were immunoblotted with the α-syn polyclonal antibody. β-actin served as the loading control. (D) Quantitative analysis of the total protein level of α-syn from (C). Data are presented as the mean ± SD (one-way ANOVA) ***p
    Figure Legend Snippet: The Aptamers of F5R1 and F5R2 Enhanced Lysosomal Degradation of α-syn and Rescued the Cell Defects (A) SK-N-SH cells pre-treated with F5R1, F5R2 or random DNA sequence were transfected the α-syn or vector control vectors and incubated for 24 hr. The extracts were separated by SDS-PAGE and blotted onto PVDF membrane. The membrane was blocked and probed with the α-syn specific polyclonal antibody. β-actin served as the loading control. (B) Quantitative analysis of the total protein level of α-syn from (A). (C) SK-N-SH cells were similarly treated as in (A) except for incubation time (48 hr). The cell extracts were immunoblotted with the α-syn polyclonal antibody. β-actin served as the loading control. (D) Quantitative analysis of the total protein level of α-syn from (C). Data are presented as the mean ± SD (one-way ANOVA) ***p

    Techniques Used: Sequencing, Transfection, Plasmid Preparation, Incubation, SDS Page

    10) Product Images from "Scavenger receptor-C acts as a receptor for Bacillus thuringiensis vegetative insecticidal protein Vip3Aa and mediates the internalization of Vip3Aa via endocytosis"

    Article Title: Scavenger receptor-C acts as a receptor for Bacillus thuringiensis vegetative insecticidal protein Vip3Aa and mediates the internalization of Vip3Aa via endocytosis

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007347

    Vip3Aa interacts with Sf-SR-C in vitr o. (A) Confocal microscopy images of Sf9 cells treated with RFP or Vip3Aa-RFP (10 μg/mL) for 3 h. Nuclei are stained with DAPI (blue) and cell membrane are stained with FITC-phalloidin (green). Scale bar, 10 μm. (B) Bio-Vip3Aa was incubated with the extracts of Sf9 cell membrane proteins, immunoprecipitated with Streptavidin Mag Sepharose, and detected by Coomassie brilliant blue staining. (a): Lane 1, biotin labeled Vip3Aa; lane 2, the immune complexes. (b): Image of the band of Vip3Aa that was excised from lane 2. (C) The purified GST-Sf-SR-N and GST protein. (D) Vip3Aa-Flag or Cry1Ac were mixed with purified GST-Sf-SR-N, and then the associated complex was pulled down using GST-Sepharose affinity beads followed by immunoblotting (IB) with an anti-Flag antibody or anti-Cry1Ac antibody. (E) MST assay to measure the binding between Vip3Aa and GST-SR-C-N. The labelled Vip3Aa was kept constant at 10 nM, and the GST-SR-C-N is titrated from 0.3 nM to 10 μM. Fitted binding curves and Kd values (mean ± SD) were derived from three independent experiments. (F) Sf9 cells were transiently transfected with the plasmid pIZT-SR-C and the empty vector pIZT/V5-His respectively. 48 h after transfection, cells were collected for immunoblotting with anti-V5 antibody. (G) Sf9-pIZT-SR-C cells lysate was incubated with Vip3Aa-Flag or Cry1Ac, Sf-SR-C in the cells lysate was immunoprecipitated (IP) with anti-V5 antibody, Vip3Aa-Flag and Cry1Ac in the elution was detected by immunoblotting with anti-Flag antibody and anti-Cry1Ac antibody respectively. (H) The lysate of Sf9-pIZT-SR-C cells were subjected to SDS-PAGE, and then transferred to PVDF membranes. The PVDF membranes were probed with Vip3Aa-flag or with Vip3Aa-flag plus unlabeled Vip3Aa without Flag-tag (200-fold), followed by immunoblotting with an anti-Flag antibody.
    Figure Legend Snippet: Vip3Aa interacts with Sf-SR-C in vitr o. (A) Confocal microscopy images of Sf9 cells treated with RFP or Vip3Aa-RFP (10 μg/mL) for 3 h. Nuclei are stained with DAPI (blue) and cell membrane are stained with FITC-phalloidin (green). Scale bar, 10 μm. (B) Bio-Vip3Aa was incubated with the extracts of Sf9 cell membrane proteins, immunoprecipitated with Streptavidin Mag Sepharose, and detected by Coomassie brilliant blue staining. (a): Lane 1, biotin labeled Vip3Aa; lane 2, the immune complexes. (b): Image of the band of Vip3Aa that was excised from lane 2. (C) The purified GST-Sf-SR-N and GST protein. (D) Vip3Aa-Flag or Cry1Ac were mixed with purified GST-Sf-SR-N, and then the associated complex was pulled down using GST-Sepharose affinity beads followed by immunoblotting (IB) with an anti-Flag antibody or anti-Cry1Ac antibody. (E) MST assay to measure the binding between Vip3Aa and GST-SR-C-N. The labelled Vip3Aa was kept constant at 10 nM, and the GST-SR-C-N is titrated from 0.3 nM to 10 μM. Fitted binding curves and Kd values (mean ± SD) were derived from three independent experiments. (F) Sf9 cells were transiently transfected with the plasmid pIZT-SR-C and the empty vector pIZT/V5-His respectively. 48 h after transfection, cells were collected for immunoblotting with anti-V5 antibody. (G) Sf9-pIZT-SR-C cells lysate was incubated with Vip3Aa-Flag or Cry1Ac, Sf-SR-C in the cells lysate was immunoprecipitated (IP) with anti-V5 antibody, Vip3Aa-Flag and Cry1Ac in the elution was detected by immunoblotting with anti-Flag antibody and anti-Cry1Ac antibody respectively. (H) The lysate of Sf9-pIZT-SR-C cells were subjected to SDS-PAGE, and then transferred to PVDF membranes. The PVDF membranes were probed with Vip3Aa-flag or with Vip3Aa-flag plus unlabeled Vip3Aa without Flag-tag (200-fold), followed by immunoblotting with an anti-Flag antibody.

    Techniques Used: Confocal Microscopy, Staining, Incubation, Immunoprecipitation, Labeling, Purification, Microscale Thermophoresis, Binding Assay, Derivative Assay, Transfection, Plasmid Preparation, SDS Page, FLAG-tag

    11) Product Images from "Salmonella enterotoxin (Stn) regulates membrane composition and integrity"

    Article Title: Salmonella enterotoxin (Stn) regulates membrane composition and integrity

    Journal: Disease Models & Mechanisms

    doi: 10.1242/dmm.009324

    Interaction between Stn and OmpA. OmpA (1 μg/reaction) was separated by SDS-PAGE and transferred to PVDF membranes, which were soaked in the presence of purified TF-tag (10 μg; A) or purified TF-Stn (10 μg; B), and also in the absence of probe (C) at 4°C for 16 hours. Interaction of TF-Stn and OmpA was detected with anti-TF antibody. Asterisks indicate the non-specific signals with anti-TF antibody and arrow is the specific signal that formed as a result of the OmpA-Stn complex. Panel C was performed as a control reaction for the verification of antibody quality. TF, trigger factor-tag used as a negative control.
    Figure Legend Snippet: Interaction between Stn and OmpA. OmpA (1 μg/reaction) was separated by SDS-PAGE and transferred to PVDF membranes, which were soaked in the presence of purified TF-tag (10 μg; A) or purified TF-Stn (10 μg; B), and also in the absence of probe (C) at 4°C for 16 hours. Interaction of TF-Stn and OmpA was detected with anti-TF antibody. Asterisks indicate the non-specific signals with anti-TF antibody and arrow is the specific signal that formed as a result of the OmpA-Stn complex. Panel C was performed as a control reaction for the verification of antibody quality. TF, trigger factor-tag used as a negative control.

    Techniques Used: SDS Page, Purification, Negative Control

    12) Product Images from "Marburg virus-like particles by co-expression of glycoprotein and matrix protein in insect cells induces immune responses in mice"

    Article Title: Marburg virus-like particles by co-expression of glycoprotein and matrix protein in insect cells induces immune responses in mice

    Journal: Virology Journal

    doi: 10.1186/s12985-017-0869-3

    Western blot analysis of GP and VP40 protein expression in the MARV VLPs. 10 μg of MARV VLPs by co-expression of GP and VP40 were mixed with reducing (with β-mercaptoethanol) protein sample buffer, heated at 95 °C for 5 min, and then subjected to 10% SDS-PAGE with different gels. Two different gels were transferred onto a polyvinylidene fluoride (PVDF) membrane for the Western blot analysis, respectively. a GP was incubated with mouse anti-MARV GP polyclonal antibody (control, lane 1; MARV VLPs, lane 2) and the molecular weight were approximately 150KD. b VP40 proteins was incubated with mouse anti-MARV VP40 polyclonal antibody (MARV VLPs, lane 1; control, lane 2) and the molecular weight were approximately 38KD
    Figure Legend Snippet: Western blot analysis of GP and VP40 protein expression in the MARV VLPs. 10 μg of MARV VLPs by co-expression of GP and VP40 were mixed with reducing (with β-mercaptoethanol) protein sample buffer, heated at 95 °C for 5 min, and then subjected to 10% SDS-PAGE with different gels. Two different gels were transferred onto a polyvinylidene fluoride (PVDF) membrane for the Western blot analysis, respectively. a GP was incubated with mouse anti-MARV GP polyclonal antibody (control, lane 1; MARV VLPs, lane 2) and the molecular weight were approximately 150KD. b VP40 proteins was incubated with mouse anti-MARV VP40 polyclonal antibody (MARV VLPs, lane 1; control, lane 2) and the molecular weight were approximately 38KD

    Techniques Used: Western Blot, Expressing, SDS Page, Incubation, Molecular Weight

    Western blot analysis of GP and VP40 protein expression in the purified MARV VLPs. 10 μg of the purified MARV VLPs were mixed with reducing (with β-mercaptoethanol) protein sample buffer, heated at 95 °C for 5 min, and then subjected to 10% SDS-PAGE with different gels. Two different gels were transferred onto a polyvinylidene fluoride (PVDF) membrane for the Western blot analysis, respectively. a GP was incubated with mouse anti-MARV GP polyclonal antibody (purified MARV VLPs, lane 1; control, lane 2). b VP40 proteins was incubated with mouse anti-MARV VP40 polyclonal antibody (purified MARV VLPs, lane 1; control, lane 2)
    Figure Legend Snippet: Western blot analysis of GP and VP40 protein expression in the purified MARV VLPs. 10 μg of the purified MARV VLPs were mixed with reducing (with β-mercaptoethanol) protein sample buffer, heated at 95 °C for 5 min, and then subjected to 10% SDS-PAGE with different gels. Two different gels were transferred onto a polyvinylidene fluoride (PVDF) membrane for the Western blot analysis, respectively. a GP was incubated with mouse anti-MARV GP polyclonal antibody (purified MARV VLPs, lane 1; control, lane 2). b VP40 proteins was incubated with mouse anti-MARV VP40 polyclonal antibody (purified MARV VLPs, lane 1; control, lane 2)

    Techniques Used: Western Blot, Expressing, Purification, SDS Page, Incubation

    13) Product Images from "Scavenger receptor-C acts as a receptor for Bacillus thuringiensis vegetative insecticidal protein Vip3Aa and mediates the internalization of Vip3Aa via endocytosis"

    Article Title: Scavenger receptor-C acts as a receptor for Bacillus thuringiensis vegetative insecticidal protein Vip3Aa and mediates the internalization of Vip3Aa via endocytosis

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007347

    Vip3Aa interacts with Sf-SR-C in vitr o. (A) Confocal microscopy images of Sf9 cells treated with RFP or Vip3Aa-RFP (10 μg/mL) for 3 h. Nuclei are stained with DAPI (blue) and cell membrane are stained with FITC-phalloidin (green). Scale bar, 10 μm. (B) Bio-Vip3Aa was incubated with the extracts of Sf9 cell membrane proteins, immunoprecipitated with Streptavidin Mag Sepharose, and detected by Coomassie brilliant blue staining. (a): Lane 1, biotin labeled Vip3Aa; lane 2, the immune complexes. (b): Image of the band of Vip3Aa that was excised from lane 2. (C) The purified GST-Sf-SR-N and GST protein. (D) Vip3Aa-Flag or Cry1Ac were mixed with purified GST-Sf-SR-N, and then the associated complex was pulled down using GST-Sepharose affinity beads followed by immunoblotting (IB) with an anti-Flag antibody or anti-Cry1Ac antibody. (E) MST assay to measure the binding between Vip3Aa and GST-SR-C-N. The labelled Vip3Aa was kept constant at 10 nM, and the GST-SR-C-N is titrated from 0.3 nM to 10 μM. Fitted binding curves and Kd values (mean ± SD) were derived from three independent experiments. (F) Sf9 cells were transiently transfected with the plasmid pIZT-SR-C and the empty vector pIZT/V5-His respectively. 48 h after transfection, cells were collected for immunoblotting with anti-V5 antibody. (G) Sf9-pIZT-SR-C cells lysate was incubated with Vip3Aa-Flag or Cry1Ac, Sf-SR-C in the cells lysate was immunoprecipitated (IP) with anti-V5 antibody, Vip3Aa-Flag and Cry1Ac in the elution was detected by immunoblotting with anti-Flag antibody and anti-Cry1Ac antibody respectively. (H) The lysate of Sf9-pIZT-SR-C cells were subjected to SDS-PAGE, and then transferred to PVDF membranes. The PVDF membranes were probed with Vip3Aa-flag or with Vip3Aa-flag plus unlabeled Vip3Aa without Flag-tag (200-fold), followed by immunoblotting with an anti-Flag antibody.
    Figure Legend Snippet: Vip3Aa interacts with Sf-SR-C in vitr o. (A) Confocal microscopy images of Sf9 cells treated with RFP or Vip3Aa-RFP (10 μg/mL) for 3 h. Nuclei are stained with DAPI (blue) and cell membrane are stained with FITC-phalloidin (green). Scale bar, 10 μm. (B) Bio-Vip3Aa was incubated with the extracts of Sf9 cell membrane proteins, immunoprecipitated with Streptavidin Mag Sepharose, and detected by Coomassie brilliant blue staining. (a): Lane 1, biotin labeled Vip3Aa; lane 2, the immune complexes. (b): Image of the band of Vip3Aa that was excised from lane 2. (C) The purified GST-Sf-SR-N and GST protein. (D) Vip3Aa-Flag or Cry1Ac were mixed with purified GST-Sf-SR-N, and then the associated complex was pulled down using GST-Sepharose affinity beads followed by immunoblotting (IB) with an anti-Flag antibody or anti-Cry1Ac antibody. (E) MST assay to measure the binding between Vip3Aa and GST-SR-C-N. The labelled Vip3Aa was kept constant at 10 nM, and the GST-SR-C-N is titrated from 0.3 nM to 10 μM. Fitted binding curves and Kd values (mean ± SD) were derived from three independent experiments. (F) Sf9 cells were transiently transfected with the plasmid pIZT-SR-C and the empty vector pIZT/V5-His respectively. 48 h after transfection, cells were collected for immunoblotting with anti-V5 antibody. (G) Sf9-pIZT-SR-C cells lysate was incubated with Vip3Aa-Flag or Cry1Ac, Sf-SR-C in the cells lysate was immunoprecipitated (IP) with anti-V5 antibody, Vip3Aa-Flag and Cry1Ac in the elution was detected by immunoblotting with anti-Flag antibody and anti-Cry1Ac antibody respectively. (H) The lysate of Sf9-pIZT-SR-C cells were subjected to SDS-PAGE, and then transferred to PVDF membranes. The PVDF membranes were probed with Vip3Aa-flag or with Vip3Aa-flag plus unlabeled Vip3Aa without Flag-tag (200-fold), followed by immunoblotting with an anti-Flag antibody.

    Techniques Used: Confocal Microscopy, Staining, Incubation, Immunoprecipitation, Labeling, Purification, Microscale Thermophoresis, Binding Assay, Derivative Assay, Transfection, Plasmid Preparation, SDS Page, FLAG-tag

    14) Product Images from "Cell Membrane Expression of Cardiac Sodium Channel Nav1.5 Is Modulated by ?-Actinin-2 Interaction †"

    Article Title: Cell Membrane Expression of Cardiac Sodium Channel Nav1.5 Is Modulated by ?-Actinin-2 Interaction †

    Journal: Biochemistry

    doi: 10.1021/bi901086v

    α-Actinin-2 and Na v 1.5/LIII–IV interacted in vitro . (A) Blot overlay assay showing the in vitro interaction of α-actinin-2 with the His 6 -LIII–IV fusion protein. Increasing amounts (15, 25, and 45 µg) of cDNA encoding α-actinin-2 transiently expressed in tsA201 cells were separated by SDS–PAGE and transferred to PVDF membranes. The blots were used to detect the expression of α-actinin-2 proteins (whole cell extracts, bottom panel) or were incubated with either 1 µg/mL purified LIII–IV fusion protein (overlay assay, top panel) or 1 µg/mL BSA (negative control, middle panel) followed by anti-LIII–IV (top and middle panels) or anti-α-actinin-2 antibody (bottom panel). The LIII–IV fusion protein bound specifically to α-actinin-2 in a dose-dependent manner (top panel, lanes 1–3). No binding was detected in the control lane containing extracts from untransfected tsA201 cells (top panel, lane 4) or in the blot incubated with BSA (middle panel). (B) A His pulldown experiment was performed as described in Materials and Methods. tsA201 cell extracts transiently expressing α-actinin-2 were incubated with either His 6 -LIII–IV fusion protein prebound to Ni 2+ -NTA beads or Ni 2+ -NTA beads alone (negative control). After being extensively washed, bound proteins were eluted and separated via 10% SDS–PAGE which were then blotted with anti-α-actinin-2 antibody (top panel) or anti-LIII–IV antibody (bottom panel): lanes 1 and 2, extract starting material; lane 3, His 6 -LIII–IV fusion protein retained from extract; and lane 4, Ni 2+ -NTA beads alone (negative control). The asterisk indicates that the band detected in lane 3 may be a degradation product of the His 6 -LIII–IV fusion protein. These experiments were repeated four times with similar results.
    Figure Legend Snippet: α-Actinin-2 and Na v 1.5/LIII–IV interacted in vitro . (A) Blot overlay assay showing the in vitro interaction of α-actinin-2 with the His 6 -LIII–IV fusion protein. Increasing amounts (15, 25, and 45 µg) of cDNA encoding α-actinin-2 transiently expressed in tsA201 cells were separated by SDS–PAGE and transferred to PVDF membranes. The blots were used to detect the expression of α-actinin-2 proteins (whole cell extracts, bottom panel) or were incubated with either 1 µg/mL purified LIII–IV fusion protein (overlay assay, top panel) or 1 µg/mL BSA (negative control, middle panel) followed by anti-LIII–IV (top and middle panels) or anti-α-actinin-2 antibody (bottom panel). The LIII–IV fusion protein bound specifically to α-actinin-2 in a dose-dependent manner (top panel, lanes 1–3). No binding was detected in the control lane containing extracts from untransfected tsA201 cells (top panel, lane 4) or in the blot incubated with BSA (middle panel). (B) A His pulldown experiment was performed as described in Materials and Methods. tsA201 cell extracts transiently expressing α-actinin-2 were incubated with either His 6 -LIII–IV fusion protein prebound to Ni 2+ -NTA beads or Ni 2+ -NTA beads alone (negative control). After being extensively washed, bound proteins were eluted and separated via 10% SDS–PAGE which were then blotted with anti-α-actinin-2 antibody (top panel) or anti-LIII–IV antibody (bottom panel): lanes 1 and 2, extract starting material; lane 3, His 6 -LIII–IV fusion protein retained from extract; and lane 4, Ni 2+ -NTA beads alone (negative control). The asterisk indicates that the band detected in lane 3 may be a degradation product of the His 6 -LIII–IV fusion protein. These experiments were repeated four times with similar results.

    Techniques Used: In Vitro, Overlay Assay, SDS Page, Expressing, Incubation, Purification, Negative Control, Binding Assay

    15) Product Images from "Photoconductivity of acid exfoliated and flash-light-processed MoS2 films"

    Article Title: Photoconductivity of acid exfoliated and flash-light-processed MoS2 films

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-21688-0

    ( a ) Schematic drawing of experimental procedures. MoS 2 was first exfoliated in a pH 1.0 nitric acid solution and then collected on a PVDF membrane by filtration, followed by flash-light processing and polishing. Gold electrodes with a designed pattern were then deposited onto the film, and the photoresponse was measured under illumination. ( b ) TEM image of exfoliated MoS2 sheets. The insert is the electron diffraction pattern of the flake. ( c ) AFM image of MoS 2 sheets, ( d ) A photograph of exfoliated MoS 2 after two months of settling and the UV/Vis absorbance of exfoliated MoS 2 in water. Low ( e ) and high ( f ) magnification SEM images of MoS 2 film deposited onto a PVDF membrane after flash-light processing and polishing. ( g ) Cross-section SEM image of MoS 2 film.
    Figure Legend Snippet: ( a ) Schematic drawing of experimental procedures. MoS 2 was first exfoliated in a pH 1.0 nitric acid solution and then collected on a PVDF membrane by filtration, followed by flash-light processing and polishing. Gold electrodes with a designed pattern were then deposited onto the film, and the photoresponse was measured under illumination. ( b ) TEM image of exfoliated MoS2 sheets. The insert is the electron diffraction pattern of the flake. ( c ) AFM image of MoS 2 sheets, ( d ) A photograph of exfoliated MoS 2 after two months of settling and the UV/Vis absorbance of exfoliated MoS 2 in water. Low ( e ) and high ( f ) magnification SEM images of MoS 2 film deposited onto a PVDF membrane after flash-light processing and polishing. ( g ) Cross-section SEM image of MoS 2 film.

    Techniques Used: Filtration, Transmission Electron Microscopy

    16) Product Images from "Novel DNA Aptamers for Parkinson’s Disease Treatment Inhibit α-Synuclein Aggregation and Facilitate its Degradation"

    Article Title: Novel DNA Aptamers for Parkinson’s Disease Treatment Inhibit α-Synuclein Aggregation and Facilitate its Degradation

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2018.02.011

    The Aptamers of F5R1 and F5R2 Enhanced Lysosomal Degradation of α-syn and Rescued the Cell Defects (A) SK-N-SH cells pre-treated with F5R1, F5R2 or random DNA sequence were transfected the α-syn or vector control vectors and incubated for 24 hr. The extracts were separated by SDS-PAGE and blotted onto PVDF membrane. The membrane was blocked and probed with the α-syn specific polyclonal antibody. β-actin served as the loading control. (B) Quantitative analysis of the total protein level of α-syn from (A). (C) SK-N-SH cells were similarly treated as in (A) except for incubation time (48 hr). The cell extracts were immunoblotted with the α-syn polyclonal antibody. β-actin served as the loading control. (D) Quantitative analysis of the total protein level of α-syn from (C). Data are presented as the mean ± SD (one-way ANOVA) ***p
    Figure Legend Snippet: The Aptamers of F5R1 and F5R2 Enhanced Lysosomal Degradation of α-syn and Rescued the Cell Defects (A) SK-N-SH cells pre-treated with F5R1, F5R2 or random DNA sequence were transfected the α-syn or vector control vectors and incubated for 24 hr. The extracts were separated by SDS-PAGE and blotted onto PVDF membrane. The membrane was blocked and probed with the α-syn specific polyclonal antibody. β-actin served as the loading control. (B) Quantitative analysis of the total protein level of α-syn from (A). (C) SK-N-SH cells were similarly treated as in (A) except for incubation time (48 hr). The cell extracts were immunoblotted with the α-syn polyclonal antibody. β-actin served as the loading control. (D) Quantitative analysis of the total protein level of α-syn from (C). Data are presented as the mean ± SD (one-way ANOVA) ***p

    Techniques Used: Sequencing, Transfection, Plasmid Preparation, Incubation, SDS Page

    17) Product Images from "Investigation of Copper Cysteamine Nanoparticles as a New Type of Radiosensitiers for Colorectal Carcinoma Treatment"

    Article Title: Investigation of Copper Cysteamine Nanoparticles as a New Type of Radiosensitiers for Colorectal Carcinoma Treatment

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-09375-y

    The protein expression levels of Bax, Bcl-2, LC3B, and P62 was detected by western blot analysis. SW620 cells were seeded into a 6-well plate at a density of 5 × 10 5 cells per well, cultured overnight and treated with or without Cu-Cy-PDT. The cells were lysed for 30 minutes in 1 × RIPA buffer that contained protease and phosphatase inhibitors. Samples containing equal amounts of protein (25 μg) were resolved on SDS-PAGE in a 10–15% gel and then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was then blocked in 5% non-fat milk for 2 h at room temperature, incubated with primary antibody Bax, Bcl-2, ATG7, LC3B, and Actin antibody (Cell Signaling, Danvers, MA, USA) overnight at 4 °C, washed with TBST, and incubated with a secondary antibody for 2 h at room temperature. The immunoblots of the incubated membranes were visualized with an enhanced chemiluminescence (ECL) Kit (CW Bio).
    Figure Legend Snippet: The protein expression levels of Bax, Bcl-2, LC3B, and P62 was detected by western blot analysis. SW620 cells were seeded into a 6-well plate at a density of 5 × 10 5 cells per well, cultured overnight and treated with or without Cu-Cy-PDT. The cells were lysed for 30 minutes in 1 × RIPA buffer that contained protease and phosphatase inhibitors. Samples containing equal amounts of protein (25 μg) were resolved on SDS-PAGE in a 10–15% gel and then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was then blocked in 5% non-fat milk for 2 h at room temperature, incubated with primary antibody Bax, Bcl-2, ATG7, LC3B, and Actin antibody (Cell Signaling, Danvers, MA, USA) overnight at 4 °C, washed with TBST, and incubated with a secondary antibody for 2 h at room temperature. The immunoblots of the incubated membranes were visualized with an enhanced chemiluminescence (ECL) Kit (CW Bio).

    Techniques Used: Expressing, Western Blot, Cell Culture, SDS Page, Incubation

    18) Product Images from "Hepatitis B spliced protein (HBSP) promotes the carcinogenic effects of benzo [alpha] pyrene by interacting with microsomal epoxide hydrolase and enhancing its hydrolysis activity"

    Article Title: Hepatitis B spliced protein (HBSP) promotes the carcinogenic effects of benzo [alpha] pyrene by interacting with microsomal epoxide hydrolase and enhancing its hydrolysis activity

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-14-282

    Effects of HBSP on the proliferation of B [alpha] P- treated Huh- 7 hepatoma cells. (A) HBSP expression in Huh-7/HBSP/B[alpha]P, Huh-7/GFP/B[alpha]P or Huh-7/HBSP. 30 μg of cellular proteins were subjected to 12% SDS-PAGE, transfered to a PVDF membrane, and probed with anti-mEH. β-actin served as a loading control. (B) Detection of BPDE-DNA in Huh-7/HBSP/B[alpha]P, Huh-7/GFP/B[alpha]P or Huh-7/HBSP cells. Cells on sterilized glass coverslips were detected for BPDE-DNA by immunocytochemistry assay. Images were taken at × 400 magnification. (C) Cell proliferation of Huh-7/HBSP/B[alpha]P, Huh-7/GFP/B[alpha]P, Huh-7/HBSP or Huh-7/GFP cells. Cells were seeded in 96-well plates at 2 × 10 3 /well, cell proliferation was determined daily in triplicate for 9 days by BrdU assay. The optical density (OD) was measured at 450 nm using a microplate reader. The analyses were repeated three times, and the results were expressed as mean ± SD.
    Figure Legend Snippet: Effects of HBSP on the proliferation of B [alpha] P- treated Huh- 7 hepatoma cells. (A) HBSP expression in Huh-7/HBSP/B[alpha]P, Huh-7/GFP/B[alpha]P or Huh-7/HBSP. 30 μg of cellular proteins were subjected to 12% SDS-PAGE, transfered to a PVDF membrane, and probed with anti-mEH. β-actin served as a loading control. (B) Detection of BPDE-DNA in Huh-7/HBSP/B[alpha]P, Huh-7/GFP/B[alpha]P or Huh-7/HBSP cells. Cells on sterilized glass coverslips were detected for BPDE-DNA by immunocytochemistry assay. Images were taken at × 400 magnification. (C) Cell proliferation of Huh-7/HBSP/B[alpha]P, Huh-7/GFP/B[alpha]P, Huh-7/HBSP or Huh-7/GFP cells. Cells were seeded in 96-well plates at 2 × 10 3 /well, cell proliferation was determined daily in triplicate for 9 days by BrdU assay. The optical density (OD) was measured at 450 nm using a microplate reader. The analyses were repeated three times, and the results were expressed as mean ± SD.

    Techniques Used: Expressing, SDS Page, Immunocytochemistry, BrdU Staining

    19) Product Images from "Phosphorylation of bamboo mosaic virus satellite RNA (satBaMV)-encoded protein P20 downregulates the formation of satBaMV-P20 ribonucleoprotein complex"

    Article Title: Phosphorylation of bamboo mosaic virus satellite RNA (satBaMV)-encoded protein P20 downregulates the formation of satBaMV-P20 ribonucleoprotein complex

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr705

    Protein interactions of wild-type and mutant rP20. ( A ) Overlay assay. Purified WT, S11A or S11D rP20 was dot-blotted onto a PVDF membrane. After blocking with 1% BSA (w/v) in 140 mM NaCl, 10 mM Tris–HCl, pH 7.4, 2 mM EDTA, 0.1% Tween 20 (v/v) and 2 mM DTT at room temperature, the membrane was overlayed at 4°C with 35 [S]-Met-labeled WT, S11A or S11D translated in vitro. After washing the membrane in Tris-buffered saline buffer containing 0.05% Tween 20 (v/v), protein interactions were detected by autoradiography. Bovine serum albumin was used as a control. ( B ) Glutaraldehyde cross-linking assay. Total protein extracted from N. benthamiana leaves co-infected with BaMV and WT, S11A or S11D satBaMV was cross-linked with 0.025% glutaraldehyde (v/v) for indicated times at 30°C, resolved on 12.5% SDS–PAGE and immunodetected with anti-P20 serum.
    Figure Legend Snippet: Protein interactions of wild-type and mutant rP20. ( A ) Overlay assay. Purified WT, S11A or S11D rP20 was dot-blotted onto a PVDF membrane. After blocking with 1% BSA (w/v) in 140 mM NaCl, 10 mM Tris–HCl, pH 7.4, 2 mM EDTA, 0.1% Tween 20 (v/v) and 2 mM DTT at room temperature, the membrane was overlayed at 4°C with 35 [S]-Met-labeled WT, S11A or S11D translated in vitro. After washing the membrane in Tris-buffered saline buffer containing 0.05% Tween 20 (v/v), protein interactions were detected by autoradiography. Bovine serum albumin was used as a control. ( B ) Glutaraldehyde cross-linking assay. Total protein extracted from N. benthamiana leaves co-infected with BaMV and WT, S11A or S11D satBaMV was cross-linked with 0.025% glutaraldehyde (v/v) for indicated times at 30°C, resolved on 12.5% SDS–PAGE and immunodetected with anti-P20 serum.

    Techniques Used: Mutagenesis, Overlay Assay, Purification, Blocking Assay, Labeling, In Vitro, Autoradiography, Infection, SDS Page

    20) Product Images from "The Involvement of Wheat F-Box Protein Gene TaFBA1 in the Oxidative Stress Tolerance of Plants"

    Article Title: The Involvement of Wheat F-Box Protein Gene TaFBA1 in the Oxidative Stress Tolerance of Plants

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0122117

    Expression of TaFBA1 in wheat under MV-induced oxidative stress. Wheat seedlings with one leaf were subjected to 10-μM MV treatments. Seedlings treated with sterile water were chosen as controls. Seedlings were harvested at different time points for analysis. (A) Expression of TaFBA1 at the mRNA transcript level in shoots, as shown by qPCR; tubulin cDNA was used as a control reference; (B) Expression of TaFBA1 at the protein level in shoots as shown by western blot. After 12.5% SDS-PAGE, protein samples were electro-transferred onto a PVDF membrane and probed with the TaFBA1 antibody produced in our laboratory. The Rubisco large subunit was used as a loading control.
    Figure Legend Snippet: Expression of TaFBA1 in wheat under MV-induced oxidative stress. Wheat seedlings with one leaf were subjected to 10-μM MV treatments. Seedlings treated with sterile water were chosen as controls. Seedlings were harvested at different time points for analysis. (A) Expression of TaFBA1 at the mRNA transcript level in shoots, as shown by qPCR; tubulin cDNA was used as a control reference; (B) Expression of TaFBA1 at the protein level in shoots as shown by western blot. After 12.5% SDS-PAGE, protein samples were electro-transferred onto a PVDF membrane and probed with the TaFBA1 antibody produced in our laboratory. The Rubisco large subunit was used as a loading control.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, SDS Page, Produced

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    Article Snippet: .. The cell lysates were prepared, ran on SDS-PAGE, transferred to PVDF membrane, and incubated with monoclonal anti-GFP antibody (1:10,000) (Sigma) or anti-myc antibody (1:1000) (Santa Cruz). .. Membranes were washed with TBS buffer and incubated with corresponding secondary antibody (Santa Cruz) tagged with horseradish peroxidase for 1 h. Proteins were visualized with enhanced chemiluminescence (ECL) reagents (Cell Signaling).

    Article Title: The inhibition of the apoptosis pathway by the Coxiella burnetii effector protein CaeA requires the EK repetition motif, but is independent of survivin
    Article Snippet: .. After 6h incubation at 37°C in 5%CO2 samples were separated by SDS-PAGE and transferred to a PVDF membrane (Millipore). .. The membranes were probed with antibodies directed against Bcl-2 (2870), Bcl-xL (2764), Mcl-1 (4572), cleaved caspase 7 (9491), cleaved caspase 9 (9501), XIAP (2045) and survivin (2808) all from Cell Signaling, cleaved PARP (611038, BD Biosciences) and actin (A2066, Sigma-Aldrich).

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    Millipore millipore pvdf membrane
    Dependence of thermally on-off ratio on the cross-linker amounts in the range from 0.1-2.0 mol% in for <t>PNIPAAm-PVDF</t> <t>Millipore</t> membrane (P=1.4 bar). For all the membranes, the NIPAAm concentration for polymerization solution was 5 wt%. Data was corrected with viscosity and normalized by permeability at 30°C.
    Millipore Pvdf Membrane, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 358 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Dependence of thermally on-off ratio on the cross-linker amounts in the range from 0.1-2.0 mol% in for PNIPAAm-PVDF Millipore membrane (P=1.4 bar). For all the membranes, the NIPAAm concentration for polymerization solution was 5 wt%. Data was corrected with viscosity and normalized by permeability at 30°C.

    Journal: Journal of membrane science

    Article Title: Development of Bench and Full-Scale Temperature and pH Responsive Functionalized PVDF Membranes with Tunable Properties

    doi: 10.1016/j.memsci.2014.01.033

    Figure Lengend Snippet: Dependence of thermally on-off ratio on the cross-linker amounts in the range from 0.1-2.0 mol% in for PNIPAAm-PVDF Millipore membrane (P=1.4 bar). For all the membranes, the NIPAAm concentration for polymerization solution was 5 wt%. Data was corrected with viscosity and normalized by permeability at 30°C.

    Article Snippet: It should be noted that unlike the Millipore PVDF membrane, the functionalization of full-scale membrane was by dip-coating method.

    Techniques: Concentration Assay, Permeability

    ATR-FTIR spectrum of blank PVDF, PNIPAAm functionalized PVDF Millipore membrane and PNIPAAm-FPAA-PVDFHE Sepro membrane.

    Journal: Journal of membrane science

    Article Title: Development of Bench and Full-Scale Temperature and pH Responsive Functionalized PVDF Membranes with Tunable Properties

    doi: 10.1016/j.memsci.2014.01.033

    Figure Lengend Snippet: ATR-FTIR spectrum of blank PVDF, PNIPAAm functionalized PVDF Millipore membrane and PNIPAAm-FPAA-PVDFHE Sepro membrane.

    Article Snippet: It should be noted that unlike the Millipore PVDF membrane, the functionalization of full-scale membrane was by dip-coating method.

    Techniques:

    Dynamic and Reversible flux response versus ramp change in feed temperature above LCST and below LCST through PNIPAAm-PVDF Millipore membrane at P = 1.4 bar. The inset is the experimental temperature step change approximation. For the polymerization, the NIPAAm concentration was 5 wt%, cross-linker was 0.1mol%.

    Journal: Journal of membrane science

    Article Title: Development of Bench and Full-Scale Temperature and pH Responsive Functionalized PVDF Membranes with Tunable Properties

    doi: 10.1016/j.memsci.2014.01.033

    Figure Lengend Snippet: Dynamic and Reversible flux response versus ramp change in feed temperature above LCST and below LCST through PNIPAAm-PVDF Millipore membrane at P = 1.4 bar. The inset is the experimental temperature step change approximation. For the polymerization, the NIPAAm concentration was 5 wt%, cross-linker was 0.1mol%.

    Article Snippet: It should be noted that unlike the Millipore PVDF membrane, the functionalization of full-scale membrane was by dip-coating method.

    Techniques: Concentration Assay

    The effects of temperature and monomer concentration on dextran rejection with PNIPAAm-PVDF Millipore membrane (5mol% cross-linker) (Mw=2,000,000 g/mol; Stokes radius r s = 26.1nm, calculated from r s =0.27·M w 0.498 ).

    Journal: Journal of membrane science

    Article Title: Development of Bench and Full-Scale Temperature and pH Responsive Functionalized PVDF Membranes with Tunable Properties

    doi: 10.1016/j.memsci.2014.01.033

    Figure Lengend Snippet: The effects of temperature and monomer concentration on dextran rejection with PNIPAAm-PVDF Millipore membrane (5mol% cross-linker) (Mw=2,000,000 g/mol; Stokes radius r s = 26.1nm, calculated from r s =0.27·M w 0.498 ).

    Article Snippet: It should be noted that unlike the Millipore PVDF membrane, the functionalization of full-scale membrane was by dip-coating method.

    Techniques: Concentration Assay

    SEM images of blank PVDF (A) and PNIPAAm-PVDF Millipore membranes (B: 25°C(below LCST); C: 40 °C (above LCST))

    Journal: Journal of membrane science

    Article Title: Development of Bench and Full-Scale Temperature and pH Responsive Functionalized PVDF Membranes with Tunable Properties

    doi: 10.1016/j.memsci.2014.01.033

    Figure Lengend Snippet: SEM images of blank PVDF (A) and PNIPAAm-PVDF Millipore membranes (B: 25°C(below LCST); C: 40 °C (above LCST))

    Article Snippet: It should be noted that unlike the Millipore PVDF membrane, the functionalization of full-scale membrane was by dip-coating method.

    Techniques:

    Effect of monomer (NIPAAm) concentration on water flux at 1.4 bar and calculated effective pore size for PNIPAAm-PVDF Millipore membrane. cross-linker concentration = 1mol%.

    Journal: Journal of membrane science

    Article Title: Development of Bench and Full-Scale Temperature and pH Responsive Functionalized PVDF Membranes with Tunable Properties

    doi: 10.1016/j.memsci.2014.01.033

    Figure Lengend Snippet: Effect of monomer (NIPAAm) concentration on water flux at 1.4 bar and calculated effective pore size for PNIPAAm-PVDF Millipore membrane. cross-linker concentration = 1mol%.

    Article Snippet: It should be noted that unlike the Millipore PVDF membrane, the functionalization of full-scale membrane was by dip-coating method.

    Techniques: Concentration Assay

    p27 BBP/eIF6 is present in all cell lines, and in a cytoskeletal associated pool. (Left) 30 μg of total protein extracts from established cell lines and primary cultures were run on 12% acrylamide gels, transferred on immobilon-P membranes and blotted with the p27 BBP/eIF6 antiserum, followed by ECL detection. The arrow points at the p27 BBP/eIF6 band. The cell lines were mouse NIH/3T3 fibroblasts, human A431 epidermoid carcinoma, human HeLa epitheloid carcinoma, human pancreatic carcinoma FG2, human Jurkat T cells, transformed human keratinocytes HaCaT, human insulinoma cells Rin2A, and human neuroblastoma SK-N-MC. Primary cultures were mouse resting splenocytes and human fibroblasts. (Right) p27 BBP/eIF6 in Xenopus oocytes extracted either in Laemmli buffer (total), or according to the nuclear matrix/intermediate filaments procedure of He et al. (1990, see Materials and Methods). The lane marked soluble contains cytosolic proteins extracted with an isotonic Triton X-100 buffer. The lane marked insoluble contains nuclear matrix/intermediate filament-associated proteins left after sequential extraction with ammonium sulphate, nuclease, and high salt.

    Journal: The Journal of Cell Biology

    Article Title: The ?4 Integrin Interactor p27BBP/eIF6 Is an Essential Nuclear Matrix Protein Involved in 60S Ribosomal Subunit Assembly

    doi:

    Figure Lengend Snippet: p27 BBP/eIF6 is present in all cell lines, and in a cytoskeletal associated pool. (Left) 30 μg of total protein extracts from established cell lines and primary cultures were run on 12% acrylamide gels, transferred on immobilon-P membranes and blotted with the p27 BBP/eIF6 antiserum, followed by ECL detection. The arrow points at the p27 BBP/eIF6 band. The cell lines were mouse NIH/3T3 fibroblasts, human A431 epidermoid carcinoma, human HeLa epitheloid carcinoma, human pancreatic carcinoma FG2, human Jurkat T cells, transformed human keratinocytes HaCaT, human insulinoma cells Rin2A, and human neuroblastoma SK-N-MC. Primary cultures were mouse resting splenocytes and human fibroblasts. (Right) p27 BBP/eIF6 in Xenopus oocytes extracted either in Laemmli buffer (total), or according to the nuclear matrix/intermediate filaments procedure of He et al. (1990, see Materials and Methods). The lane marked soluble contains cytosolic proteins extracted with an isotonic Triton X-100 buffer. The lane marked insoluble contains nuclear matrix/intermediate filament-associated proteins left after sequential extraction with ammonium sulphate, nuclease, and high salt.

    Article Snippet: Western Blot Analysis All samples were denatured before loading in Laemmli buffer ( ) and run on denaturing 12% SDS–acrylamide gel, transferred to Immobilon P membranes ( Millipore Corp. ), and blotted with the rabbit p27BBP/eIF6 antiserum at 1:1,000 dilution as previously described ( ).

    Techniques: Transformation Assay

    p27 BBP/eIF6 cosediments with 60S and 80S ribosomal subunits. Fractions collected after the sucrose gradient centrifugation (top) of extracts from strain ySP664 ( iih1 Δ GAL-Hsp27 BBP/eIF6 ), logarithmically growing in galactose-containing medium, were precipitated by TCA, and equal amounts of protein extracts were run on 12% acrylamide gels, transferred on immobilon-P membranes and probed with the anti–p27 BBP/eIF6 antiserum, followed by the ECL detection method (bottom). Note that p27 BBP/eIF6 (arrow) is highly enriched in the free 60S and 80S fractions, but clearly absent from the polysome fraction. The high molecular weight band in lane 1 is a nonspecific product recognized by secondary antibodies.

    Journal: The Journal of Cell Biology

    Article Title: The ?4 Integrin Interactor p27BBP/eIF6 Is an Essential Nuclear Matrix Protein Involved in 60S Ribosomal Subunit Assembly

    doi:

    Figure Lengend Snippet: p27 BBP/eIF6 cosediments with 60S and 80S ribosomal subunits. Fractions collected after the sucrose gradient centrifugation (top) of extracts from strain ySP664 ( iih1 Δ GAL-Hsp27 BBP/eIF6 ), logarithmically growing in galactose-containing medium, were precipitated by TCA, and equal amounts of protein extracts were run on 12% acrylamide gels, transferred on immobilon-P membranes and probed with the anti–p27 BBP/eIF6 antiserum, followed by the ECL detection method (bottom). Note that p27 BBP/eIF6 (arrow) is highly enriched in the free 60S and 80S fractions, but clearly absent from the polysome fraction. The high molecular weight band in lane 1 is a nonspecific product recognized by secondary antibodies.

    Article Snippet: Western Blot Analysis All samples were denatured before loading in Laemmli buffer ( ) and run on denaturing 12% SDS–acrylamide gel, transferred to Immobilon P membranes ( Millipore Corp. ), and blotted with the rabbit p27BBP/eIF6 antiserum at 1:1,000 dilution as previously described ( ).

    Techniques: Gradient Centrifugation, Molecular Weight

    Western blot analysis of nuclear extracts from macrophages of pIpC-treated PPARγ-MXCre + mice and similarly treated PPARγ-MXCre − mice. A total of 10 μg of protein from nuclear extracts of macrophages and 10 μg of total protein from Hepa-1 cells transfected with an expression vector for PPARγ1 (pSG5-PPARγ cDNA) were subjected to electrophoresis on a 4 to 15% Tris-HCl gradient gel (Bio-Rad), transferred to Immobilon-P membranes (Millipore), and probed as recommended by the manufacturer with anti-PPARγ antibodies (Santa Cruz Biotechnologies) specific for the N terminus (E-8, PPARγ antibody) (A) and the C terminus (H-100, PPARγ antibody) (B) of the PPARγ protein. Detection of immunoreactive proteins was done by using an enhanced chemiluminescence blot detection system (Amersham).

    Journal: Molecular and Cellular Biology

    Article Title: Conditional Disruption of the Peroxisome Proliferator-Activated Receptor ? Gene in Mice Results in Lowered Expression of ABCA1, ABCG1, and apoE in Macrophages and Reduced Cholesterol Efflux

    doi: 10.1128/MCB.22.8.2607-2619.2002

    Figure Lengend Snippet: Western blot analysis of nuclear extracts from macrophages of pIpC-treated PPARγ-MXCre + mice and similarly treated PPARγ-MXCre − mice. A total of 10 μg of protein from nuclear extracts of macrophages and 10 μg of total protein from Hepa-1 cells transfected with an expression vector for PPARγ1 (pSG5-PPARγ cDNA) were subjected to electrophoresis on a 4 to 15% Tris-HCl gradient gel (Bio-Rad), transferred to Immobilon-P membranes (Millipore), and probed as recommended by the manufacturer with anti-PPARγ antibodies (Santa Cruz Biotechnologies) specific for the N terminus (E-8, PPARγ antibody) (A) and the C terminus (H-100, PPARγ antibody) (B) of the PPARγ protein. Detection of immunoreactive proteins was done by using an enhanced chemiluminescence blot detection system (Amersham).

    Article Snippet: Then, 10 μg of total protein from Hepa-1 cells and 10 μg of protein from nuclear extracts of macrophages were subjected to electrophoresis on a 4 to 15% Tris-HCl gradient gel (Bio-Rad, Hercules, Calif.), transferred to Immobilon-P membranes (Millipore, Bedford, Mass.), and probed according to the manufacturer's recommendations with anti-PPARγ antibodies (E-8 and H-100; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.) as indicated.

    Techniques: Western Blot, Mouse Assay, Transfection, Expressing, Plasmid Preparation, Electrophoresis

    GSK-3β binds to D1 subunits and preferentially phosphorylates cyclin D1 in complexes with CDK4. ( A ) Sf9 cells infected with baculoviruses encoding cyclin D1 or cyclin D1 plus CDK4 with or without wild-type GSK-3β as indicated were metabolically labeled with [ 32 P]orthophosphate. Lysates were subjected to precipitation with NRS (lane 1 ), antibody to cyclin D1 (lanes 2 , 3 ), or antiserum specific for the carboxyl terminus of CDK4 (lanes 4 , 5 ) as indicated. Phosphorylated proteins were resolved on denaturing polyacrylamide gels and transferred to a nitrocellulose membrane. Following autoradiography, the membrane was blotted with the antibody to cyclin D1 ( bottom ) and sites of antibody binding were visualized by enhanced chemiluminescence. ( B ) Sf9 cells infected with baculoviruses encoding cyclin D1 and CDK4 together with wild-type (wt) or kinase-defective (kd) GSK-3β were labeled with [ 32 P]orthophosphate. Lysates were subjected to precipitation with NRS or anti-D1 as indicated, and phosphorylated proteins were resolved on a denaturing polyacrylamide gel followed by transfer to Immobilon-P membrane (autoradiographic exposure time 12 hr; top ). Following autoradiography, the membrane was blotted with the antibody to cyclin D1 as in A . The relative ratio of 32 P-labeled cyclin D1 versus total cyclin D1 in each lysate (densitometric scanning) is indicated between the two autoradiographs. ( C ) Sf9 lysates infected with baculoviruses encoding the proteins indicated were precipitated with the indicated antibodies and blotted with a monoclonal antibody to GSK-3β. Sites of antibody binding were visualized by enhanced chemiluminesence.

    Journal: Genes & Development

    Article Title: Glycogen synthase kinase-3? regulates cyclin D1 proteolysis and subcellular localization

    doi:

    Figure Lengend Snippet: GSK-3β binds to D1 subunits and preferentially phosphorylates cyclin D1 in complexes with CDK4. ( A ) Sf9 cells infected with baculoviruses encoding cyclin D1 or cyclin D1 plus CDK4 with or without wild-type GSK-3β as indicated were metabolically labeled with [ 32 P]orthophosphate. Lysates were subjected to precipitation with NRS (lane 1 ), antibody to cyclin D1 (lanes 2 , 3 ), or antiserum specific for the carboxyl terminus of CDK4 (lanes 4 , 5 ) as indicated. Phosphorylated proteins were resolved on denaturing polyacrylamide gels and transferred to a nitrocellulose membrane. Following autoradiography, the membrane was blotted with the antibody to cyclin D1 ( bottom ) and sites of antibody binding were visualized by enhanced chemiluminescence. ( B ) Sf9 cells infected with baculoviruses encoding cyclin D1 and CDK4 together with wild-type (wt) or kinase-defective (kd) GSK-3β were labeled with [ 32 P]orthophosphate. Lysates were subjected to precipitation with NRS or anti-D1 as indicated, and phosphorylated proteins were resolved on a denaturing polyacrylamide gel followed by transfer to Immobilon-P membrane (autoradiographic exposure time 12 hr; top ). Following autoradiography, the membrane was blotted with the antibody to cyclin D1 as in A . The relative ratio of 32 P-labeled cyclin D1 versus total cyclin D1 in each lysate (densitometric scanning) is indicated between the two autoradiographs. ( C ) Sf9 lysates infected with baculoviruses encoding the proteins indicated were precipitated with the indicated antibodies and blotted with a monoclonal antibody to GSK-3β. Sites of antibody binding were visualized by enhanced chemiluminesence.

    Article Snippet: Phosphorylated proteins were resolved on denaturing polyacrylamide gels, transferred to Immobilon-P membranes (Millipore), and visualized by autoradiographic exposure.

    Techniques: Infection, Metabolic Labelling, Labeling, Autoradiography, Binding Assay

    GSK-3β phosphorylates cyclin D1 on Thr-286. ( A ) Cyclin D1 (odd lanes) or cyclin D1-(T286A) (even lanes) immunoprecipitated from Sf9 cells infected with baculoviruses encoding D1 and CDK4 were mixed with recombinant GSK-3β (lanes 1 , 2 ), ERK2 (lanes 3 , 4 ), SAPK (lanes 5 , 6 ), or cyclin E–CDK2 (lanes 7 , 8 ) plus [γ- 32 P]ATP. After incubation at 30°C for 30 min, phosphorylated proteins were separated on a denaturing polyacrylamide gel, transferred to an Immobilon-P membrane, and visualized by autoradiography. The position of phosphorylated cyclin D1 is indicated. ( B ) Membrane slices containing cyclin D1 phosphorylated by GSK-3β in vitro ( left ), phosphorylated by endogenous Sf9 kinases ( middle ), or by a mixture of the two ( right ).

    Journal: Genes & Development

    Article Title: Glycogen synthase kinase-3? regulates cyclin D1 proteolysis and subcellular localization

    doi:

    Figure Lengend Snippet: GSK-3β phosphorylates cyclin D1 on Thr-286. ( A ) Cyclin D1 (odd lanes) or cyclin D1-(T286A) (even lanes) immunoprecipitated from Sf9 cells infected with baculoviruses encoding D1 and CDK4 were mixed with recombinant GSK-3β (lanes 1 , 2 ), ERK2 (lanes 3 , 4 ), SAPK (lanes 5 , 6 ), or cyclin E–CDK2 (lanes 7 , 8 ) plus [γ- 32 P]ATP. After incubation at 30°C for 30 min, phosphorylated proteins were separated on a denaturing polyacrylamide gel, transferred to an Immobilon-P membrane, and visualized by autoradiography. The position of phosphorylated cyclin D1 is indicated. ( B ) Membrane slices containing cyclin D1 phosphorylated by GSK-3β in vitro ( left ), phosphorylated by endogenous Sf9 kinases ( middle ), or by a mixture of the two ( right ).

    Article Snippet: Phosphorylated proteins were resolved on denaturing polyacrylamide gels, transferred to Immobilon-P membranes (Millipore), and visualized by autoradiographic exposure.

    Techniques: Immunoprecipitation, Infection, Recombinant, Incubation, Autoradiography, In Vitro