rabbit anti phospho ack1  (Millipore)


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  • 93
    Name:
    Anti alpha Tubulin antibody
    Description:
    Anti α Tubulin antibody Mouse monoclonal mouse IgG1 isotype is derived from the hybridoma DM1A produced by the fusion of mouse myeloma cells NS1 and splenocytes from BALB c mice immunized with purified chick brain tubulin The isotype is determined by a double diffusion immunoassay using Mouse Monoclonal Antibody Isotyping Reagents Product Number ISO2 Tubulin is the major building block of microtubules This intracellular cylindrical filamentous structure is present in almost all eukaryotic cells Microtubules function as structural and mobile elements in mitosis intracellular transport flagellar movement and the cytoskeleton Tubulin is a heterodimer that consists of α tubulin and β tubulin Both subunits have a molecular weight of approx 50 kDa and share considerable homology In addition to α and β tubulin several other tubulins have been identified bringing the number of distinct tubulin classes to seven Most of these tubulins have distinct subcellular localization and an emerging diverse set of functions Out of the seven different tubulins four new members of the tubulin family were identified recently which consist of δ ξ η and ε tubulin η and ε tubulins were discovered by database searches Microtubular systems contain at least three α tubulin isoforms Two isoforms are coded by two α tubulin genes which are both transcribed and code for extremely similar proteins The third isoform is generated by post translational modification At least three modifications of tubulin subunits have been described the phosphorylation of β tubulin from brain the removal of the carboxy terminal tyrosine form a tubulin in vertebrate tissues and the acetylation of the amino group of lysine s in α tubulin α tubulin also called tubulin α 4a TUBA4A is mapped to human chromosome 2q35 The gene codes for a member of the α tubulin family and contains 448 amino acids α subunit of tubulin has molecular weight of 50 000 Monoclonal antibodies recognizing α tubulin together with monoclonal antibodies to other tubulin types β β tubulin isotype I II β tubulin isotype III tyrosine tubulin and the acetylated form of α tubulin provide a specific and useful tool in studying the intracellular distribution of tubulin and the static and dynamic aspects of cytoskeleton
    Catalog Number:
    t6199
    Price:
    None
    Applications:
    Anti-α-Tubulin antibody, Mouse monoclonal has been used in immunocytofluorescence and western blot analysis.
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    Structured Review

    Millipore rabbit anti phospho ack1
    Anti alpha Tubulin antibody
    Anti α Tubulin antibody Mouse monoclonal mouse IgG1 isotype is derived from the hybridoma DM1A produced by the fusion of mouse myeloma cells NS1 and splenocytes from BALB c mice immunized with purified chick brain tubulin The isotype is determined by a double diffusion immunoassay using Mouse Monoclonal Antibody Isotyping Reagents Product Number ISO2 Tubulin is the major building block of microtubules This intracellular cylindrical filamentous structure is present in almost all eukaryotic cells Microtubules function as structural and mobile elements in mitosis intracellular transport flagellar movement and the cytoskeleton Tubulin is a heterodimer that consists of α tubulin and β tubulin Both subunits have a molecular weight of approx 50 kDa and share considerable homology In addition to α and β tubulin several other tubulins have been identified bringing the number of distinct tubulin classes to seven Most of these tubulins have distinct subcellular localization and an emerging diverse set of functions Out of the seven different tubulins four new members of the tubulin family were identified recently which consist of δ ξ η and ε tubulin η and ε tubulins were discovered by database searches Microtubular systems contain at least three α tubulin isoforms Two isoforms are coded by two α tubulin genes which are both transcribed and code for extremely similar proteins The third isoform is generated by post translational modification At least three modifications of tubulin subunits have been described the phosphorylation of β tubulin from brain the removal of the carboxy terminal tyrosine form a tubulin in vertebrate tissues and the acetylation of the amino group of lysine s in α tubulin α tubulin also called tubulin α 4a TUBA4A is mapped to human chromosome 2q35 The gene codes for a member of the α tubulin family and contains 448 amino acids α subunit of tubulin has molecular weight of 50 000 Monoclonal antibodies recognizing α tubulin together with monoclonal antibodies to other tubulin types β β tubulin isotype I II β tubulin isotype III tyrosine tubulin and the acetylated form of α tubulin provide a specific and useful tool in studying the intracellular distribution of tubulin and the static and dynamic aspects of cytoskeleton
    https://www.bioz.com/result/rabbit anti phospho ack1/product/Millipore
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    Images

    1) Product Images from "Synthetic lethality of TNK2 inhibition in PTPN11-mutant leukemia"

    Article Title: Synthetic lethality of TNK2 inhibition in PTPN11-mutant leukemia

    Journal: Science signaling

    doi: 10.1126/scisignal.aao5617

    Working model: Synthetic lethality of TNK2 inhibition in PTPN11-mutant leukemia.
    Figure Legend Snippet: Working model: Synthetic lethality of TNK2 inhibition in PTPN11-mutant leukemia.

    Techniques Used: Inhibition, Mutagenesis

    TNK2 increases signaling through PTPN11/RAS/MAPK in cells overexpressing mutant PTPN11.
    Figure Legend Snippet: TNK2 increases signaling through PTPN11/RAS/MAPK in cells overexpressing mutant PTPN11.

    Techniques Used: Mutagenesis

    A primary patient sample containing a PTPN11 mutation demonstrates dasatinib sensitivity and over-reliance on TNK2.
    Figure Legend Snippet: A primary patient sample containing a PTPN11 mutation demonstrates dasatinib sensitivity and over-reliance on TNK2.

    Techniques Used: Mutagenesis

    Inhibition of TNK2 reduces signaling through PTPN11/RAS/MAPK.
    Figure Legend Snippet: Inhibition of TNK2 reduces signaling through PTPN11/RAS/MAPK.

    Techniques Used: Inhibition

    Functional assays show increased transformation potential and sensitivity to TNK2 inhibition.
    Figure Legend Snippet: Functional assays show increased transformation potential and sensitivity to TNK2 inhibition.

    Techniques Used: Functional Assay, Transformation Assay, Inhibition

    2) Product Images from "Regulation of Gene Transcription by Voltage-gated L-type Calcium Channel, Cav1.3 *"

    Article Title: Regulation of Gene Transcription by Voltage-gated L-type Calcium Channel, Cav1.3 *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.586883

    Subcellular localization of SK2 channels was altered in atrial myocytes isolated from homozygous Ca v 1.3 null mutant mice. The subcellular distribution of SK2 channels in atrial myocytes isolated from Ca v 1.3 −/− mice (KO) ( a ) as compared with those of the WT animals ( b ). Anti-SK2 antibodies were used for single staining. The double staining showed SK2 channels and α-actinin2 ( Actn2 ) localization patterns. The right panels in a and b show single staining at higher magnification. Scale bars are 10 μm. Atrial myocytes were obtained from three pairs of Ca v 1.3 −/− mice compared with WT littermates. All experiments were repeated independently three times and consistent data were obtained as shown.
    Figure Legend Snippet: Subcellular localization of SK2 channels was altered in atrial myocytes isolated from homozygous Ca v 1.3 null mutant mice. The subcellular distribution of SK2 channels in atrial myocytes isolated from Ca v 1.3 −/− mice (KO) ( a ) as compared with those of the WT animals ( b ). Anti-SK2 antibodies were used for single staining. The double staining showed SK2 channels and α-actinin2 ( Actn2 ) localization patterns. The right panels in a and b show single staining at higher magnification. Scale bars are 10 μm. Atrial myocytes were obtained from three pairs of Ca v 1.3 −/− mice compared with WT littermates. All experiments were repeated independently three times and consistent data were obtained as shown.

    Techniques Used: Isolation, Mutagenesis, Mouse Assay, Staining, Double Staining

    Subcellular distribution of SK2 channel proteins in atrial tissues from Ca v 1.3 −/− (KO) and WT mice. a, Western blot analyses showing similar expression levels of SK2 channel protein in whole atrial tissue lysates isolated from Ca v 1.3 −/− and WT animals. GAPDH was used as a loading control. b , summary data for SK2 protein expression levels normalized to GAPDH. c, abnormal distribution of SK2 channels was observed in Ca v 1.3 −/− mice using a discontinuous sucrose density gradient ultracentrifugation. The purity of membrane fractionation in mouse atrial tissues was tested and normalized by Na,K-ATPase as the plasma membrane marker. Each sample of atrial myocytes was isolated from 5 animals and the experiments were repeated independently three times.
    Figure Legend Snippet: Subcellular distribution of SK2 channel proteins in atrial tissues from Ca v 1.3 −/− (KO) and WT mice. a, Western blot analyses showing similar expression levels of SK2 channel protein in whole atrial tissue lysates isolated from Ca v 1.3 −/− and WT animals. GAPDH was used as a loading control. b , summary data for SK2 protein expression levels normalized to GAPDH. c, abnormal distribution of SK2 channels was observed in Ca v 1.3 −/− mice using a discontinuous sucrose density gradient ultracentrifugation. The purity of membrane fractionation in mouse atrial tissues was tested and normalized by Na,K-ATPase as the plasma membrane marker. Each sample of atrial myocytes was isolated from 5 animals and the experiments were repeated independently three times.

    Techniques Used: Mouse Assay, Western Blot, Expressing, Isolation, Fractionation, Marker

    The normal expression of MLC2 is required for proper membrane localization of SK2 channels. a, expression of mRNAs of Myl2 was significantly decreased in Ca v 1.3 −/− (KO) compared with WT mice ( A , atria). b, Western blot analysis showing a significant decrease in MLC2 protein in Ca v 1.3 −/− mice compared with WT animals ( n = 3 animals for each group were used for Western blot analyses). c, MLC2 interacted directly with the SK2 channel as assessed using Y2H assays. d , left panels show the SK2 staining pattern in atrial myocytes treated with control siRNA. Right panels : treatment of atrial myocytes with siRNA specific to MLC2 resulted in a significant decrease in MLC2 expression. Moreover, there was a significant decrease in SK2 membrane expression. Lower panels compare the SK2 staining of the regions outlined in white boxes at higher magnification. A total of three animals were used for each group. e, apamin-sensitive currents recorded from HEK293 cells transfected with human SK2 channel alone compared with SK2 channel co-expressed with hMLC2. Data from non-transfected cells are shown in the lower left panel. Right lower panel compared the current density in pA/pF of apamin-sensitive currents. A total of 6 cells were performed for each group with similar results. f, a schematic model illustrating the C terminus of Ca v 1.3 as a transcriptional regulator for MLC2 expression. MLC2 physically interacts with SK2 channels and facilitates the targeting of SK2 channels to the plasma membrane. SK2 channels are shown to interact with filamin A ( FLNA ) and α-actinin2 ( Actn2 ) cytoskeletal proteins via the N and C termini, respectively. CaMBD refers to calmodulin binding domain within the C terminus of SK2 channels.
    Figure Legend Snippet: The normal expression of MLC2 is required for proper membrane localization of SK2 channels. a, expression of mRNAs of Myl2 was significantly decreased in Ca v 1.3 −/− (KO) compared with WT mice ( A , atria). b, Western blot analysis showing a significant decrease in MLC2 protein in Ca v 1.3 −/− mice compared with WT animals ( n = 3 animals for each group were used for Western blot analyses). c, MLC2 interacted directly with the SK2 channel as assessed using Y2H assays. d , left panels show the SK2 staining pattern in atrial myocytes treated with control siRNA. Right panels : treatment of atrial myocytes with siRNA specific to MLC2 resulted in a significant decrease in MLC2 expression. Moreover, there was a significant decrease in SK2 membrane expression. Lower panels compare the SK2 staining of the regions outlined in white boxes at higher magnification. A total of three animals were used for each group. e, apamin-sensitive currents recorded from HEK293 cells transfected with human SK2 channel alone compared with SK2 channel co-expressed with hMLC2. Data from non-transfected cells are shown in the lower left panel. Right lower panel compared the current density in pA/pF of apamin-sensitive currents. A total of 6 cells were performed for each group with similar results. f, a schematic model illustrating the C terminus of Ca v 1.3 as a transcriptional regulator for MLC2 expression. MLC2 physically interacts with SK2 channels and facilitates the targeting of SK2 channels to the plasma membrane. SK2 channels are shown to interact with filamin A ( FLNA ) and α-actinin2 ( Actn2 ) cytoskeletal proteins via the N and C termini, respectively. CaMBD refers to calmodulin binding domain within the C terminus of SK2 channels.

    Techniques Used: Expressing, Mouse Assay, Western Blot, Staining, Transfection, Binding Assay

    3) Product Images from "TRF1 Ensures the Centromeric Function of Aurora-B and Proper Chromosome Segregation"

    Article Title: TRF1 Ensures the Centromeric Function of Aurora-B and Proper Chromosome Segregation

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00161-14

    Merotelic microtubule attachments are induced by TRF1 depletion. (A) Time-lapse images of HeLa-Kyoto cells coexpressing EGFP–CENP-A and EGFP–α-tubulin. Cells were transfected with the indicated siRNAs. Forty-eight hours after transfection,
    Figure Legend Snippet: Merotelic microtubule attachments are induced by TRF1 depletion. (A) Time-lapse images of HeLa-Kyoto cells coexpressing EGFP–CENP-A and EGFP–α-tubulin. Cells were transfected with the indicated siRNAs. Forty-eight hours after transfection,

    Techniques Used: Transfection

    4) Product Images from "FAM21 directs SNX27–retromer cargoes to the plasma membrane by preventing transport to the Golgi apparatus"

    Article Title: FAM21 directs SNX27–retromer cargoes to the plasma membrane by preventing transport to the Golgi apparatus

    Journal: Nature Communications

    doi: 10.1038/ncomms10939

    The interaction of SNX27 with FAM21 is required for proper recycling of its cargo to the plasma membrane (PM). ( a ) Schematic representation of SNX27 constructs used. ( b ) hTERT-RPE1 cells stably expressing empty vector or 3 × FLAG-tagged, siRNA-resistant SNX27 constructs were transfected with indicated SNX27 or control (siCTL) siRNAs, and analysed by immunoblotting. An arrow denotes endogenous SNX27. The SNX27 ΔPDZ proteins were not detected by anti-SNX27 antibodies because of a deficiency of epitope. ( c ) Myc-FAM21 was co-transfected with the indicated HA-SNX27 constructs in HEK293T cells, and lysates were immunoprecipitated with control mouse IgG or anti-haemagglutinin (HA) antibodies. Then, precipitates were immunoblotted as indicated. Asterisks (*) denote IgG heavy or light chains. ( d ) hTERT-RPE1 cells stably expressing empty vector or 3 × FLAG-tagged, siRNA-resistant SNX27 constructs were transfected with the indicated siRNAs and immunostained for FLAG epitope (green) and GLUT1 (red). Merged images with 4',6-diamidino-2-phenylindole staining (blue) are to the right. Scale bar, 10 μm. ( e ) Cells were counted based on co-localization of GLUT1 with GM130 (Golgi) or LAMP1 (lysosome) as shown in Supplementary Fig. 3 . Cells without accumulated GLUT1 signals were considered as plasma membrane (PM). Graphs express means±s.d. ( n =3; 200 cells per group). ( f ) hTERT-RPE1 cells stably expressing empty vector or 3 × FLAG-tagged, siRNA-resistant SNX27 constructs were transfected with the indicated siRNAs. Twenty-four hours later, cells were cultured in the absence or presence of 50 nM bafilomycin A1 for 24 h, and then analysed by immunoblotting. The intensities of each GLUT1 were normalized with respective GAPDH signals. Levels of GLUT1 in siSNX27-transfected cells relative to siCTL-transfected cells were measured from five independent experiments. Graphs express means±s.d. * P
    Figure Legend Snippet: The interaction of SNX27 with FAM21 is required for proper recycling of its cargo to the plasma membrane (PM). ( a ) Schematic representation of SNX27 constructs used. ( b ) hTERT-RPE1 cells stably expressing empty vector or 3 × FLAG-tagged, siRNA-resistant SNX27 constructs were transfected with indicated SNX27 or control (siCTL) siRNAs, and analysed by immunoblotting. An arrow denotes endogenous SNX27. The SNX27 ΔPDZ proteins were not detected by anti-SNX27 antibodies because of a deficiency of epitope. ( c ) Myc-FAM21 was co-transfected with the indicated HA-SNX27 constructs in HEK293T cells, and lysates were immunoprecipitated with control mouse IgG or anti-haemagglutinin (HA) antibodies. Then, precipitates were immunoblotted as indicated. Asterisks (*) denote IgG heavy or light chains. ( d ) hTERT-RPE1 cells stably expressing empty vector or 3 × FLAG-tagged, siRNA-resistant SNX27 constructs were transfected with the indicated siRNAs and immunostained for FLAG epitope (green) and GLUT1 (red). Merged images with 4',6-diamidino-2-phenylindole staining (blue) are to the right. Scale bar, 10 μm. ( e ) Cells were counted based on co-localization of GLUT1 with GM130 (Golgi) or LAMP1 (lysosome) as shown in Supplementary Fig. 3 . Cells without accumulated GLUT1 signals were considered as plasma membrane (PM). Graphs express means±s.d. ( n =3; 200 cells per group). ( f ) hTERT-RPE1 cells stably expressing empty vector or 3 × FLAG-tagged, siRNA-resistant SNX27 constructs were transfected with the indicated siRNAs. Twenty-four hours later, cells were cultured in the absence or presence of 50 nM bafilomycin A1 for 24 h, and then analysed by immunoblotting. The intensities of each GLUT1 were normalized with respective GAPDH signals. Levels of GLUT1 in siSNX27-transfected cells relative to siCTL-transfected cells were measured from five independent experiments. Graphs express means±s.d. * P

    Techniques Used: Construct, Stable Transfection, Expressing, Plasmid Preparation, Transfection, Immunoprecipitation, FLAG-tag, Staining, Cell Culture

    Depletion of FAM21 causes mis-sorting of SNX27–retromer cargoes to the Golgi apparatus. ( a – d ) hTERT-RPE1 cells transfected with the indicated specific or control (siCTL) siRNAs were immunostained for GLUT1 (green) along with LAMP1 ( a , red) or GM130 ( c , red). Co-localization of GLUT1 with LAMP1 ( b ) or GM130 ( d ) was analysed by calculation of Pearson's coefficient. Graphs express means±s.d. ( n =3; > 30 cells per group). ( e ) hTERT-RPE1 cells transfected with an siRNA targeting FAM21 were incubated with 10 μg ml −1 of Alexa 568-conjugated Tf for 20 min at 37 °C before fixation. Cells were then immunostained for GM130 and GLUT1. For comparison, representative magnified views of the Golgi were chosen and displayed. ( f ) Co-localization at the Golgi area among GLUT1, GM130 and Tf were analysed by calculation of Pearson's coefficient. Graphs express means±s.d. (30 cells per group). ( g ) hTERT-RPE1 cells transfected with the indicated siRNAs were incubated with 0.05% of dimethylsulphoxide or 5 μg ml −1 of Brefeldin A for 5 min at 37 °C before fixation and immunostained for GLUT1 (green), GM130 (red) and Rab11 (grey). Insets are magnified views of the Golgi. ( h ) hTERT-RPE1 cells stably expressing Myc-β2AR were transfected with the indicated siRNAs and immunostained for Myc epitope (green) and GM130 (red). ( i ) Cells with Myc signals accumulating at the Golgi from h were counted ( n =2; 200 cells per group). Merged images with 4',6-diamidino-2-phenylindole staining (blue) are to the right. Scale bars, 10 μm. * P
    Figure Legend Snippet: Depletion of FAM21 causes mis-sorting of SNX27–retromer cargoes to the Golgi apparatus. ( a – d ) hTERT-RPE1 cells transfected with the indicated specific or control (siCTL) siRNAs were immunostained for GLUT1 (green) along with LAMP1 ( a , red) or GM130 ( c , red). Co-localization of GLUT1 with LAMP1 ( b ) or GM130 ( d ) was analysed by calculation of Pearson's coefficient. Graphs express means±s.d. ( n =3; > 30 cells per group). ( e ) hTERT-RPE1 cells transfected with an siRNA targeting FAM21 were incubated with 10 μg ml −1 of Alexa 568-conjugated Tf for 20 min at 37 °C before fixation. Cells were then immunostained for GM130 and GLUT1. For comparison, representative magnified views of the Golgi were chosen and displayed. ( f ) Co-localization at the Golgi area among GLUT1, GM130 and Tf were analysed by calculation of Pearson's coefficient. Graphs express means±s.d. (30 cells per group). ( g ) hTERT-RPE1 cells transfected with the indicated siRNAs were incubated with 0.05% of dimethylsulphoxide or 5 μg ml −1 of Brefeldin A for 5 min at 37 °C before fixation and immunostained for GLUT1 (green), GM130 (red) and Rab11 (grey). Insets are magnified views of the Golgi. ( h ) hTERT-RPE1 cells stably expressing Myc-β2AR were transfected with the indicated siRNAs and immunostained for Myc epitope (green) and GM130 (red). ( i ) Cells with Myc signals accumulating at the Golgi from h were counted ( n =2; 200 cells per group). Merged images with 4',6-diamidino-2-phenylindole staining (blue) are to the right. Scale bars, 10 μm. * P

    Techniques Used: Transfection, Incubation, Stable Transfection, Expressing, Staining

    Mis-sorting of GLUT1 into the Golgi in FAM21-depleted cells is caused by an elevation of PI(4)P levels at the Golgi. ( a ) hTERT-RPE1 cells transfected with indicated FAM21 or control (siCTL) siRNAs were immunostained for GM130 (red) and TGN46 (green). ( b ) The area of the Golgi was measured by staining of GM130 or TGN46 from a . Graphs express means±s.d. ( n =3; 20 cells per group). ( c , d ) hTERT-RPE1 cells transfected with indicated siRNAs were immunostained for GM130 (red) along with PI(4)P ( c , green) or PI4KB ( d , green). ( e ) The intensities of PI4KB at the Golgi were measured from 30 cells of three independent experiments from d . Total intensities of PI4KB were divided by the Golgi area. To calculate a fold increase between two groups, averages from FAM21-depleted cells were divided by those of control cells. Graphs express means±s.d. ( n =3; 30 cells per group). ( f ) hTERT-RPE1 cells transfected with indicated siRNAs were lysed and subjected to immunoblotting. ( g ) hTERT-RPE1 cells transfected with indicated siRNAs were immunostained for SNX27 (green) and GLUT1 (red). ( h ) Co-localization between GLUT1 and GM130 was analysed by calculation of Pearson's coefficient. Graphs express means±s.d. ( n =3; > 30 cells per group). ( i ) hTERT-RPE1 cells transfected with indicated siRNAs were immunostained for SNX6 (green) and GM130 (red). ( j ) The Golgi area was drawn based on GM130 staining, and SNX6 signals in the corresponding area were measured. The area of SNX6 signal per cell was divided by the area of Golgi to calculate the accumulation of SNX6 at the Golgi. Graphs express means±s.d. (20 cells per group). Merged images with 4',6-diamidino-2-phenylindole staining are to the right. Insets are magnified views of the Golgi area. Scale bars, 10 μm. * P
    Figure Legend Snippet: Mis-sorting of GLUT1 into the Golgi in FAM21-depleted cells is caused by an elevation of PI(4)P levels at the Golgi. ( a ) hTERT-RPE1 cells transfected with indicated FAM21 or control (siCTL) siRNAs were immunostained for GM130 (red) and TGN46 (green). ( b ) The area of the Golgi was measured by staining of GM130 or TGN46 from a . Graphs express means±s.d. ( n =3; 20 cells per group). ( c , d ) hTERT-RPE1 cells transfected with indicated siRNAs were immunostained for GM130 (red) along with PI(4)P ( c , green) or PI4KB ( d , green). ( e ) The intensities of PI4KB at the Golgi were measured from 30 cells of three independent experiments from d . Total intensities of PI4KB were divided by the Golgi area. To calculate a fold increase between two groups, averages from FAM21-depleted cells were divided by those of control cells. Graphs express means±s.d. ( n =3; 30 cells per group). ( f ) hTERT-RPE1 cells transfected with indicated siRNAs were lysed and subjected to immunoblotting. ( g ) hTERT-RPE1 cells transfected with indicated siRNAs were immunostained for SNX27 (green) and GLUT1 (red). ( h ) Co-localization between GLUT1 and GM130 was analysed by calculation of Pearson's coefficient. Graphs express means±s.d. ( n =3; > 30 cells per group). ( i ) hTERT-RPE1 cells transfected with indicated siRNAs were immunostained for SNX6 (green) and GM130 (red). ( j ) The Golgi area was drawn based on GM130 staining, and SNX6 signals in the corresponding area were measured. The area of SNX6 signal per cell was divided by the area of Golgi to calculate the accumulation of SNX6 at the Golgi. Graphs express means±s.d. (20 cells per group). Merged images with 4',6-diamidino-2-phenylindole staining are to the right. Insets are magnified views of the Golgi area. Scale bars, 10 μm. * P

    Techniques Used: Transfection, Staining

    FAM21 regulates a precise localization of SNX27 at an endosomal subdomain through their interaction. ( a ) hTERT-RPE1 cells transfected with the indicated SNX27 or control (siCTL) siRNAs were immunostained for SNX27 (green) and GM130 (red). Insets are magnified views of the Golgi area. Merged images with 4',6-diamidino-2-phenylindole staining (blue) are to the right. ( b ) To measure the size of each punctum, 30 cells of each group were analysed by ImageJ using SNX27 signals from a . ( c ) hTERT-RPE1 cells transfected with the indicated siRNAs were immunoblotted. ( d ) The levels of SNX27 mRNA in hTERT-RPE1 cells transfected with the indicated siRNAs were determined by quantitative real-time PCR. ( e , f ) hTERT-RPE1 cells stably expressing FLAG-SNX27, WT or ΔFERM mutant were immunostained for FLAG epitope (green) along with EEA1 ( e , red) or FAM21 ( f , red). Merged images with 4',6-diamidino-2-phenylindole (blue) staining and magnified views of insets are to the right. ( g ) To measure the total size of puncta visualized by FLAG signals from e , 30 cells of each group were analysed with ImageJ. Graphs express means±s.d. ** P
    Figure Legend Snippet: FAM21 regulates a precise localization of SNX27 at an endosomal subdomain through their interaction. ( a ) hTERT-RPE1 cells transfected with the indicated SNX27 or control (siCTL) siRNAs were immunostained for SNX27 (green) and GM130 (red). Insets are magnified views of the Golgi area. Merged images with 4',6-diamidino-2-phenylindole staining (blue) are to the right. ( b ) To measure the size of each punctum, 30 cells of each group were analysed by ImageJ using SNX27 signals from a . ( c ) hTERT-RPE1 cells transfected with the indicated siRNAs were immunoblotted. ( d ) The levels of SNX27 mRNA in hTERT-RPE1 cells transfected with the indicated siRNAs were determined by quantitative real-time PCR. ( e , f ) hTERT-RPE1 cells stably expressing FLAG-SNX27, WT or ΔFERM mutant were immunostained for FLAG epitope (green) along with EEA1 ( e , red) or FAM21 ( f , red). Merged images with 4',6-diamidino-2-phenylindole (blue) staining and magnified views of insets are to the right. ( g ) To measure the total size of puncta visualized by FLAG signals from e , 30 cells of each group were analysed with ImageJ. Graphs express means±s.d. ** P

    Techniques Used: Transfection, Staining, Real-time Polymerase Chain Reaction, Stable Transfection, Expressing, Mutagenesis, FLAG-tag

    FAM21 has a distinct function in recycling of SNX27–retromer cargoes from other WASH complex components. ( a ) hTERT-RPE1 cells transfected with the indicated specific or control (siCTL) siRNAs were immunostained for GLUT1 (green) and GM130 (red). ( b ) Co-localization between GLUT1 and GM130 was analysed by calculation of Pearson's coefficient. Graphs express means±s.d. ( n =3; > 30 cells per group). ( c ) hTERT-RPE1 cells transfected with indicated siRNAs were immunostained for PI4KB (green) and GM130 (red). ( d ) Intensities of PI4KB at the Golgi were measured from 30 cells per group from c . Total intensities of PI4KB were divided by the Golgi area. To calculate a fold-increase from control cells, averages of experimental groups were divided by those of control cells. Graphs express means±s.d. ( e ) hTERT-RPE1 cells transfected with the indicated siRNAs were immunoblotted. ( f – h ) hTERT-RPE1 cells transfected with the indicated siRNAs were immunostained as shown. Insets are magnified views of the Golgi area in g . Merged images with 4',6-diamidino-2-phenylindole staining are to the right. Scale bars, 10 μm. * P
    Figure Legend Snippet: FAM21 has a distinct function in recycling of SNX27–retromer cargoes from other WASH complex components. ( a ) hTERT-RPE1 cells transfected with the indicated specific or control (siCTL) siRNAs were immunostained for GLUT1 (green) and GM130 (red). ( b ) Co-localization between GLUT1 and GM130 was analysed by calculation of Pearson's coefficient. Graphs express means±s.d. ( n =3; > 30 cells per group). ( c ) hTERT-RPE1 cells transfected with indicated siRNAs were immunostained for PI4KB (green) and GM130 (red). ( d ) Intensities of PI4KB at the Golgi were measured from 30 cells per group from c . Total intensities of PI4KB were divided by the Golgi area. To calculate a fold-increase from control cells, averages of experimental groups were divided by those of control cells. Graphs express means±s.d. ( e ) hTERT-RPE1 cells transfected with the indicated siRNAs were immunoblotted. ( f – h ) hTERT-RPE1 cells transfected with the indicated siRNAs were immunostained as shown. Insets are magnified views of the Golgi area in g . Merged images with 4',6-diamidino-2-phenylindole staining are to the right. Scale bars, 10 μm. * P

    Techniques Used: Transfection, Staining

    SNX27 interacts directly with FAM21 among the WASH complex components. ( a ) HEK293T cells were transfected with 3 × FLAG-tagged-WASH complex proteins. Lysates were incubated with GST or GST-SNX27 proteins and precipitated with Glutathione Sepharose 4B beads. Precipitates were then immunoblotted as shown. ( b ) Schematic representation of FAM21 constructs used. LFa motifs and α-helices are indicated. ( c , d ) HA-SNX27 was co-transfected with the indicated Myc-tagged constructs in HEK293T cells, and lysates were immunoprecipitated with anti-haemagglutinin (HA) antibodies and immunoblotted. Asterisks (*) denote IgG heavy chains. ( e ) GST or GST-SNX27 fusion proteins were incubated with indicated His-tagged FAM21 proteins and precipitated using Glutathione Sepharose 4B beads. Precipitates were immunoblotted as indicated. ( f ) Biotinylated peptides corresponding to FAM21 residues 40–79 and 592–600 were incubated with CBP-SNX27 and precipitated using streptavidin-conjugated beads. Precipitates were immunoblotted for CBP.
    Figure Legend Snippet: SNX27 interacts directly with FAM21 among the WASH complex components. ( a ) HEK293T cells were transfected with 3 × FLAG-tagged-WASH complex proteins. Lysates were incubated with GST or GST-SNX27 proteins and precipitated with Glutathione Sepharose 4B beads. Precipitates were then immunoblotted as shown. ( b ) Schematic representation of FAM21 constructs used. LFa motifs and α-helices are indicated. ( c , d ) HA-SNX27 was co-transfected with the indicated Myc-tagged constructs in HEK293T cells, and lysates were immunoprecipitated with anti-haemagglutinin (HA) antibodies and immunoblotted. Asterisks (*) denote IgG heavy chains. ( e ) GST or GST-SNX27 fusion proteins were incubated with indicated His-tagged FAM21 proteins and precipitated using Glutathione Sepharose 4B beads. Precipitates were immunoblotted as indicated. ( f ) Biotinylated peptides corresponding to FAM21 residues 40–79 and 592–600 were incubated with CBP-SNX27 and precipitated using streptavidin-conjugated beads. Precipitates were immunoblotted for CBP.

    Techniques Used: Transfection, Incubation, Construct, Immunoprecipitation

    Model for roles of the SNX27–retromer–WASH complex in cargo sorting. After internalization, cargoes harbouring a PDZ-binding motif such as β2AR and GLUT1 bind to a PDZ domain of SNX27. SNX27 serves as an adaptor linking its cargoes to the endosomal tubules through its interaction. Interaction of its PDZ domain with VPS26 of retromer prevents lysosomal entry of cargoes, whereas the interaction of its FERM domain with FAM21 of the WASH complex prevents its transport to the Golgi apparatus. Therefore, these interactions within the SNX27–retromer–WASH complex direct the cargoes towards recycling to the plasma membrane. The WASH complex, in particular, functions in localizing SNX27 at a specialized endosomal subdomain. In addition, FAM21 controls PI4KB levels at the Golgi, resulting in maintaining proper levels of PI(4)P at the Golgi, which prevents dissociation of cargoes from the complex at the Golgi.
    Figure Legend Snippet: Model for roles of the SNX27–retromer–WASH complex in cargo sorting. After internalization, cargoes harbouring a PDZ-binding motif such as β2AR and GLUT1 bind to a PDZ domain of SNX27. SNX27 serves as an adaptor linking its cargoes to the endosomal tubules through its interaction. Interaction of its PDZ domain with VPS26 of retromer prevents lysosomal entry of cargoes, whereas the interaction of its FERM domain with FAM21 of the WASH complex prevents its transport to the Golgi apparatus. Therefore, these interactions within the SNX27–retromer–WASH complex direct the cargoes towards recycling to the plasma membrane. The WASH complex, in particular, functions in localizing SNX27 at a specialized endosomal subdomain. In addition, FAM21 controls PI4KB levels at the Golgi, resulting in maintaining proper levels of PI(4)P at the Golgi, which prevents dissociation of cargoes from the complex at the Golgi.

    Techniques Used: Binding Assay

    5) Product Images from "IL-13 dampens human airway epithelial innate immunity through induction of IL-1 receptor-associated kinase M"

    Article Title: IL-13 dampens human airway epithelial innate immunity through induction of IL-1 receptor-associated kinase M

    Journal: The Journal of Allergy and Clinical Immunology

    doi: 10.1016/j.jaci.2011.10.043

    IL-13 induces IRAK-M protein expression in cultured human brushed bronchial epithelial cells. Upper panel , IRAK-M protein quantitative data (means ± SEM) in healthy subjects (n = 4) and asthmatic patients (n = 6). Lower panel , Representative IRAK-M and GAPDH Western blots.
    Figure Legend Snippet: IL-13 induces IRAK-M protein expression in cultured human brushed bronchial epithelial cells. Upper panel , IRAK-M protein quantitative data (means ± SEM) in healthy subjects (n = 4) and asthmatic patients (n = 6). Lower panel , Representative IRAK-M and GAPDH Western blots.

    Techniques Used: Expressing, Cell Culture, Western Blot

    PI3K activation is required for IL-13–induced IRAK-M expression. A, Representative phospho-Akt (pAkt) and total Akt Western blots (n = 3 independent experiments). B, Upper level , IRAK-M protein quantitative data (means ± SEMs) are from 4 independent experiments. Lower panel , Representative IRAK-M and GAPDH Western blots. DMSO , Dimethyl sulfoxide; Wort , wortmannin.
    Figure Legend Snippet: PI3K activation is required for IL-13–induced IRAK-M expression. A, Representative phospho-Akt (pAkt) and total Akt Western blots (n = 3 independent experiments). B, Upper level , IRAK-M protein quantitative data (means ± SEMs) are from 4 independent experiments. Lower panel , Representative IRAK-M and GAPDH Western blots. DMSO , Dimethyl sulfoxide; Wort , wortmannin.

    Techniques Used: Activation Assay, Expressing, Western Blot

    IL-4 induces IRAK-M protein expression in cultured normal human tracheobronchial epithelial cells. Representative IRAK-M and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Western blots of 4 replicates are shown.
    Figure Legend Snippet: IL-4 induces IRAK-M protein expression in cultured normal human tracheobronchial epithelial cells. Representative IRAK-M and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Western blots of 4 replicates are shown.

    Techniques Used: Expressing, Cell Culture, Western Blot

    Role of IRAK-M in IL-13–mediated impairment of epithelial TLR2 signaling. A , IRAK-M shRNA (shIRAK-M) significantly reduced IRAK-M mRNA (left panel) and protein (right panel) expression compared with the control (firefly luciferase shRNA [shLUC] ) in normal human brushed bronchial epithelial cells (n = 4). B , NF-κB p65 activity, TLR2 protein, and human hBD2 peptide levels in normal human brushed bronchial epithelial cells that were transduced with shLUC or shIRAK-M, followed by IL-13, Pam2CSK4 (Pam2) , or both treatments (n = 4). Short thick horizontal lines represent means.
    Figure Legend Snippet: Role of IRAK-M in IL-13–mediated impairment of epithelial TLR2 signaling. A , IRAK-M shRNA (shIRAK-M) significantly reduced IRAK-M mRNA (left panel) and protein (right panel) expression compared with the control (firefly luciferase shRNA [shLUC] ) in normal human brushed bronchial epithelial cells (n = 4). B , NF-κB p65 activity, TLR2 protein, and human hBD2 peptide levels in normal human brushed bronchial epithelial cells that were transduced with shLUC or shIRAK-M, followed by IL-13, Pam2CSK4 (Pam2) , or both treatments (n = 4). Short thick horizontal lines represent means.

    Techniques Used: shRNA, Expressing, Luciferase, Activity Assay, Transduction

    c-Jun directly binds to the IRAK-M gene promoter through PI3K activation on IL-13 stimulation. A , Representative phospho–c-Jun and GAPDH Western blots (n = 4 independent experiments). B , Phospho– c-Jun activity data (means ± SEM) are from 3 independent experiments. C , Chromatin immunoprecipitation assay. Left panel , Increased c-Jun binding to IRAK-M gene promoter after IL-13. Data (means ± SEM) are from 3 independent experiments. Right panel , Representative agarose gel electrophoresis. Ab , Antibody; DMSO , dimethyl sulfoxide; Wort , wortmannin.
    Figure Legend Snippet: c-Jun directly binds to the IRAK-M gene promoter through PI3K activation on IL-13 stimulation. A , Representative phospho–c-Jun and GAPDH Western blots (n = 4 independent experiments). B , Phospho– c-Jun activity data (means ± SEM) are from 3 independent experiments. C , Chromatin immunoprecipitation assay. Left panel , Increased c-Jun binding to IRAK-M gene promoter after IL-13. Data (means ± SEM) are from 3 independent experiments. Right panel , Representative agarose gel electrophoresis. Ab , Antibody; DMSO , dimethyl sulfoxide; Wort , wortmannin.

    Techniques Used: Activation Assay, Western Blot, Activity Assay, Chromatin Immunoprecipitation, Binding Assay, Agarose Gel Electrophoresis

    Increased IRAK-M protein expression in airway epithelial cells from asthmatic patients. Upper panel , Airway epithelial IRAK-M protein quantitative data in endobronchial biopsy specimens of healthy subjects (n = 4) and asthmatic patients (n = 6). Lower panel , Representative IRAK-M immunohistochemistry staining in bronchial epithelial cells and submucosal inflammatory cells ( black arrows , original magnification ×200). Data are presented as means (thick horizontal lines) ± SEMs.
    Figure Legend Snippet: Increased IRAK-M protein expression in airway epithelial cells from asthmatic patients. Upper panel , Airway epithelial IRAK-M protein quantitative data in endobronchial biopsy specimens of healthy subjects (n = 4) and asthmatic patients (n = 6). Lower panel , Representative IRAK-M immunohistochemistry staining in bronchial epithelial cells and submucosal inflammatory cells ( black arrows , original magnification ×200). Data are presented as means (thick horizontal lines) ± SEMs.

    Techniques Used: Expressing, Immunohistochemistry, Staining

    IRAK-M overexpression decreases TLR2 signaling activation in NCIH292 cells. A , Overexpression of human IRAK-M protein was confirmed by using Western blotting. B , NF-κB p65 activity. C , IL-8 protein levels in cell supernatants. Data are from 3 independent experiments and presented as means ± SEMs. EV , Empty vector; hIRAK-MV , human IRAK-M vector.
    Figure Legend Snippet: IRAK-M overexpression decreases TLR2 signaling activation in NCIH292 cells. A , Overexpression of human IRAK-M protein was confirmed by using Western blotting. B , NF-κB p65 activity. C , IL-8 protein levels in cell supernatants. Data are from 3 independent experiments and presented as means ± SEMs. EV , Empty vector; hIRAK-MV , human IRAK-M vector.

    Techniques Used: Over Expression, Activation Assay, Western Blot, Activity Assay, Plasmid Preparation

    6) Product Images from "Vimentin Regulates Scribble Activity by Protecting It from Proteasomal Degradation"

    Article Title: Vimentin Regulates Scribble Activity by Protecting It from Proteasomal Degradation

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E08-02-0199

    Silencing of Scrib or vimentin expression in MDCK cells leads to defects in cell morphology and Golgi complex orientation during directed cell migration. (A) Aberrant morphology. Monolayers of cells treated with nontargeting (a, e, and i), vimentin (b, f, and j), Scrib (c, g, and k), or Scrib and vimentin (d, h, and l) siRNA were wounded and stained with an antibody to ZO-2 (a–d) to visualize the cell outline. Scrib (e–h) and vimentin (i–l) were stained to monitor the effectiveness of the siRNA treatment. Note how in control cells the long axis of the cells is directed toward the wound edge (bottom of the images), whereas it is random in cells treated with the specific siRNAs. (B) Monolayers of cells treated with nontargeting (a), vimentin (b), Scrib (c), or Scrib and vimentin (d) siRNA were wounded and stained with an antibody to the cis -Golgi marker GM130 (red) and DAPI (blue) to label nuclei. The wound edge is demarcated with a white line. (e) Golgi complex orientation relative to the nucleus and the migration front was quantified as described in Materials and Methods . Shown is the fraction of leading edge cellswith correctly polarized Golgi complexes that position in front of the nucleus, facing the wound. Results represent the means of three independent experiments, in which at least 400 cells where scored for each condition. Error bars, SD of the mean. A red line indicates basal levels for a random orientation of 33%. (f) Schematic representation of Golgi complex orientation. The position of Golgi complex relative to the nucleus (blue) and wound edge was determined for ∼30 individual cells for each siRNA treatment and plotted. The shaded sector from 30° to 150° faces the wound edge and is bisected perpendicular to this edge. Note how the positioning of the Golgi complex of most control siRNA-treated cells falls within this sector, whereas that of cells where vimentin, Scrib, or both had been silenced is randomized.
    Figure Legend Snippet: Silencing of Scrib or vimentin expression in MDCK cells leads to defects in cell morphology and Golgi complex orientation during directed cell migration. (A) Aberrant morphology. Monolayers of cells treated with nontargeting (a, e, and i), vimentin (b, f, and j), Scrib (c, g, and k), or Scrib and vimentin (d, h, and l) siRNA were wounded and stained with an antibody to ZO-2 (a–d) to visualize the cell outline. Scrib (e–h) and vimentin (i–l) were stained to monitor the effectiveness of the siRNA treatment. Note how in control cells the long axis of the cells is directed toward the wound edge (bottom of the images), whereas it is random in cells treated with the specific siRNAs. (B) Monolayers of cells treated with nontargeting (a), vimentin (b), Scrib (c), or Scrib and vimentin (d) siRNA were wounded and stained with an antibody to the cis -Golgi marker GM130 (red) and DAPI (blue) to label nuclei. The wound edge is demarcated with a white line. (e) Golgi complex orientation relative to the nucleus and the migration front was quantified as described in Materials and Methods . Shown is the fraction of leading edge cellswith correctly polarized Golgi complexes that position in front of the nucleus, facing the wound. Results represent the means of three independent experiments, in which at least 400 cells where scored for each condition. Error bars, SD of the mean. A red line indicates basal levels for a random orientation of 33%. (f) Schematic representation of Golgi complex orientation. The position of Golgi complex relative to the nucleus (blue) and wound edge was determined for ∼30 individual cells for each siRNA treatment and plotted. The shaded sector from 30° to 150° faces the wound edge and is bisected perpendicular to this edge. Note how the positioning of the Golgi complex of most control siRNA-treated cells falls within this sector, whereas that of cells where vimentin, Scrib, or both had been silenced is randomized.

    Techniques Used: Expressing, Migration, Staining, Marker

    Slower wound closure rates due to a less directed migration of MDCK cells treated with Scrib or vimentin siRNA. (A) Wound closure. Monolayers of cells treated with nontargeting (a and e), vimentin (b and f), Scrib (c and g), or Scrib and vimentin (d and h) siRNA were wounded and allowed to migrate for 16 h. Images were taken after wounding (0 h; s a–d) or 16 h of migration (e–h). The black marks at the bottom of the dishes allow alignment of the wounds. Panels shown are representative of at least three independent experiments. (B) Quantification of cell migration directionality using live cell tracking. The X-Y graphs represent migration coordinates of 10 different cells at the wound edge treated with nontargeting (a), vimentin (b), Scrib (c), or vimentin and Scrib (d) siRNA, tracked over time 4 d after siRNA transfection. Start points for the different cells were adjusted to (0,0) coordinates. Results are representative of at least three independent experiments. (e) Tortuosity was scored for at least 30 individual cells for each siRNA treatment (n = 3; p
    Figure Legend Snippet: Slower wound closure rates due to a less directed migration of MDCK cells treated with Scrib or vimentin siRNA. (A) Wound closure. Monolayers of cells treated with nontargeting (a and e), vimentin (b and f), Scrib (c and g), or Scrib and vimentin (d and h) siRNA were wounded and allowed to migrate for 16 h. Images were taken after wounding (0 h; s a–d) or 16 h of migration (e–h). The black marks at the bottom of the dishes allow alignment of the wounds. Panels shown are representative of at least three independent experiments. (B) Quantification of cell migration directionality using live cell tracking. The X-Y graphs represent migration coordinates of 10 different cells at the wound edge treated with nontargeting (a), vimentin (b), Scrib (c), or vimentin and Scrib (d) siRNA, tracked over time 4 d after siRNA transfection. Start points for the different cells were adjusted to (0,0) coordinates. Results are representative of at least three independent experiments. (e) Tortuosity was scored for at least 30 individual cells for each siRNA treatment (n = 3; p

    Techniques Used: Migration, Cell Tracking Assay, Transfection

    siRNA-mediated depletion of endogenous vimentin and Scrib in MDCK cells. (A) Silencing of Scrib and vimentin monitored by immunofluorescence microscopy. Scrib (a–d, red) and vimentin (e–h, white) were visualized in MDCK cells treated for 3 d with a nontargeting siRNA (a and e) or siRNAs to vimentin (b and f), Scrib (c and g), or both Scrib and vimentin (d and h). (B) Silencing of Scrib and vimentin monitored by Western blot analysis. Scrib and vimentin protein levels in lysates of cells treated with siRNA over a 6-d period were monitored by Western blot on days 2, 4, and 6. Keratin 18 was detected to monitor for equal cell lysate loading.
    Figure Legend Snippet: siRNA-mediated depletion of endogenous vimentin and Scrib in MDCK cells. (A) Silencing of Scrib and vimentin monitored by immunofluorescence microscopy. Scrib (a–d, red) and vimentin (e–h, white) were visualized in MDCK cells treated for 3 d with a nontargeting siRNA (a and e) or siRNAs to vimentin (b and f), Scrib (c and g), or both Scrib and vimentin (d and h). (B) Silencing of Scrib and vimentin monitored by Western blot analysis. Scrib and vimentin protein levels in lysates of cells treated with siRNA over a 6-d period were monitored by Western blot on days 2, 4, and 6. Keratin 18 was detected to monitor for equal cell lysate loading.

    Techniques Used: Immunofluorescence, Microscopy, Western Blot

    Proteasome-dependent degradation of Scrib is inhibited by its interaction with vimentin. (A) Vimentin expression in MDCK cells was silenced using siRNA over 3 d. Cells were subsequently reseeded to sparse and confluent cultures, and Scrib protein levels were monitored by Western blot analysis on day 4. K18 was detected to check for e qual cell lysate loading. (B) Quantitative representation of Scrib down-regulation relative to levels of vimentin silencing in MDCK cells. (C) MDCK cells expressing EGFP-hScrib WT (∼250 kDa), LRR (∼130 kDa), PDZ (∼150 kDa), or, as a negative control, EGFP alone, were treated with vimentin (+) or nontargeting (−) siRNA. hScrib expression was analyzed by Western blot using antibodies to GFP. GAPDH served as a control for equal lysate input. (D) MDCK cells expressing EGFP-hScrib WT (a–d), LRR (e–h), or PDZ (i–l) were treated with nontargeting (a, b, e, f, I, and j) or vimentin (c, d, g, h, k, and l) siRNA and EGFP-hScrib (b, f, j, d, h, and l; green) and vimentin (a, e, i, c, g, and k; red) expression was visualized by fluorescence microscopy. (E) MDCK cells exogenously expressing EGFP vimentin, ECFP-K8, EYFP-K18, or EGFP alone were analyzed by Western blot for expression of Scrib. GAPDH served as a control for equal lysate input. (F–H) Effect of a proteasome inhibitor on Scrib turnover. (F) Western blot. MDCK cells expressing EGFP-hScrib WT were treated with vimentin (+) or nontargeting (−) siRNA for 3 d and subsequently in the presence of a proteasome inhibitor for 0, 3, 6, or 9 h. Scrib levels and vimentin expression levels were then analyzed by Western blot. Note how in vimentin depleted cells, EGFP-hScrib (250 kDa) as well as endogenous Scrib (220 kDa) degradation is blocked by the proteasome inhibitor (also see H). Actin served as a control for equal lysate input. (G) Immunofluorescence microscopy. MDCK cells expressing EGFP-hScrib WT were treated with vimentin siRNA and subsequently, a proteasome inhibitor for 0 h (a and b) or 9 h (c and d) and EGFP-hScrib (a and c; green) and vimentin (b and d; red) expression was visualized by fluorescence microscopy. (H) Western blot for endogenous Scrib. MDCK cells were treated with vimentin (+) or nontargeting (−) siRNA and subsequently in the presence of a proteasome inhibitor for 0, 3, 6, or 9 h. Endogenous levels of canine Scrib and vimentin were then analyzed by Western blot. GAPDH served as a control for equal lysate input. (I) hScrib-EGFP of nontargeting or vimentin siRNA-treated MDCK cells in the 9 h presence (+) or absence (−) of proteasome inhibitor was immunoprecipitated and ubiquitinylated hScrib detected by Western blot. Normal IgG served as a negative control.
    Figure Legend Snippet: Proteasome-dependent degradation of Scrib is inhibited by its interaction with vimentin. (A) Vimentin expression in MDCK cells was silenced using siRNA over 3 d. Cells were subsequently reseeded to sparse and confluent cultures, and Scrib protein levels were monitored by Western blot analysis on day 4. K18 was detected to check for e qual cell lysate loading. (B) Quantitative representation of Scrib down-regulation relative to levels of vimentin silencing in MDCK cells. (C) MDCK cells expressing EGFP-hScrib WT (∼250 kDa), LRR (∼130 kDa), PDZ (∼150 kDa), or, as a negative control, EGFP alone, were treated with vimentin (+) or nontargeting (−) siRNA. hScrib expression was analyzed by Western blot using antibodies to GFP. GAPDH served as a control for equal lysate input. (D) MDCK cells expressing EGFP-hScrib WT (a–d), LRR (e–h), or PDZ (i–l) were treated with nontargeting (a, b, e, f, I, and j) or vimentin (c, d, g, h, k, and l) siRNA and EGFP-hScrib (b, f, j, d, h, and l; green) and vimentin (a, e, i, c, g, and k; red) expression was visualized by fluorescence microscopy. (E) MDCK cells exogenously expressing EGFP vimentin, ECFP-K8, EYFP-K18, or EGFP alone were analyzed by Western blot for expression of Scrib. GAPDH served as a control for equal lysate input. (F–H) Effect of a proteasome inhibitor on Scrib turnover. (F) Western blot. MDCK cells expressing EGFP-hScrib WT were treated with vimentin (+) or nontargeting (−) siRNA for 3 d and subsequently in the presence of a proteasome inhibitor for 0, 3, 6, or 9 h. Scrib levels and vimentin expression levels were then analyzed by Western blot. Note how in vimentin depleted cells, EGFP-hScrib (250 kDa) as well as endogenous Scrib (220 kDa) degradation is blocked by the proteasome inhibitor (also see H). Actin served as a control for equal lysate input. (G) Immunofluorescence microscopy. MDCK cells expressing EGFP-hScrib WT were treated with vimentin siRNA and subsequently, a proteasome inhibitor for 0 h (a and b) or 9 h (c and d) and EGFP-hScrib (a and c; green) and vimentin (b and d; red) expression was visualized by fluorescence microscopy. (H) Western blot for endogenous Scrib. MDCK cells were treated with vimentin (+) or nontargeting (−) siRNA and subsequently in the presence of a proteasome inhibitor for 0, 3, 6, or 9 h. Endogenous levels of canine Scrib and vimentin were then analyzed by Western blot. GAPDH served as a control for equal lysate input. (I) hScrib-EGFP of nontargeting or vimentin siRNA-treated MDCK cells in the 9 h presence (+) or absence (−) of proteasome inhibitor was immunoprecipitated and ubiquitinylated hScrib detected by Western blot. Normal IgG served as a negative control.

    Techniques Used: Expressing, Western Blot, Negative Control, Fluorescence, Microscopy, Immunofluorescence, Immunoprecipitation

    Silencing of Scrib and vimentin expression affects cell–cell aggregation and spreading. (A) Cell aggregation. MDCK cells treated with nontargeting (a and e), vimentin (b and f), Scrib (c and g), or Scrib and vimentin (d and h) siRNA were allowed to aggregate in a hanging drop and photographed (a–d). (B) Cell spreading. Cell aggregates were transferred from the hanging drop onto coverslips and allowed to adhere and spread (e–h). Note how cells treated with specific siRNAs form less compact aggregates (b–d) and show enhanced spreading (f–h) compared with control cells (a and e, respectively). Assays were carried out 4 d after siRNA transfection.
    Figure Legend Snippet: Silencing of Scrib and vimentin expression affects cell–cell aggregation and spreading. (A) Cell aggregation. MDCK cells treated with nontargeting (a and e), vimentin (b and f), Scrib (c and g), or Scrib and vimentin (d and h) siRNA were allowed to aggregate in a hanging drop and photographed (a–d). (B) Cell spreading. Cell aggregates were transferred from the hanging drop onto coverslips and allowed to adhere and spread (e–h). Note how cells treated with specific siRNAs form less compact aggregates (b–d) and show enhanced spreading (f–h) compared with control cells (a and e, respectively). Assays were carried out 4 d after siRNA transfection.

    Techniques Used: Expressing, Transfection

    7) Product Images from "Cells Lacking the Fumarase Tumor Suppressor Are Protected from Apoptosis through a Hypoxia-Inducible Factor-Independent, AMPK-Dependent Mechanism"

    Article Title: Cells Lacking the Fumarase Tumor Suppressor Are Protected from Apoptosis through a Hypoxia-Inducible Factor-Independent, AMPK-Dependent Mechanism

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.06160-11

    Study of Bcl-2 family members in FH-defective cells. Pro- and antiapoptotic activities of Bcl-2 family members are regulated at either the transcriptional or posttranscriptional level. Thus, expression (A and B) and phosphorylation (C and D) of a number of them were studied by Western blot analysis of FH-defective (FH shRNA) and FH-proficient (untransduced) HK-2 cells before and after treatment with 40 μM CDDP for the indicated times. (A) After treatment, both the prosurvival proteins MCL-1 and BCL-X L were less decreased in FH-defective than in FH-proficient cells. (B) Expression of the proapoptotic proteins BMF and BAX was similarly induced by CDDP. (C) BCL-2 was comparably phosphorylated after treatment with CDDP in FH-defective and FH-proficient cells. (D) Conversely, the phosphorylation of BAD was higher in FH-defective HK-2 cells than in FH-proficient cells and was not modified by CDDP treatment. All blots were reprobed with either antibody against the total protein or a monoclonal vinculin antibody to show equal loading. (E) Treatment of FH-proficient HK-2 cells (untransduced) with AICAR, a cell-permeating AMPK activator, resulted in increased AMPK and BAD phosphorylation.
    Figure Legend Snippet: Study of Bcl-2 family members in FH-defective cells. Pro- and antiapoptotic activities of Bcl-2 family members are regulated at either the transcriptional or posttranscriptional level. Thus, expression (A and B) and phosphorylation (C and D) of a number of them were studied by Western blot analysis of FH-defective (FH shRNA) and FH-proficient (untransduced) HK-2 cells before and after treatment with 40 μM CDDP for the indicated times. (A) After treatment, both the prosurvival proteins MCL-1 and BCL-X L were less decreased in FH-defective than in FH-proficient cells. (B) Expression of the proapoptotic proteins BMF and BAX was similarly induced by CDDP. (C) BCL-2 was comparably phosphorylated after treatment with CDDP in FH-defective and FH-proficient cells. (D) Conversely, the phosphorylation of BAD was higher in FH-defective HK-2 cells than in FH-proficient cells and was not modified by CDDP treatment. All blots were reprobed with either antibody against the total protein or a monoclonal vinculin antibody to show equal loading. (E) Treatment of FH-proficient HK-2 cells (untransduced) with AICAR, a cell-permeating AMPK activator, resulted in increased AMPK and BAD phosphorylation.

    Techniques Used: Expressing, Western Blot, shRNA, Modification

    8) Product Images from "Tamoxifen inhibits malignant peripheral nerve sheath tumor growth in an estrogen receptor-independent manner"

    Article Title: Tamoxifen inhibits malignant peripheral nerve sheath tumor growth in an estrogen receptor-independent manner

    Journal: Neuro-Oncology

    doi: 10.1093/neuonc/noq146

    MPNST cells and MPNSTs express estrogen receptors. (A) Immunoblotted lysates of MCF-7 breast carcinoma cells and 6 human MPNST cell lines (STS-26T, ST88-14, NMS-2PC, 90-8, S462, and T265-2c cells) were probed for ERα, ERβ, and GPER. Blots were reprobed for GAPDH to compare loading. (B) Immunoblotted lysates of 4 surgically resected MPNSTs and non-neoplastic sciatic nerve were probed for ERα, ERβ, and GPER. Blots were reprobed for GAPDH to compare loading. (C) Real-time quantitative PCR comparing the expression of ERβ (left panel) and GPER (right panel) mRNA in dermal neurofibromas, plexiform neurofibromas, and MPNSTs. Bars indicate relative levels of expression. Note that values are expressed on a log 10 scale. 95% confidence intervals are indicated. Numbers above the bars indicate the fold change converted from log 10 .
    Figure Legend Snippet: MPNST cells and MPNSTs express estrogen receptors. (A) Immunoblotted lysates of MCF-7 breast carcinoma cells and 6 human MPNST cell lines (STS-26T, ST88-14, NMS-2PC, 90-8, S462, and T265-2c cells) were probed for ERα, ERβ, and GPER. Blots were reprobed for GAPDH to compare loading. (B) Immunoblotted lysates of 4 surgically resected MPNSTs and non-neoplastic sciatic nerve were probed for ERα, ERβ, and GPER. Blots were reprobed for GAPDH to compare loading. (C) Real-time quantitative PCR comparing the expression of ERβ (left panel) and GPER (right panel) mRNA in dermal neurofibromas, plexiform neurofibromas, and MPNSTs. Bars indicate relative levels of expression. Note that values are expressed on a log 10 scale. 95% confidence intervals are indicated. Numbers above the bars indicate the fold change converted from log 10 .

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    MPNST proliferation, survival, and tamoxifen responsiveness is ER independent. (A) Immunoblot analyses indicate that ERβ (left panels) and GPER (right panels) expression is ablated in ST88-14 cells stably transfected with plasmids expressing shRNAs targeting mRNAs encoding these receptors, but not in the parent line or ST88-14 cells stably transfected with a plasmid expressing a nonsense control shRNA (Control). Numbers above lanes indicate the transfected shRNA plasmid, with individual sublines identified by a letter following the plasmid number. Blots were reprobed for GAPDH to verify equal loading. (B) Estrogen response element–firefly luciferase reporters were transiently transfected into ST88-14 cells or ST88-14 cells stably transfected with an ERβ shRNA (583B). 48 hours later, cells were stimulated with 0–25 nM 17β-estradiol. Firefly luciferase activity was assayed and normalized to levels of Renilla luciferase expressed from a cotransfected constitutively active plasmid. (C) ST88-14 cells and ST88-14 cells stably transfected with a GPER shRNA were challenged for the indicated times with the GPER-specific agonist G1. Lysates of these cultures were immunoblotted and probed for phosphorylated ERK 1/2. Blots were reprobed for GAPDH to verify equal loading. (D) Average 3 H-thymidine incorporation and standard errors are indicated for ST88-14 cells (Parent) and sublines transfected with a nonsense control (Control) or shRNAs targeting ERβ or GPER. Levels of 3 H-thymidine incorporation are normalized to levels in the parent ST88-14 line. (E) ST88-14 cells (Parent) and sublines transfected with a nonsense control (Control), ERβ shRNA (583A) or GPER shRNA (584A) were challenged with vehicle (0) or 10 μM 4-hydroxy-tamoxifen for 24 hours and their ability to cleave calcein AM was then measured. Average calcein AM cleavage and standard errors are indicated and normalized to levels in the parent ST88-14 line.
    Figure Legend Snippet: MPNST proliferation, survival, and tamoxifen responsiveness is ER independent. (A) Immunoblot analyses indicate that ERβ (left panels) and GPER (right panels) expression is ablated in ST88-14 cells stably transfected with plasmids expressing shRNAs targeting mRNAs encoding these receptors, but not in the parent line or ST88-14 cells stably transfected with a plasmid expressing a nonsense control shRNA (Control). Numbers above lanes indicate the transfected shRNA plasmid, with individual sublines identified by a letter following the plasmid number. Blots were reprobed for GAPDH to verify equal loading. (B) Estrogen response element–firefly luciferase reporters were transiently transfected into ST88-14 cells or ST88-14 cells stably transfected with an ERβ shRNA (583B). 48 hours later, cells were stimulated with 0–25 nM 17β-estradiol. Firefly luciferase activity was assayed and normalized to levels of Renilla luciferase expressed from a cotransfected constitutively active plasmid. (C) ST88-14 cells and ST88-14 cells stably transfected with a GPER shRNA were challenged for the indicated times with the GPER-specific agonist G1. Lysates of these cultures were immunoblotted and probed for phosphorylated ERK 1/2. Blots were reprobed for GAPDH to verify equal loading. (D) Average 3 H-thymidine incorporation and standard errors are indicated for ST88-14 cells (Parent) and sublines transfected with a nonsense control (Control) or shRNAs targeting ERβ or GPER. Levels of 3 H-thymidine incorporation are normalized to levels in the parent ST88-14 line. (E) ST88-14 cells (Parent) and sublines transfected with a nonsense control (Control), ERβ shRNA (583A) or GPER shRNA (584A) were challenged with vehicle (0) or 10 μM 4-hydroxy-tamoxifen for 24 hours and their ability to cleave calcein AM was then measured. Average calcein AM cleavage and standard errors are indicated and normalized to levels in the parent ST88-14 line.

    Techniques Used: Expressing, Stable Transfection, Transfection, Plasmid Preparation, shRNA, Luciferase, Activity Assay

    9) Product Images from "DNA Mismatch Repair-dependent Activation of c-Abl/p73?/GADD45?-mediated Apoptosis *DNA Mismatch Repair-dependent Activation of c-Abl/p73?/GADD45?-mediated Apoptosis * S⃞"

    Article Title: DNA Mismatch Repair-dependent Activation of c-Abl/p73?/GADD45?-mediated Apoptosis *DNA Mismatch Repair-dependent Activation of c-Abl/p73?/GADD45?-mediated Apoptosis * S⃞

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M709954200

    hMLH1/c-Abl/p73α/GADD45α signaling controls G 2 arrest, apoptosis, and survival. Shown is the MMR detection of O 6 -methylguanine ( O 6 -MeG ) DNA lesions created after MNNG signals through c-Abl to increase p73α and GADD45α expression. c-Abl/GADD45α controls G 2 arrest, apoptosis, and survival, whereas c-Abl/p73α is needed in apoptosis and survival.
    Figure Legend Snippet: hMLH1/c-Abl/p73α/GADD45α signaling controls G 2 arrest, apoptosis, and survival. Shown is the MMR detection of O 6 -methylguanine ( O 6 -MeG ) DNA lesions created after MNNG signals through c-Abl to increase p73α and GADD45α expression. c-Abl/GADD45α controls G 2 arrest, apoptosis, and survival, whereas c-Abl/p73α is needed in apoptosis and survival.

    Techniques Used: Expressing

    10) Product Images from "ALG-2-interacting Tubby-like protein superfamily member PLSCR3 is secreted by an exosomal pathway and taken up by recipient cultured cells"

    Article Title: ALG-2-interacting Tubby-like protein superfamily member PLSCR3 is secreted by an exosomal pathway and taken up by recipient cultured cells

    Journal: Bioscience Reports

    doi: 10.1042/BSR20120123

    Secretion of Scr3 into culture medium ( A ) HEK-293/Scr3 cells and Scr3-expressing ALG-2 knockdown HEK-293 cells (HEK-293/Scr3/ALG-2 KD ) were cultured for 2 days, and each CM was centrifuged at 10000 g for 15 min. The obtained culture supernatant (Culture Sup) and TCL were subjected to WB using anti-Scr3 mAb (top panel), anti-ALG-2 pAb (middle panel) and anti-GAPDH mAb (bottom panel). ( B ) CM of HEK-293/Scr3 cells was fractionated by sequential centrifugations at different gravities, and the supernatants and pellets were analysed for Scr3 by WB. S 1 , supernatant of 1000 g , 10 min; P 1 , pellets of 1000 g , 10 min; S 2 , supernatant of centrifugation of S 1 at 10000 g , 15 min; P 2 , pellets of centrifugation of S 1 at 10000 g , 15 min; S 3 , supernatant of centrifugation of S 2 at 100000 g , 60 min; P 3 , pellets of centrifugation of S 2 at 100000 g , 60 min. ( C ) TCL and pellets of centrifugation of S 2 at 100000 g for 60 min of each CM-P 3 from HEK-293, HEK-293/ALG-2 KD , HEK-293/Scr3 and HEK-293/Scr3/ALG-2 KD cells were analysed for Scr3, Alix, ALG-2, Rab5B and GAPDH by WB using specific antibodies. Representative data obtained from three ( A ) and two ( B , C ) independent experiments are shown.
    Figure Legend Snippet: Secretion of Scr3 into culture medium ( A ) HEK-293/Scr3 cells and Scr3-expressing ALG-2 knockdown HEK-293 cells (HEK-293/Scr3/ALG-2 KD ) were cultured for 2 days, and each CM was centrifuged at 10000 g for 15 min. The obtained culture supernatant (Culture Sup) and TCL were subjected to WB using anti-Scr3 mAb (top panel), anti-ALG-2 pAb (middle panel) and anti-GAPDH mAb (bottom panel). ( B ) CM of HEK-293/Scr3 cells was fractionated by sequential centrifugations at different gravities, and the supernatants and pellets were analysed for Scr3 by WB. S 1 , supernatant of 1000 g , 10 min; P 1 , pellets of 1000 g , 10 min; S 2 , supernatant of centrifugation of S 1 at 10000 g , 15 min; P 2 , pellets of centrifugation of S 1 at 10000 g , 15 min; S 3 , supernatant of centrifugation of S 2 at 100000 g , 60 min; P 3 , pellets of centrifugation of S 2 at 100000 g , 60 min. ( C ) TCL and pellets of centrifugation of S 2 at 100000 g for 60 min of each CM-P 3 from HEK-293, HEK-293/ALG-2 KD , HEK-293/Scr3 and HEK-293/Scr3/ALG-2 KD cells were analysed for Scr3, Alix, ALG-2, Rab5B and GAPDH by WB using specific antibodies. Representative data obtained from three ( A ) and two ( B , C ) independent experiments are shown.

    Techniques Used: Expressing, Cell Culture, Western Blot, Centrifugation

    11) Product Images from "HSPB7 interacts with dimerized FLNC and its absence results in progressive myopathy in skeletal muscles"

    Article Title: HSPB7 interacts with dimerized FLNC and its absence results in progressive myopathy in skeletal muscles

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.179887

    Loss of HSPB7 results in FLNC and γ-sarcoglycan protein aggregation. Confocal micrographs of longitudinal sections (A) and cross-sections (B,C) of the diaphragm of 12-week-old mice. Specific antibodies were used to identify the distributions of the sarcomeric components α-actinin (Z-line), FLNC and γ-sarcoglycan as indicated. FLNC (arrows in A,C) and γ-sarcoglycan (arrows in B) aggregation were observed in the diaphragm muscle. The arrangement of α-actinin was normal (arrowheads in A). However, Z-line disarray was also observed in the diaphragm of CKO mice (asterisks in A). FLNC and γ-sarcoglycan were discontinuous or missing in some regions of the sarcolemma (arrowheads in B,C). Extracellular matrix accumulation was labeled with wheat germ agglutinin (WGA). FLNC mislocalizes on the sarcolemma (arrowheads in C). The nucleus was visualized by Hoechst 33342 staining. (D) Western blot analysis of expression levels of FLNC and α-actinin in the diaphragm muscle. The muscle homogenate supernatant (S) and pellet (P) fractions from control (Flox/Flox) and CKO mice at 12 and 36 weeks of age. GAPDH was used to verify the loading amount in the supernatant. Coomassie Blue staining (45–75 kDa) was used to verify the loading amount in the pellet. The graph shows the mean±s.d. densitometry results ( n =3). * P
    Figure Legend Snippet: Loss of HSPB7 results in FLNC and γ-sarcoglycan protein aggregation. Confocal micrographs of longitudinal sections (A) and cross-sections (B,C) of the diaphragm of 12-week-old mice. Specific antibodies were used to identify the distributions of the sarcomeric components α-actinin (Z-line), FLNC and γ-sarcoglycan as indicated. FLNC (arrows in A,C) and γ-sarcoglycan (arrows in B) aggregation were observed in the diaphragm muscle. The arrangement of α-actinin was normal (arrowheads in A). However, Z-line disarray was also observed in the diaphragm of CKO mice (asterisks in A). FLNC and γ-sarcoglycan were discontinuous or missing in some regions of the sarcolemma (arrowheads in B,C). Extracellular matrix accumulation was labeled with wheat germ agglutinin (WGA). FLNC mislocalizes on the sarcolemma (arrowheads in C). The nucleus was visualized by Hoechst 33342 staining. (D) Western blot analysis of expression levels of FLNC and α-actinin in the diaphragm muscle. The muscle homogenate supernatant (S) and pellet (P) fractions from control (Flox/Flox) and CKO mice at 12 and 36 weeks of age. GAPDH was used to verify the loading amount in the supernatant. Coomassie Blue staining (45–75 kDa) was used to verify the loading amount in the pellet. The graph shows the mean±s.d. densitometry results ( n =3). * P

    Techniques Used: Mouse Assay, Labeling, Whole Genome Amplification, Staining, Western Blot, Expressing

    Expression and localization of HSPB7 in skeletal muscle. (A) Western blot analysis of calf muscle showing HSPB7 expression from embryonic stages to adulthood (E14.5 to P56). (B) Immunohistochemical analysis of HSPB7 and MHC expression at E13.5 (arrows, muscle tissue; arrowheads, heart). (C) Western blot analysis of HSPB7 expression in the heart and various skeletal muscles. GAPDH was used as loading control. EDL, extensor digitorum longorum; TA, tibialis anterior. (D) Subcellular localization of HSPB7 in the soleus muscle of adult mice. The muscle sections were co-immunostained with antibodies against HSPB7 (red) and desmin (green), sarcomeric α-actinin (green) or γ-sarcoglycan (green). The nucleus was visualized by Hoechst 33342 staining. The arrows in A and C represent the expression of HSPB7. Scale bars: 500 μm (B, upper and middle panel); 200 μm (B, lower panel); 10 μm (D).
    Figure Legend Snippet: Expression and localization of HSPB7 in skeletal muscle. (A) Western blot analysis of calf muscle showing HSPB7 expression from embryonic stages to adulthood (E14.5 to P56). (B) Immunohistochemical analysis of HSPB7 and MHC expression at E13.5 (arrows, muscle tissue; arrowheads, heart). (C) Western blot analysis of HSPB7 expression in the heart and various skeletal muscles. GAPDH was used as loading control. EDL, extensor digitorum longorum; TA, tibialis anterior. (D) Subcellular localization of HSPB7 in the soleus muscle of adult mice. The muscle sections were co-immunostained with antibodies against HSPB7 (red) and desmin (green), sarcomeric α-actinin (green) or γ-sarcoglycan (green). The nucleus was visualized by Hoechst 33342 staining. The arrows in A and C represent the expression of HSPB7. Scale bars: 500 μm (B, upper and middle panel); 200 μm (B, lower panel); 10 μm (D).

    Techniques Used: Expressing, Western Blot, Immunohistochemistry, Mouse Assay, Staining

    12) Product Images from "The EBNA3 Family of Epstein-Barr Virus Nuclear Proteins Associates with the USP46/USP12 Deubiquitination Complexes to Regulate Lymphoblastoid Cell Line Growth"

    Article Title: The EBNA3 Family of Epstein-Barr Virus Nuclear Proteins Associates with the USP46/USP12 Deubiquitination Complexes to Regulate Lymphoblastoid Cell Line Growth

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004822

    EBNA3 proteins preferentially bind the WDR48 subunit of the USP46 DUB complex. (A) Immunoprecipitation assay in 293T cells demonstrating association of flag tagged EBNA3 proteins (F-E3A, F-E3B, and F-E3C) with WDR48 (left) and WDR20 (right). (B) Immunoprecipitation assay demonstrating WDR48 cotransfection enhanced USP46 association with EBNA3s (right panel, compare lanes 3–5 with 8–10). Epitope tagged BNRF1 (F-HA-BNRF1), an EBV protein of approximately the same size as the EBNA3 is included as an additional negative control. One percent (panel A) or two percent (panel B) of the input are shown for comparison.
    Figure Legend Snippet: EBNA3 proteins preferentially bind the WDR48 subunit of the USP46 DUB complex. (A) Immunoprecipitation assay in 293T cells demonstrating association of flag tagged EBNA3 proteins (F-E3A, F-E3B, and F-E3C) with WDR48 (left) and WDR20 (right). (B) Immunoprecipitation assay demonstrating WDR48 cotransfection enhanced USP46 association with EBNA3s (right panel, compare lanes 3–5 with 8–10). Epitope tagged BNRF1 (F-HA-BNRF1), an EBV protein of approximately the same size as the EBNA3 is included as an additional negative control. One percent (panel A) or two percent (panel B) of the input are shown for comparison.

    Techniques Used: Immunoprecipitation, Cotransfection, Negative Control

    Purified EBNA3 complexes exhibit DUB activity. (A) Tandem affinity purification was performed on wild type or EBNA3-F-HA expressing LCLs. Purified complexes from wild type (X), E3A-F-HA (closed circle), E3B-F-HA (closed triangle), or E3C-F-HA (closed square) LCLs were assayed for DUB activity by fluorometric assay using Ub-AMC as a substrate. (B) Western blot of purified EBNA3 complexes used in (A) demonstrating selective precipitation of tagged EBNA3 protein complexes and co-purification of RBPJ and USP46 proteins. Lysates derived from the equivalent of approximately 1.5x10 6 cells (input), 2.4x10 6 cells (Flag elution), or 10x10 6 cells (HA elution) were loaded on each lane, separated by SDS PAGE, and probed with EBNA3s, Flag, EBNA3A, and HA antibodies.
    Figure Legend Snippet: Purified EBNA3 complexes exhibit DUB activity. (A) Tandem affinity purification was performed on wild type or EBNA3-F-HA expressing LCLs. Purified complexes from wild type (X), E3A-F-HA (closed circle), E3B-F-HA (closed triangle), or E3C-F-HA (closed square) LCLs were assayed for DUB activity by fluorometric assay using Ub-AMC as a substrate. (B) Western blot of purified EBNA3 complexes used in (A) demonstrating selective precipitation of tagged EBNA3 protein complexes and co-purification of RBPJ and USP46 proteins. Lysates derived from the equivalent of approximately 1.5x10 6 cells (input), 2.4x10 6 cells (Flag elution), or 10x10 6 cells (HA elution) were loaded on each lane, separated by SDS PAGE, and probed with EBNA3s, Flag, EBNA3A, and HA antibodies.

    Techniques Used: Purification, Activity Assay, Affinity Purification, Expressing, Western Blot, Copurification, Derivative Assay, SDS Page

    Inability to derive USP46 null LCLs using CRISPR/ Cas9 mediated gene editing. (A) Western blot for USP46 in 721 LCLs cells transfected with a plasmid expressing either of two guide RNAs targeting different USP46 exons (CRISPR 1 or CRISPR 2) and a hygromycin resistance gene. Prior to harvesting, cells were subjected to hygromycin selection for one month (resulting in 6 clones for CRISPR 1 and 8 clones CRISPR 2). Untransfected 721 cells are also shown (WT). As a loading control, lysates were probed for beta actin (bottom panel). (B) Western blots of 293T cells that were transfected same CRISPR plasmids as in panel A and also subjected to one month of hygromycin selection. Hygromycin resistance cells were harvested and blotted for USP46 (specific band is indicated by arrowhead). Untransfected 293T cells are also shown (WT).
    Figure Legend Snippet: Inability to derive USP46 null LCLs using CRISPR/ Cas9 mediated gene editing. (A) Western blot for USP46 in 721 LCLs cells transfected with a plasmid expressing either of two guide RNAs targeting different USP46 exons (CRISPR 1 or CRISPR 2) and a hygromycin resistance gene. Prior to harvesting, cells were subjected to hygromycin selection for one month (resulting in 6 clones for CRISPR 1 and 8 clones CRISPR 2). Untransfected 721 cells are also shown (WT). As a loading control, lysates were probed for beta actin (bottom panel). (B) Western blots of 293T cells that were transfected same CRISPR plasmids as in panel A and also subjected to one month of hygromycin selection. Hygromycin resistance cells were harvested and blotted for USP46 (specific band is indicated by arrowhead). Untransfected 293T cells are also shown (WT).

    Techniques Used: CRISPR, Western Blot, Transfection, Plasmid Preparation, Expressing, Selection, Clone Assay

    EBNA3C associates with the WDR48/USP46 complex in EBNA3C-F-HA LCLs. Immunoprecipitation assay using Flag agarose to retrieve protein complexes from EBNA3C-F-HA LCLs (E3C-F-HA) is compared to flag immunoprecipiates from untagged wildtype (WT) LCLs. One percent of total cell lysate (Input) or immunoprepicitated specimens using Flag agarose (Flag IP) were separated by SDS PAGE and probed using antibodies to EBNA3C, RBPJ, CtBP1, WDR48, WDR20, USP46, or NF-kB p65.
    Figure Legend Snippet: EBNA3C associates with the WDR48/USP46 complex in EBNA3C-F-HA LCLs. Immunoprecipitation assay using Flag agarose to retrieve protein complexes from EBNA3C-F-HA LCLs (E3C-F-HA) is compared to flag immunoprecipiates from untagged wildtype (WT) LCLs. One percent of total cell lysate (Input) or immunoprepicitated specimens using Flag agarose (Flag IP) were separated by SDS PAGE and probed using antibodies to EBNA3C, RBPJ, CtBP1, WDR48, WDR20, USP46, or NF-kB p65.

    Techniques Used: Immunoprecipitation, SDS Page

    ChIP assay for WDR48 at the p14ARF promoter. Chromatin immunoprecipitation (ChIP) assays were performed using antibodies for WDR48 (A) from EBNA3C-HT LCLs that were grown in the presence of 4HT (dark gray) or after 14 days of growth in the absence of 4HT (light gray). Amount of genomic DNA was measured by real time PCR using primers specific to the EBNA3C binding site in the p14 ARF promoters or sites near the EIF2AK3 and PPIA genes which bind cell transcription factors but not EBNA3C. The bar graph represents the amount of DNA precipitated relative to the amount of DNA in the corresponding input sample. The experiment shown is representative of four independent experiments and error bars indicating standard error of the mean within this experiment. Asterisk denotes that the difference in ChIP signal seen at the p14 ARF promoter is statistically significant (p = 0.01). (B) Western blot for USP46, WDR48, and tubulin levels in whole cell lysates from EBNA3CHT LCLs grown in the presence of 4HT or after 14 days of growth in the absence of 4HT.
    Figure Legend Snippet: ChIP assay for WDR48 at the p14ARF promoter. Chromatin immunoprecipitation (ChIP) assays were performed using antibodies for WDR48 (A) from EBNA3C-HT LCLs that were grown in the presence of 4HT (dark gray) or after 14 days of growth in the absence of 4HT (light gray). Amount of genomic DNA was measured by real time PCR using primers specific to the EBNA3C binding site in the p14 ARF promoters or sites near the EIF2AK3 and PPIA genes which bind cell transcription factors but not EBNA3C. The bar graph represents the amount of DNA precipitated relative to the amount of DNA in the corresponding input sample. The experiment shown is representative of four independent experiments and error bars indicating standard error of the mean within this experiment. Asterisk denotes that the difference in ChIP signal seen at the p14 ARF promoter is statistically significant (p = 0.01). (B) Western blot for USP46, WDR48, and tubulin levels in whole cell lysates from EBNA3CHT LCLs grown in the presence of 4HT or after 14 days of growth in the absence of 4HT.

    Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Western Blot

    Subcellular localization of USP46 complexes in LCLs. Wild type LCLs were extracted into five subcellular fractions [cytoplasm (C), membrane (M), soluble nuclear (Nu), chromatin (Ch), cytoskeleton (CS)], resolved by SDS-PAGE and probed for EBNA3 proteins, RBPJ, and components of the USP46 complex (USP46, WDR20, and WDR48). Fraction purity was assessed by probing for tubulin, BRG1, Histone H2B, and LaminB.
    Figure Legend Snippet: Subcellular localization of USP46 complexes in LCLs. Wild type LCLs were extracted into five subcellular fractions [cytoplasm (C), membrane (M), soluble nuclear (Nu), chromatin (Ch), cytoskeleton (CS)], resolved by SDS-PAGE and probed for EBNA3 proteins, RBPJ, and components of the USP46 complex (USP46, WDR20, and WDR48). Fraction purity was assessed by probing for tubulin, BRG1, Histone H2B, and LaminB.

    Techniques Used: SDS Page

    13) Product Images from "A Buffered Alcohol-Based Fixative for Histomorphologic and Molecular Applications"

    Article Title: A Buffered Alcohol-Based Fixative for Histomorphologic and Molecular Applications

    Journal: Journal of Histochemistry and Cytochemistry

    doi: 10.1369/0022155416649579

    Protein quantity and quality of buffered ethanol 70% (BE70) fixative. (A) Amount of protein extracted from each condition was measured using the bicinchoninic acid (BCA) Protein Assay Kit. The protein extraction yield was expressed as the mean of three replicated samples (mean ± SD). (B) Protein integrity of different fixative solutions was assessed by western blotting. Proteins extracted from different fixative solutions were separated by 4% to 12% reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), electroblotted to nitrocellulose membrane, and probed with anti-aquaporin 1 (AQP1; 1:1000). (C) Western blotting by anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody. Relative GAPDH signal of each entity was normalized to neutral-buffered formalin (NBF). Abbreviations: E, 70% ethanol.
    Figure Legend Snippet: Protein quantity and quality of buffered ethanol 70% (BE70) fixative. (A) Amount of protein extracted from each condition was measured using the bicinchoninic acid (BCA) Protein Assay Kit. The protein extraction yield was expressed as the mean of three replicated samples (mean ± SD). (B) Protein integrity of different fixative solutions was assessed by western blotting. Proteins extracted from different fixative solutions were separated by 4% to 12% reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), electroblotted to nitrocellulose membrane, and probed with anti-aquaporin 1 (AQP1; 1:1000). (C) Western blotting by anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody. Relative GAPDH signal of each entity was normalized to neutral-buffered formalin (NBF). Abbreviations: E, 70% ethanol.

    Techniques Used: BIA-KA, Protein Extraction, Western Blot, Polyacrylamide Gel Electrophoresis, SDS Page

    14) Product Images from "Cardiac-Myocyte-Specific Excision of the Vinculin Gene Disrupts Cellular Junctions, Causing Sudden Death or Dilated Cardiomyopathy ▿"

    Article Title: Cardiac-Myocyte-Specific Excision of the Vinculin Gene Disrupts Cellular Junctions, Causing Sudden Death or Dilated Cardiomyopathy ▿

    Journal:

    doi: 10.1128/MCB.00728-07

    The expression of metavinculin transcript and protein is significantly reduced in heart, and vinculin/metavinculin immunoreactive expression is reduced in cardiac myocytes. (A and B) Whole-heart samples from 12-week-old mice were analyzed by using either
    Figure Legend Snippet: The expression of metavinculin transcript and protein is significantly reduced in heart, and vinculin/metavinculin immunoreactive expression is reduced in cardiac myocytes. (A and B) Whole-heart samples from 12-week-old mice were analyzed by using either

    Techniques Used: Expressing, Mouse Assay

    Cardiac myocytes null for vinculin (Vin) show lack of cadherin (Cad) expression and abnormal distribution of connexin 43. Tissue from physiologically normal cVclKO and control hearts was obtained at 8 weeks of age and examined by deconvolution microscopy.
    Figure Legend Snippet: Cardiac myocytes null for vinculin (Vin) show lack of cadherin (Cad) expression and abnormal distribution of connexin 43. Tissue from physiologically normal cVclKO and control hearts was obtained at 8 weeks of age and examined by deconvolution microscopy.

    Techniques Used: Expressing, Microscopy

    Construction of floxed Vcl mice and proof of cardiac-myocyte-specific Vcl exon 3 excision. (A) Construction of “floxed” vinculin mice. In the targeting construct, loxP recognition sites were positioned surrounding exon 3 (E3) of the Vcl
    Figure Legend Snippet: Construction of floxed Vcl mice and proof of cardiac-myocyte-specific Vcl exon 3 excision. (A) Construction of “floxed” vinculin mice. In the targeting construct, loxP recognition sites were positioned surrounding exon 3 (E3) of the Vcl

    Techniques Used: Mouse Assay, Construct

    Cardiac-myocyte-specific excision of the vinculin gene causes high mortality rates in cVclKO mice. (A) Male survival curves. (B) Female survival curves. Gender affects the mortality rate in cVclKO mice. By 14 weeks of age, only 51% of male cVclKO
    Figure Legend Snippet: Cardiac-myocyte-specific excision of the vinculin gene causes high mortality rates in cVclKO mice. (A) Male survival curves. (B) Female survival curves. Gender affects the mortality rate in cVclKO mice. By 14 weeks of age, only 51% of male cVclKO

    Techniques Used: Mouse Assay

    Cardiac-myocyte vinculin deficiency leads to high mortality rates.
    Figure Legend Snippet: Cardiac-myocyte vinculin deficiency leads to high mortality rates.

    Techniques Used:

    15) Product Images from "HSPB7 interacts with dimerized FLNC and its absence results in progressive myopathy in skeletal muscles"

    Article Title: HSPB7 interacts with dimerized FLNC and its absence results in progressive myopathy in skeletal muscles

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.179887

    HSPB7 interacts and colocalizes with FLNC. (A) Diaphragm muscle from adult Hspb7 Flox/Flox and wild-type (WT) mice was lysed and incubated with anti-FLAG M2 affinity gel for co-immunoprecipitation (IP) of proteins binding to HSPB7, and analyzed by western blotting. (B) The colocalization of HSPB7 and FLNC was assessed by confocal microscopy. The muscle sections were co-immunostained with antibodies against HSPB7 (red) and FLNC (green). The nucleus was visualized by Hoechst 33342 staining. (C) A construct of FLNC lacking the dimerization region (HA–FLNC-CΔ24) was overexpressed in HEK293T cells. The domains mediating interaction between HSPB7 and FLNC were assessed through co-immunoprecipitation in vitro . (D) The dimerization region deletion and mutations of FLNC-C (HA–W2711X and HA–M2670D) were overexpressed in HEK293T cells. The domains mediating interaction between HSPB7 and FLNC were assessed through co-immunoprecipitation in vitro . Scale bars: 10 μm.
    Figure Legend Snippet: HSPB7 interacts and colocalizes with FLNC. (A) Diaphragm muscle from adult Hspb7 Flox/Flox and wild-type (WT) mice was lysed and incubated with anti-FLAG M2 affinity gel for co-immunoprecipitation (IP) of proteins binding to HSPB7, and analyzed by western blotting. (B) The colocalization of HSPB7 and FLNC was assessed by confocal microscopy. The muscle sections were co-immunostained with antibodies against HSPB7 (red) and FLNC (green). The nucleus was visualized by Hoechst 33342 staining. (C) A construct of FLNC lacking the dimerization region (HA–FLNC-CΔ24) was overexpressed in HEK293T cells. The domains mediating interaction between HSPB7 and FLNC were assessed through co-immunoprecipitation in vitro . (D) The dimerization region deletion and mutations of FLNC-C (HA–W2711X and HA–M2670D) were overexpressed in HEK293T cells. The domains mediating interaction between HSPB7 and FLNC were assessed through co-immunoprecipitation in vitro . Scale bars: 10 μm.

    Techniques Used: Mouse Assay, Incubation, Immunoprecipitation, Binding Assay, Western Blot, Confocal Microscopy, Staining, Construct, In Vitro

    16) Product Images from "Autoprocessing of human immunodeficiency virus type 1 protease miniprecursor fusions in mammalian cells"

    Article Title: Autoprocessing of human immunodeficiency virus type 1 protease miniprecursor fusions in mammalian cells

    Journal: AIDS Research and Therapy

    doi: 10.1186/1742-6405-7-27

    Known protease inhibitors block protease autoprocessing . A . HEK293T cells transfected with the indicated pEBG construct were incubated with or without protease inhibitors at increasing concentrations. Darunavir: 0.1 μM, 1 μM and 10 μM; Indinavir: 1 μM, 10 μM and 100 μM. Post-nuclear cell lysates were prepared at 40 h post-transfection and aliquots (~20 μL) of each sample were analyzed in parallel using monoclonal mouse anti-Flag, anti-HA, anti-GAPDH primary antibodies and IR800 goat anti-mouse secondary antibody. Schematic diagrams of the full length fusion precursor and processing products are indicated at left. Molecular mass markers (kDa) are indicated at right. B . HEK293T cells that were transfected with NL4-3-derived proviruses encoding the indicated proteases were incubated with or without protease inhibitors at the same concentrations as in panel A. Post-nuclear cell lysates (Cell) and VLP particles (VLP) were prepared as described (Materials and Methods) and subjected to western blot analysis using monoclonal mouse anti-p24. The full length Gag polyprotein (p55) and p24/p25 doublet are indicated at left.
    Figure Legend Snippet: Known protease inhibitors block protease autoprocessing . A . HEK293T cells transfected with the indicated pEBG construct were incubated with or without protease inhibitors at increasing concentrations. Darunavir: 0.1 μM, 1 μM and 10 μM; Indinavir: 1 μM, 10 μM and 100 μM. Post-nuclear cell lysates were prepared at 40 h post-transfection and aliquots (~20 μL) of each sample were analyzed in parallel using monoclonal mouse anti-Flag, anti-HA, anti-GAPDH primary antibodies and IR800 goat anti-mouse secondary antibody. Schematic diagrams of the full length fusion precursor and processing products are indicated at left. Molecular mass markers (kDa) are indicated at right. B . HEK293T cells that were transfected with NL4-3-derived proviruses encoding the indicated proteases were incubated with or without protease inhibitors at the same concentrations as in panel A. Post-nuclear cell lysates (Cell) and VLP particles (VLP) were prepared as described (Materials and Methods) and subjected to western blot analysis using monoclonal mouse anti-p24. The full length Gag polyprotein (p55) and p24/p25 doublet are indicated at left.

    Techniques Used: Blocking Assay, Transfection, Construct, Incubation, Derivative Assay, Western Blot

    17) Product Images from "Cardiac-Myocyte-Specific Excision of the Vinculin Gene Disrupts Cellular Junctions, Causing Sudden Death or Dilated Cardiomyopathy ▿"

    Article Title: Cardiac-Myocyte-Specific Excision of the Vinculin Gene Disrupts Cellular Junctions, Causing Sudden Death or Dilated Cardiomyopathy ▿

    Journal:

    doi: 10.1128/MCB.00728-07

    Cardiac myocytes null for vinculin (Vin) show lack of cadherin (Cad) expression and abnormal distribution of connexin 43. Tissue from physiologically normal cVclKO and control hearts was obtained at 8 weeks of age and examined by deconvolution microscopy.
    Figure Legend Snippet: Cardiac myocytes null for vinculin (Vin) show lack of cadherin (Cad) expression and abnormal distribution of connexin 43. Tissue from physiologically normal cVclKO and control hearts was obtained at 8 weeks of age and examined by deconvolution microscopy.

    Techniques Used: Expressing, Microscopy

    18) Product Images from "FAM21 directs SNX27–retromer cargoes to the plasma membrane by preventing transport to the Golgi apparatus"

    Article Title: FAM21 directs SNX27–retromer cargoes to the plasma membrane by preventing transport to the Golgi apparatus

    Journal: Nature Communications

    doi: 10.1038/ncomms10939

    SNX27 interacts directly with FAM21 among the WASH complex components. ( a ) HEK293T cells were transfected with 3 × FLAG-tagged-WASH complex proteins. Lysates were incubated with GST or GST-SNX27 proteins and precipitated with Glutathione Sepharose 4B beads. Precipitates were then immunoblotted as shown. ( b ) Schematic representation of FAM21 constructs used. LFa motifs and α-helices are indicated. ( c , d ) HA-SNX27 was co-transfected with the indicated Myc-tagged constructs in HEK293T cells, and lysates were immunoprecipitated with anti-haemagglutinin (HA) antibodies and immunoblotted. Asterisks (*) denote IgG heavy chains. ( e ) GST or GST-SNX27 fusion proteins were incubated with indicated His-tagged FAM21 proteins and precipitated using Glutathione Sepharose 4B beads. Precipitates were immunoblotted as indicated. ( f ) Biotinylated peptides corresponding to FAM21 residues 40–79 and 592–600 were incubated with CBP-SNX27 and precipitated using streptavidin-conjugated beads. Precipitates were immunoblotted for CBP.
    Figure Legend Snippet: SNX27 interacts directly with FAM21 among the WASH complex components. ( a ) HEK293T cells were transfected with 3 × FLAG-tagged-WASH complex proteins. Lysates were incubated with GST or GST-SNX27 proteins and precipitated with Glutathione Sepharose 4B beads. Precipitates were then immunoblotted as shown. ( b ) Schematic representation of FAM21 constructs used. LFa motifs and α-helices are indicated. ( c , d ) HA-SNX27 was co-transfected with the indicated Myc-tagged constructs in HEK293T cells, and lysates were immunoprecipitated with anti-haemagglutinin (HA) antibodies and immunoblotted. Asterisks (*) denote IgG heavy chains. ( e ) GST or GST-SNX27 fusion proteins were incubated with indicated His-tagged FAM21 proteins and precipitated using Glutathione Sepharose 4B beads. Precipitates were immunoblotted as indicated. ( f ) Biotinylated peptides corresponding to FAM21 residues 40–79 and 592–600 were incubated with CBP-SNX27 and precipitated using streptavidin-conjugated beads. Precipitates were immunoblotted for CBP.

    Techniques Used: Transfection, Incubation, Construct, Immunoprecipitation

    19) Product Images from "HSPB7 interacts with dimerized FLNC and its absence results in progressive myopathy in skeletal muscles"

    Article Title: HSPB7 interacts with dimerized FLNC and its absence results in progressive myopathy in skeletal muscles

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.179887

    Loss of HSPB7 results in FLNC and γ-sarcoglycan protein aggregation. Confocal micrographs of longitudinal sections (A) and cross-sections (B,C) of the diaphragm of 12-week-old mice. Specific antibodies were used to identify the distributions of the sarcomeric components α-actinin (Z-line), FLNC and γ-sarcoglycan as indicated. FLNC (arrows in A,C) and γ-sarcoglycan (arrows in B) aggregation were observed in the diaphragm muscle. The arrangement of α-actinin was normal (arrowheads in A). However, Z-line disarray was also observed in the diaphragm of CKO mice (asterisks in A). FLNC and γ-sarcoglycan were discontinuous or missing in some regions of the sarcolemma (arrowheads in B,C). Extracellular matrix accumulation was labeled with wheat germ agglutinin (WGA). FLNC mislocalizes on the sarcolemma (arrowheads in C). The nucleus was visualized by Hoechst 33342 staining. (D) Western blot analysis of expression levels of FLNC and α-actinin in the diaphragm muscle. The muscle homogenate supernatant (S) and pellet (P) fractions from control (Flox/Flox) and CKO mice at 12 and 36 weeks of age. GAPDH was used to verify the loading amount in the supernatant. Coomassie Blue staining (45–75 kDa) was used to verify the loading amount in the pellet. The graph shows the mean±s.d. densitometry results ( n =3). * P
    Figure Legend Snippet: Loss of HSPB7 results in FLNC and γ-sarcoglycan protein aggregation. Confocal micrographs of longitudinal sections (A) and cross-sections (B,C) of the diaphragm of 12-week-old mice. Specific antibodies were used to identify the distributions of the sarcomeric components α-actinin (Z-line), FLNC and γ-sarcoglycan as indicated. FLNC (arrows in A,C) and γ-sarcoglycan (arrows in B) aggregation were observed in the diaphragm muscle. The arrangement of α-actinin was normal (arrowheads in A). However, Z-line disarray was also observed in the diaphragm of CKO mice (asterisks in A). FLNC and γ-sarcoglycan were discontinuous or missing in some regions of the sarcolemma (arrowheads in B,C). Extracellular matrix accumulation was labeled with wheat germ agglutinin (WGA). FLNC mislocalizes on the sarcolemma (arrowheads in C). The nucleus was visualized by Hoechst 33342 staining. (D) Western blot analysis of expression levels of FLNC and α-actinin in the diaphragm muscle. The muscle homogenate supernatant (S) and pellet (P) fractions from control (Flox/Flox) and CKO mice at 12 and 36 weeks of age. GAPDH was used to verify the loading amount in the supernatant. Coomassie Blue staining (45–75 kDa) was used to verify the loading amount in the pellet. The graph shows the mean±s.d. densitometry results ( n =3). * P

    Techniques Used: Mouse Assay, Labeling, Whole Genome Amplification, Staining, Western Blot, Expressing

    Expression and localization of HSPB7 in skeletal muscle. (A) Western blot analysis of calf muscle showing HSPB7 expression from embryonic stages to adulthood (E14.5 to P56). (B) Immunohistochemical analysis of HSPB7 and MHC expression at E13.5 (arrows, muscle tissue; arrowheads, heart). (C) Western blot analysis of HSPB7 expression in the heart and various skeletal muscles. GAPDH was used as loading control. EDL, extensor digitorum longorum; TA, tibialis anterior. (D) Subcellular localization of HSPB7 in the soleus muscle of adult mice. The muscle sections were co-immunostained with antibodies against HSPB7 (red) and desmin (green), sarcomeric α-actinin (green) or γ-sarcoglycan (green). The nucleus was visualized by Hoechst 33342 staining. The arrows in A and C represent the expression of HSPB7. Scale bars: 500 μm (B, upper and middle panel); 200 μm (B, lower panel); 10 μm (D).
    Figure Legend Snippet: Expression and localization of HSPB7 in skeletal muscle. (A) Western blot analysis of calf muscle showing HSPB7 expression from embryonic stages to adulthood (E14.5 to P56). (B) Immunohistochemical analysis of HSPB7 and MHC expression at E13.5 (arrows, muscle tissue; arrowheads, heart). (C) Western blot analysis of HSPB7 expression in the heart and various skeletal muscles. GAPDH was used as loading control. EDL, extensor digitorum longorum; TA, tibialis anterior. (D) Subcellular localization of HSPB7 in the soleus muscle of adult mice. The muscle sections were co-immunostained with antibodies against HSPB7 (red) and desmin (green), sarcomeric α-actinin (green) or γ-sarcoglycan (green). The nucleus was visualized by Hoechst 33342 staining. The arrows in A and C represent the expression of HSPB7. Scale bars: 500 μm (B, upper and middle panel); 200 μm (B, lower panel); 10 μm (D).

    Techniques Used: Expressing, Western Blot, Immunohistochemistry, Mouse Assay, Staining

    20) Product Images from "The Phosphatase PTPL1 Is Required for PTEN-Mediated Regulation of Apical Membrane Size"

    Article Title: The Phosphatase PTPL1 Is Required for PTEN-Mediated Regulation of Apical Membrane Size

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00102-18

    PTEN knockout (k.o.) W4 cells cannot restrict apical membrane formation. (A) Western blot of W4 cell and PTEN k.o. cell lysates probed for PTEN and β-catenin. (B) Localization of the actin marker Lifeact-Ruby in polarized W4 cells and PTEN k.o. cells. Scale bars, 5 μm. (C) Quantification of apical membrane size in doxycycline-stimulated W4 cells and PTEN k.o. cells. Red bars represent the average. Error bars represent the standard error of the mean (SEM) ( n > 19). *, P
    Figure Legend Snippet: PTEN knockout (k.o.) W4 cells cannot restrict apical membrane formation. (A) Western blot of W4 cell and PTEN k.o. cell lysates probed for PTEN and β-catenin. (B) Localization of the actin marker Lifeact-Ruby in polarized W4 cells and PTEN k.o. cells. Scale bars, 5 μm. (C) Quantification of apical membrane size in doxycycline-stimulated W4 cells and PTEN k.o. cells. Red bars represent the average. Error bars represent the standard error of the mean (SEM) ( n > 19). *, P

    Techniques Used: Knock-Out, Western Blot, Marker

    21) Product Images from "Ubiquitin-Specific Protease USP6 Regulates the Stability of the c-Jun Protein"

    Article Title: Ubiquitin-Specific Protease USP6 Regulates the Stability of the c-Jun Protein

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00320-17

    The enzyme activity of USP6 is important for its ability to regulate the stability of the c-Jun protein. (A) An empty vector or a plasmid expressing USP6 or USP6 C541S was transfected into HeLa cells, and the cells were harvested 36 h later. Whole-cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. (B) The same assay as in panel A, except that the plasmids were transfected into MCF7 cells. (C) The same assay as in panel A, except that the empty vector or USP6-expressing plasmid was transfected into HeLa cells. (D) The indicated plasmids were transfected into MCF7 cells, and the cells were harvested 24 h later. Whole-cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. All experiments were performed at least 3 times.
    Figure Legend Snippet: The enzyme activity of USP6 is important for its ability to regulate the stability of the c-Jun protein. (A) An empty vector or a plasmid expressing USP6 or USP6 C541S was transfected into HeLa cells, and the cells were harvested 36 h later. Whole-cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. (B) The same assay as in panel A, except that the plasmids were transfected into MCF7 cells. (C) The same assay as in panel A, except that the empty vector or USP6-expressing plasmid was transfected into HeLa cells. (D) The indicated plasmids were transfected into MCF7 cells, and the cells were harvested 24 h later. Whole-cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. All experiments were performed at least 3 times.

    Techniques Used: Activity Assay, Plasmid Preparation, Expressing, Transfection, SDS Page

    USP6 interacts with c-Jun and mediates c-Jun deubiquitination. (A) 293T cells were transfected with the indicated plasmids. The cell lysates were immunoprecipitated with an anti-Flag antibody. The lysates (TCL) and immunoprecipitates were immunoblotted. (B) 293T cells were transfected with the indicated plasmids. The cell lysates were immunoprecipitated with an anti-HA antibody. The lysates and immunoprecipitates were immunoblotted. (C) The empty vector or a plasmid expressing USP6 or USP6 C541S was cotransfected with c-Jun–HA and Flag-ubiquitin expression plasmids into 293T cells. The cells were treated with MG132 (10 μM) for 4 h, and then the cells were lysed in RIPA buffer and the cell lysates were immunoprecipitated with anti-HA antibody. The lysates and immunoprecipitates were immunoblotted with the indicated antibodies. (D) 293T cells were cotransfected with HA–c-Jun and Flag-Ub. After 4 h of MG132 treatment, c-Jun was purified using anti-HA antibody and then incubated with GST or GST-tagged recombinant USP6 or USP6-C541S. c-Jun ubiquitylation was detected by immunoblotting with anti-FLAG. All experiments were performed three times.
    Figure Legend Snippet: USP6 interacts with c-Jun and mediates c-Jun deubiquitination. (A) 293T cells were transfected with the indicated plasmids. The cell lysates were immunoprecipitated with an anti-Flag antibody. The lysates (TCL) and immunoprecipitates were immunoblotted. (B) 293T cells were transfected with the indicated plasmids. The cell lysates were immunoprecipitated with an anti-HA antibody. The lysates and immunoprecipitates were immunoblotted. (C) The empty vector or a plasmid expressing USP6 or USP6 C541S was cotransfected with c-Jun–HA and Flag-ubiquitin expression plasmids into 293T cells. The cells were treated with MG132 (10 μM) for 4 h, and then the cells were lysed in RIPA buffer and the cell lysates were immunoprecipitated with anti-HA antibody. The lysates and immunoprecipitates were immunoblotted with the indicated antibodies. (D) 293T cells were cotransfected with HA–c-Jun and Flag-Ub. After 4 h of MG132 treatment, c-Jun was purified using anti-HA antibody and then incubated with GST or GST-tagged recombinant USP6 or USP6-C541S. c-Jun ubiquitylation was detected by immunoblotting with anti-FLAG. All experiments were performed three times.

    Techniques Used: Transfection, Immunoprecipitation, Plasmid Preparation, Expressing, Purification, Incubation, Recombinant

    c-Jun is potentially involved in USP6 translocation nodular fasciitis disease. (A) The T statistics ( y axis) of all the genes measured ( x axis) in the data set were plotted. The dashed lines represent the threshold (FDR
    Figure Legend Snippet: c-Jun is potentially involved in USP6 translocation nodular fasciitis disease. (A) The T statistics ( y axis) of all the genes measured ( x axis) in the data set were plotted. The dashed lines represent the threshold (FDR

    Techniques Used: Translocation Assay

    USP6 regulates c-Jun downstream signaling. (A) Cells transfected with the empty vector or USP6 expression plasmid were harvested, and nuclear and cytoplasmic fractions were generated as described in Materials and Methods. In order to detect c-Jun protein, the nuclear fraction was not diluted as much as the cytoplasmic fraction. The final volumes of the nuclear fraction samples were about 1/8 those of the cytoplasmic fraction samples. M, protein marker; #, the histone 3 antibody or the goat anti-rabbit secondary antibody might cross-react with marker proteins (lane 3). (B) Luciferase activity of HeLa or MCF7 cells transfected with AP-1 luciferase reporter and a Renilla luciferase plasmid, together with the empty vector or a plasmid expressing USP6 or USP6 C541S. (C) Levels of MMP9 mRNA in HeLa cells transfected with the empty vector or a plasmid expressing USP6 or USP6 C541S determined using RT-PCR. (D) HeLa cells were infected with lentivirus carrying sh GFP or USP6 shRNAs. Forty-eight hours later, the RNA levels of MMP10 were determined using RT-PCR. (E) HeLa cells were infected with lentivirus carrying sh GFP or c- Jun shRNAs. Forty-eight hours later, the cells were harvested, subjected to SDS-PAGE, and analyzed by immunoblotting with the indicated antibodies. (F) The empty vector or a plasmid expressing USP6 was transfected into MCF7-sh GFP or c- Jun knockdown stable cell lines. The RNA levels of MMP10 were determined using RT-PCR. (G) Levels of the MMP9 mRNAs in MCF7-sh GFP and c- Jun knockdown stable cell lines transfected with the empty vector or a plasmid expressing USP6 were determined using RT-PCR. (H) A plasmid expressing USP6-Flag was transfected into MCF7-sh GFP or c- Jun knockdown stable cell lines; 36 h later, the cells were harvested, subjected to SDS-PAGE, and analyzed by immunoblotting with the indicated antibodies. (I) A plasmid expressing USP6 or an AP-1 luciferase reporter and a Renilla luciferase plasmid were transfected into MCF7-shGFP or c-Jun knockdown stable cell lines, and 24 h later, luciferase activities were determined. All experiments were performed at least 3 times. (B to D and F to I) The data are presented as means and SEM for three triplicate samples and were analyzed by t test. *, P
    Figure Legend Snippet: USP6 regulates c-Jun downstream signaling. (A) Cells transfected with the empty vector or USP6 expression plasmid were harvested, and nuclear and cytoplasmic fractions were generated as described in Materials and Methods. In order to detect c-Jun protein, the nuclear fraction was not diluted as much as the cytoplasmic fraction. The final volumes of the nuclear fraction samples were about 1/8 those of the cytoplasmic fraction samples. M, protein marker; #, the histone 3 antibody or the goat anti-rabbit secondary antibody might cross-react with marker proteins (lane 3). (B) Luciferase activity of HeLa or MCF7 cells transfected with AP-1 luciferase reporter and a Renilla luciferase plasmid, together with the empty vector or a plasmid expressing USP6 or USP6 C541S. (C) Levels of MMP9 mRNA in HeLa cells transfected with the empty vector or a plasmid expressing USP6 or USP6 C541S determined using RT-PCR. (D) HeLa cells were infected with lentivirus carrying sh GFP or USP6 shRNAs. Forty-eight hours later, the RNA levels of MMP10 were determined using RT-PCR. (E) HeLa cells were infected with lentivirus carrying sh GFP or c- Jun shRNAs. Forty-eight hours later, the cells were harvested, subjected to SDS-PAGE, and analyzed by immunoblotting with the indicated antibodies. (F) The empty vector or a plasmid expressing USP6 was transfected into MCF7-sh GFP or c- Jun knockdown stable cell lines. The RNA levels of MMP10 were determined using RT-PCR. (G) Levels of the MMP9 mRNAs in MCF7-sh GFP and c- Jun knockdown stable cell lines transfected with the empty vector or a plasmid expressing USP6 were determined using RT-PCR. (H) A plasmid expressing USP6-Flag was transfected into MCF7-sh GFP or c- Jun knockdown stable cell lines; 36 h later, the cells were harvested, subjected to SDS-PAGE, and analyzed by immunoblotting with the indicated antibodies. (I) A plasmid expressing USP6 or an AP-1 luciferase reporter and a Renilla luciferase plasmid were transfected into MCF7-shGFP or c-Jun knockdown stable cell lines, and 24 h later, luciferase activities were determined. All experiments were performed at least 3 times. (B to D and F to I) The data are presented as means and SEM for three triplicate samples and were analyzed by t test. *, P

    Techniques Used: Transfection, Plasmid Preparation, Expressing, Generated, Marker, Luciferase, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Infection, SDS Page, Stable Transfection

    USP6 regulates the stability of the c-Jun protein. (A) An empty vector or USP6 expression plasmid was transfected into MCF7 or HeLa cells. Thirty-six hours later, the cells were harvested. Whole-cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting (IB) with the indicated antibodies. (B) RNA levels of c-Jun in MCF7 cells transfected with the empty vector and a plasmid expressing USP6. (C) HeLa cells were infected with lentivirus that carried shRNAs targeted to GFP or USP6 . Thirty-six hours later, RNA levels of USP6 were determined using RT-PCR. (D) A USP6 expression plasmid was cotransfected with shGFP (shRNA targeted to GFP ) or USP6 shRNA plasmids into HeLa cells. Forty-eight hours later, the cells were harvested, subjected to SDS-PAGE, and analyzed by immunoblotting with the indicated antibodies. (E) Cell extracts of HeLa-shGFP or HeLa-USP6 knockdown stable cell lines were harvested and subjected to Western blotting to examine the levels of the indicated proteins. (F) A c-Jun–HA expression plasmid was cotransfected with the empty vector or a USP6 expression plasmid into HeLa cells. Twenty-four hours later, the cells were treated with CHX for the indicated times. Then, cell extracts were subjected to Western blotting to examine the levels of the indicated proteins. The intensity of c-Jun–HA expression for each time point was quantified by densitometry and plotted. The experiment was repeated three times, and a representative experiment is presented. (G) The indicated plasmids were transfected into MCF7 or HeLa cells. Twenty-four hours later, the cells were harvested, subjected to SDS-PAGE, and analyzed by immunoblotting with the indicated antibodies. (H) HeLa cells were transfected with the indicated plasmids; 24 h later, whole-cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. All experiments were performed at least 3 times. (B, C, and F) The data are presented as means ± standard errors of the mean (SEM) for three triplicate samples and were analyzed by t test. *, P
    Figure Legend Snippet: USP6 regulates the stability of the c-Jun protein. (A) An empty vector or USP6 expression plasmid was transfected into MCF7 or HeLa cells. Thirty-six hours later, the cells were harvested. Whole-cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting (IB) with the indicated antibodies. (B) RNA levels of c-Jun in MCF7 cells transfected with the empty vector and a plasmid expressing USP6. (C) HeLa cells were infected with lentivirus that carried shRNAs targeted to GFP or USP6 . Thirty-six hours later, RNA levels of USP6 were determined using RT-PCR. (D) A USP6 expression plasmid was cotransfected with shGFP (shRNA targeted to GFP ) or USP6 shRNA plasmids into HeLa cells. Forty-eight hours later, the cells were harvested, subjected to SDS-PAGE, and analyzed by immunoblotting with the indicated antibodies. (E) Cell extracts of HeLa-shGFP or HeLa-USP6 knockdown stable cell lines were harvested and subjected to Western blotting to examine the levels of the indicated proteins. (F) A c-Jun–HA expression plasmid was cotransfected with the empty vector or a USP6 expression plasmid into HeLa cells. Twenty-four hours later, the cells were treated with CHX for the indicated times. Then, cell extracts were subjected to Western blotting to examine the levels of the indicated proteins. The intensity of c-Jun–HA expression for each time point was quantified by densitometry and plotted. The experiment was repeated three times, and a representative experiment is presented. (G) The indicated plasmids were transfected into MCF7 or HeLa cells. Twenty-four hours later, the cells were harvested, subjected to SDS-PAGE, and analyzed by immunoblotting with the indicated antibodies. (H) HeLa cells were transfected with the indicated plasmids; 24 h later, whole-cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. All experiments were performed at least 3 times. (B, C, and F) The data are presented as means ± standard errors of the mean (SEM) for three triplicate samples and were analyzed by t test. *, P

    Techniques Used: Plasmid Preparation, Expressing, Transfection, SDS Page, Infection, Reverse Transcription Polymerase Chain Reaction, shRNA, Stable Transfection, Western Blot

    USP6 promotes cell migration and invasion. (A) HeLa cells were infected with the indicated lentivirus to generate stable cell lines, and 2 × 10 4 HeLa cells were seeded into the upper chamber of each Transwell. Twenty-four hours later, the cells were subjected to transwell assays to measure cell migration. (B) Approximately 2 × 10 4 of the indicated HeLa cells were seeded into the upper chamber of a Transwell containing Matrigel. Twenty-four hours later, the cells were subjected to an in vitro invasion assay. A zymography assay was performed to determine MMP activity as described in Materials and Methods. (C) HeLa cells were infected with lentivirus carrying sh GFP or USP6 shRNAs to generate stable cell lines, and 2 × 10 4 of the HeLa cells were seeded into the upper chamber of a Transwell containing Matrigel. Thirty-six hours later, the cells were subjected to an in vitro invasion assay. (D) The indicated HeLa stable cell lines were infected with lentivirus carrying sh GFP or c- Jun shRNAs; 48 h later, approximately 2 × 10 4 of the HeLa cells were seeded into the upper chamber of a Transwell containing Matrigel. Thirty-six hours later, the cells were subjected to an in vitro invasion assay. All experiments were performed 3 times. In all panels data are presented as means and SEM for three triplicate samples and were analyzed by t test. *, P
    Figure Legend Snippet: USP6 promotes cell migration and invasion. (A) HeLa cells were infected with the indicated lentivirus to generate stable cell lines, and 2 × 10 4 HeLa cells were seeded into the upper chamber of each Transwell. Twenty-four hours later, the cells were subjected to transwell assays to measure cell migration. (B) Approximately 2 × 10 4 of the indicated HeLa cells were seeded into the upper chamber of a Transwell containing Matrigel. Twenty-four hours later, the cells were subjected to an in vitro invasion assay. A zymography assay was performed to determine MMP activity as described in Materials and Methods. (C) HeLa cells were infected with lentivirus carrying sh GFP or USP6 shRNAs to generate stable cell lines, and 2 × 10 4 of the HeLa cells were seeded into the upper chamber of a Transwell containing Matrigel. Thirty-six hours later, the cells were subjected to an in vitro invasion assay. (D) The indicated HeLa stable cell lines were infected with lentivirus carrying sh GFP or c- Jun shRNAs; 48 h later, approximately 2 × 10 4 of the HeLa cells were seeded into the upper chamber of a Transwell containing Matrigel. Thirty-six hours later, the cells were subjected to an in vitro invasion assay. All experiments were performed 3 times. In all panels data are presented as means and SEM for three triplicate samples and were analyzed by t test. *, P

    Techniques Used: Migration, Infection, Stable Transfection, In Vitro, Invasion Assay, Zymography, Activity Assay

    22) Product Images from "The Phosphatase PTPL1 Is Required for PTEN-Mediated Regulation of Apical Membrane Size"

    Article Title: The Phosphatase PTPL1 Is Required for PTEN-Mediated Regulation of Apical Membrane Size

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00102-18

    PTEN knockout (k.o.) W4 cells cannot restrict apical membrane formation. (A) Western blot of W4 cell and PTEN k.o. cell lysates probed for PTEN and β-catenin. (B) Localization of the actin marker Lifeact-Ruby in polarized W4 cells and PTEN k.o. cells. Scale bars, 5 μm. (C) Quantification of apical membrane size in doxycycline-stimulated W4 cells and PTEN k.o. cells. Red bars represent the average. Error bars represent the standard error of the mean (SEM) ( n > 19). *, P
    Figure Legend Snippet: PTEN knockout (k.o.) W4 cells cannot restrict apical membrane formation. (A) Western blot of W4 cell and PTEN k.o. cell lysates probed for PTEN and β-catenin. (B) Localization of the actin marker Lifeact-Ruby in polarized W4 cells and PTEN k.o. cells. Scale bars, 5 μm. (C) Quantification of apical membrane size in doxycycline-stimulated W4 cells and PTEN k.o. cells. Red bars represent the average. Error bars represent the standard error of the mean (SEM) ( n > 19). *, P

    Techniques Used: Knock-Out, Western Blot, Marker

    23) Product Images from "A Novel Regulatory Mechanism of the Bone Morphogenetic Protein (BMP) Signaling Pathway Involving the Carboxyl-Terminal Tail Domain of BMP Type II Receptor ▿"

    Article Title: A Novel Regulatory Mechanism of the Bone Morphogenetic Protein (BMP) Signaling Pathway Involving the Carboxyl-Terminal Tail Domain of BMP Type II Receptor ▿

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00218-07

    Trb3 is essential for the BMP-mediated signal in PASMCs. (A) PASMCs were transiently transfected with 250 nM of siRNA directed against human Trb3 (hTrb3) or nontargeting siRNA (control) for 48 h, followed by stimulation with (+BMP4) or without (none) BMP4 (3 nM) for 48 h. Cells were stained with FITC-conjugated antibodies against SMC markers, SMA (αSM-Actin), or calponin (αCalponin; green) and DAPI (blue). Downregulation of endogenous Trb3 in PASMCs by siRNA was confirmed by FITC-anti-Trb3 (αTrb3) antibody staining. (B) PASMCs were transfected with 250 nM siRNA directed against human Trb3 (hTrb3) or nontargeting siRNA (control) for 48 h, followed by extraction of total RNA and RT-PCR analysis. SMA and Trb3 mRNA expression relative to GAPDH mRNA expression was measured by real-time PCR. Data are plotted as means ± standard errors for three independent experiments. *, P
    Figure Legend Snippet: Trb3 is essential for the BMP-mediated signal in PASMCs. (A) PASMCs were transiently transfected with 250 nM of siRNA directed against human Trb3 (hTrb3) or nontargeting siRNA (control) for 48 h, followed by stimulation with (+BMP4) or without (none) BMP4 (3 nM) for 48 h. Cells were stained with FITC-conjugated antibodies against SMC markers, SMA (αSM-Actin), or calponin (αCalponin; green) and DAPI (blue). Downregulation of endogenous Trb3 in PASMCs by siRNA was confirmed by FITC-anti-Trb3 (αTrb3) antibody staining. (B) PASMCs were transfected with 250 nM siRNA directed against human Trb3 (hTrb3) or nontargeting siRNA (control) for 48 h, followed by extraction of total RNA and RT-PCR analysis. SMA and Trb3 mRNA expression relative to GAPDH mRNA expression was measured by real-time PCR. Data are plotted as means ± standard errors for three independent experiments. *, P

    Techniques Used: Transfection, Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction

    24) Product Images from "Mitochondrial control of cell death induced by hyperosmotic stress"

    Article Title: Mitochondrial control of cell death induced by hyperosmotic stress

    Journal: Apoptosis

    doi: 10.1007/s10495-006-0328-x

    Involvement of the Bcl-2 protein family in hyperosmotic stressinduced death of A549 cells . (a) A549 cells were treated with the indicated dose of sorbitol for 6 h, followed by the cytofluorometric assessment of mitochondrial membrane potential (DiOC 6 (3) staining) and viability (PI staining). White columns illustrate the percentage of cells with a low mitochondrial membrane potential but still viable (DiOC 6 (3) low ). Black columns indicate the percentage of cells with disrupted plasma membrane (PI + ). Data are mean of duplicate experiments ± SEM. (b, c) A549 cells were transfected with empty liposomes (Mock) or with siRNAs targeting Bax, Bak, Bcl-2, Bcl-X L , p53, the voltage-dependent anion channel 1 (VDAC1) or an irrelevant “unrelated” control (UNR). (b) To check for the effects of siRNAs, total proteins were purified from transfected A549 cells at different time points (12, 24 or 48 h), separated according to molecular weight on mono-dimensional SDS-PAGE, and finally analyzed by immunoblotting with the indicated antibodies. Antibodies specific for Mcl-1 and Bcl-X L or for GAPDH were employed as loading controls. (c) A549 cells transfected for 48 h with the indicated siRNAs were treated with 600 mM sorbitol for additional 6 h, then analyzed at FACS for mitochondrial membrane potential (DiOC 6 (3) staining) and viability (PI staining). White columns depict the percentage of cells that have dissipated the mitochondrial membrane potential but are still viable (DiOC 6 (3) low ). The percentage of cells with disrupted plasma membrane (PI + ) is illustrated by black columns. Data are mean of duplicate experiments ± SEM. Dashed lines indicate the range of statistical insignificance from control cells ( p > 0.05, ± 3 SEM). For additional details please see also “Materials and methods”
    Figure Legend Snippet: Involvement of the Bcl-2 protein family in hyperosmotic stressinduced death of A549 cells . (a) A549 cells were treated with the indicated dose of sorbitol for 6 h, followed by the cytofluorometric assessment of mitochondrial membrane potential (DiOC 6 (3) staining) and viability (PI staining). White columns illustrate the percentage of cells with a low mitochondrial membrane potential but still viable (DiOC 6 (3) low ). Black columns indicate the percentage of cells with disrupted plasma membrane (PI + ). Data are mean of duplicate experiments ± SEM. (b, c) A549 cells were transfected with empty liposomes (Mock) or with siRNAs targeting Bax, Bak, Bcl-2, Bcl-X L , p53, the voltage-dependent anion channel 1 (VDAC1) or an irrelevant “unrelated” control (UNR). (b) To check for the effects of siRNAs, total proteins were purified from transfected A549 cells at different time points (12, 24 or 48 h), separated according to molecular weight on mono-dimensional SDS-PAGE, and finally analyzed by immunoblotting with the indicated antibodies. Antibodies specific for Mcl-1 and Bcl-X L or for GAPDH were employed as loading controls. (c) A549 cells transfected for 48 h with the indicated siRNAs were treated with 600 mM sorbitol for additional 6 h, then analyzed at FACS for mitochondrial membrane potential (DiOC 6 (3) staining) and viability (PI staining). White columns depict the percentage of cells that have dissipated the mitochondrial membrane potential but are still viable (DiOC 6 (3) low ). The percentage of cells with disrupted plasma membrane (PI + ) is illustrated by black columns. Data are mean of duplicate experiments ± SEM. Dashed lines indicate the range of statistical insignificance from control cells ( p > 0.05, ± 3 SEM). For additional details please see also “Materials and methods”

    Techniques Used: Staining, Transfection, Purification, Molecular Weight, SDS Page, FACS

    25) Product Images from "The EBNA3 Family of Epstein-Barr Virus Nuclear Proteins Associates with the USP46/USP12 Deubiquitination Complexes to Regulate Lymphoblastoid Cell Line Growth"

    Article Title: The EBNA3 Family of Epstein-Barr Virus Nuclear Proteins Associates with the USP46/USP12 Deubiquitination Complexes to Regulate Lymphoblastoid Cell Line Growth

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004822

    EBNA3 proteins preferentially bind the WDR48 subunit of the USP46 DUB complex. (A) Immunoprecipitation assay in 293T cells demonstrating association of flag tagged EBNA3 proteins (F-E3A, F-E3B, and F-E3C) with WDR48 (left) and WDR20 (right). (B) Immunoprecipitation assay demonstrating WDR48 cotransfection enhanced USP46 association with EBNA3s (right panel, compare lanes 3–5 with 8–10). Epitope tagged BNRF1 (F-HA-BNRF1), an EBV protein of approximately the same size as the EBNA3 is included as an additional negative control. One percent (panel A) or two percent (panel B) of the input are shown for comparison.
    Figure Legend Snippet: EBNA3 proteins preferentially bind the WDR48 subunit of the USP46 DUB complex. (A) Immunoprecipitation assay in 293T cells demonstrating association of flag tagged EBNA3 proteins (F-E3A, F-E3B, and F-E3C) with WDR48 (left) and WDR20 (right). (B) Immunoprecipitation assay demonstrating WDR48 cotransfection enhanced USP46 association with EBNA3s (right panel, compare lanes 3–5 with 8–10). Epitope tagged BNRF1 (F-HA-BNRF1), an EBV protein of approximately the same size as the EBNA3 is included as an additional negative control. One percent (panel A) or two percent (panel B) of the input are shown for comparison.

    Techniques Used: Immunoprecipitation, Cotransfection, Negative Control

    EBNA3A1-919, which associates with CtBP1 but not WDR48, is impaired for LCL growth maintenance. (A) Transcomplementation assay comparing growth of EBNA3A-HT cells transfected with EBNA3A WT (closed diamond), EBNA3A 1–826 (open square), EBNA3A mCtBP1 (open diamond), EBNA3A mRBPJ (open triangle), or EBNA3A 1–919 (X) in the absence of 4HT. EBNA3A-HT cells were also transfected with a control GFP expression plasmid, split, and maintained in either the presence (closed square) or absence (open circle) of 4HT. Cells were counted every 3 to 4 days, and diluted in fresh media to maintain a concentration of 200,000 cells/mL. Based on dilution factors, total cell number was calculated and is plotted on the Y-axis versus time. (B) Wild type EBNA3A, not mutant EBNA3A, suppresses p16 expression level in trans-complemented cells. After 15 days of EBNA3A WT or EBNA3A mutant (1–826, mCtBP1, mRBPJ, or 1–919) transfection, cells were harvested and protein expression was detected by immunoblotting with p16 antibody and actin as an internal control. As a control experiment, GFP expression plasmid was transfected into the cells and cultured with or without 4HT for 15 days. The ration of the p16 and actin bands was quantified and is indicated in the bottom panel.
    Figure Legend Snippet: EBNA3A1-919, which associates with CtBP1 but not WDR48, is impaired for LCL growth maintenance. (A) Transcomplementation assay comparing growth of EBNA3A-HT cells transfected with EBNA3A WT (closed diamond), EBNA3A 1–826 (open square), EBNA3A mCtBP1 (open diamond), EBNA3A mRBPJ (open triangle), or EBNA3A 1–919 (X) in the absence of 4HT. EBNA3A-HT cells were also transfected with a control GFP expression plasmid, split, and maintained in either the presence (closed square) or absence (open circle) of 4HT. Cells were counted every 3 to 4 days, and diluted in fresh media to maintain a concentration of 200,000 cells/mL. Based on dilution factors, total cell number was calculated and is plotted on the Y-axis versus time. (B) Wild type EBNA3A, not mutant EBNA3A, suppresses p16 expression level in trans-complemented cells. After 15 days of EBNA3A WT or EBNA3A mutant (1–826, mCtBP1, mRBPJ, or 1–919) transfection, cells were harvested and protein expression was detected by immunoblotting with p16 antibody and actin as an internal control. As a control experiment, GFP expression plasmid was transfected into the cells and cultured with or without 4HT for 15 days. The ration of the p16 and actin bands was quantified and is indicated in the bottom panel.

    Techniques Used: Transcomplementation Assay, Transfection, Expressing, Plasmid Preparation, Concentration Assay, Mutagenesis, Cell Culture

    EBNA3C associates with the WDR48/USP46 complex in EBNA3C-F-HA LCLs. Immunoprecipitation assay using Flag agarose to retrieve protein complexes from EBNA3C-F-HA LCLs (E3C-F-HA) is compared to flag immunoprecipiates from untagged wildtype (WT) LCLs. One percent of total cell lysate (Input) or immunoprepicitated specimens using Flag agarose (Flag IP) were separated by SDS PAGE and probed using antibodies to EBNA3C, RBPJ, CtBP1, WDR48, WDR20, USP46, or NF-kB p65.
    Figure Legend Snippet: EBNA3C associates with the WDR48/USP46 complex in EBNA3C-F-HA LCLs. Immunoprecipitation assay using Flag agarose to retrieve protein complexes from EBNA3C-F-HA LCLs (E3C-F-HA) is compared to flag immunoprecipiates from untagged wildtype (WT) LCLs. One percent of total cell lysate (Input) or immunoprepicitated specimens using Flag agarose (Flag IP) were separated by SDS PAGE and probed using antibodies to EBNA3C, RBPJ, CtBP1, WDR48, WDR20, USP46, or NF-kB p65.

    Techniques Used: Immunoprecipitation, SDS Page

    Identification of EBNA3A and EBNA3C domains that mediate WDR48 association. Immunoprecipitation assays to map WDR48 binding regions within EBNA3A (A) and EBNA3C (B). 293T cells were co-transfected with Xpress tagged WDR48 and flag tagged full length EBNA3A, EBNA3C, or the indicated EBNA3A or EBNA3C deletion mutants. Cell lysates were immunoprecipitated with Flag agarose, separated by SDS PAGE, and probed for WDR48 (anti-Xpress) and EBNA3 proteins (anti-Flag). (C) Comparison of WDR48 binding results (from B) with previously published RBPJ binding results and LCL growth phenotype for each EBNA3C mutant [ 21 ].
    Figure Legend Snippet: Identification of EBNA3A and EBNA3C domains that mediate WDR48 association. Immunoprecipitation assays to map WDR48 binding regions within EBNA3A (A) and EBNA3C (B). 293T cells were co-transfected with Xpress tagged WDR48 and flag tagged full length EBNA3A, EBNA3C, or the indicated EBNA3A or EBNA3C deletion mutants. Cell lysates were immunoprecipitated with Flag agarose, separated by SDS PAGE, and probed for WDR48 (anti-Xpress) and EBNA3 proteins (anti-Flag). (C) Comparison of WDR48 binding results (from B) with previously published RBPJ binding results and LCL growth phenotype for each EBNA3C mutant [ 21 ].

    Techniques Used: Immunoprecipitation, Binding Assay, Transfection, SDS Page, Mutagenesis

    Deletion of EBNA3A residues 920–944 disrupts WDR48 binding without affecting CtBP1 association. Co-immunoprecipitation assay to assess binding of EBNA3A mutants to WDR48 (A) and CtBP1 (B). For these assays, flag tagged full length EBNA3A (1–944), an EBNA3A CtBP1 binding mutant (mCtBP), an EBNA3A mutant lacking the C-terminal 25 residues (1–919), or vector control (pSG5) was co-transfected with Xpress-WDR48 or HA-CtBP1. Lysates were immunoprecipitated with Flag agarose (A) or HA agarose (B), separated by SDS PAGE, and probed with WDR48, RBPJ, flag, EBNA3A and HA antibodies.
    Figure Legend Snippet: Deletion of EBNA3A residues 920–944 disrupts WDR48 binding without affecting CtBP1 association. Co-immunoprecipitation assay to assess binding of EBNA3A mutants to WDR48 (A) and CtBP1 (B). For these assays, flag tagged full length EBNA3A (1–944), an EBNA3A CtBP1 binding mutant (mCtBP), an EBNA3A mutant lacking the C-terminal 25 residues (1–919), or vector control (pSG5) was co-transfected with Xpress-WDR48 or HA-CtBP1. Lysates were immunoprecipitated with Flag agarose (A) or HA agarose (B), separated by SDS PAGE, and probed with WDR48, RBPJ, flag, EBNA3A and HA antibodies.

    Techniques Used: Binding Assay, Co-Immunoprecipitation Assay, Mutagenesis, Plasmid Preparation, Transfection, Immunoprecipitation, SDS Page

    ChIP assay for WDR48 at the p14ARF promoter. Chromatin immunoprecipitation (ChIP) assays were performed using antibodies for WDR48 (A) from EBNA3C-HT LCLs that were grown in the presence of 4HT (dark gray) or after 14 days of growth in the absence of 4HT (light gray). Amount of genomic DNA was measured by real time PCR using primers specific to the EBNA3C binding site in the p14 ARF promoters or sites near the EIF2AK3 and PPIA genes which bind cell transcription factors but not EBNA3C. The bar graph represents the amount of DNA precipitated relative to the amount of DNA in the corresponding input sample. The experiment shown is representative of four independent experiments and error bars indicating standard error of the mean within this experiment. Asterisk denotes that the difference in ChIP signal seen at the p14 ARF promoter is statistically significant (p = 0.01). (B) Western blot for USP46, WDR48, and tubulin levels in whole cell lysates from EBNA3CHT LCLs grown in the presence of 4HT or after 14 days of growth in the absence of 4HT.
    Figure Legend Snippet: ChIP assay for WDR48 at the p14ARF promoter. Chromatin immunoprecipitation (ChIP) assays were performed using antibodies for WDR48 (A) from EBNA3C-HT LCLs that were grown in the presence of 4HT (dark gray) or after 14 days of growth in the absence of 4HT (light gray). Amount of genomic DNA was measured by real time PCR using primers specific to the EBNA3C binding site in the p14 ARF promoters or sites near the EIF2AK3 and PPIA genes which bind cell transcription factors but not EBNA3C. The bar graph represents the amount of DNA precipitated relative to the amount of DNA in the corresponding input sample. The experiment shown is representative of four independent experiments and error bars indicating standard error of the mean within this experiment. Asterisk denotes that the difference in ChIP signal seen at the p14 ARF promoter is statistically significant (p = 0.01). (B) Western blot for USP46, WDR48, and tubulin levels in whole cell lysates from EBNA3CHT LCLs grown in the presence of 4HT or after 14 days of growth in the absence of 4HT.

    Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Western Blot

    WDR48 coimmunoprecipitates with RBPJ in EBV infected cells. Co-immunoprecipitation assays comparing the association of RBPJ with WDR48 in LCLs with that observed in EBV negative BL41 cells. Cell lysates were immunoprecipitated with polyclonal RBPJ sera, separated by SDS PAGE, and probed for EBNA3A, EBNA3C, WDR48, and RBPJ (as indicated).
    Figure Legend Snippet: WDR48 coimmunoprecipitates with RBPJ in EBV infected cells. Co-immunoprecipitation assays comparing the association of RBPJ with WDR48 in LCLs with that observed in EBV negative BL41 cells. Cell lysates were immunoprecipitated with polyclonal RBPJ sera, separated by SDS PAGE, and probed for EBNA3A, EBNA3C, WDR48, and RBPJ (as indicated).

    Techniques Used: Infection, Immunoprecipitation, SDS Page

    WDR48 SLD2 mediates binding to EBNA3B and EBNA3C, but is not required for EBNA3A binding. Immunoprecipitation assays were performed to assess effect of deleting the WDR48 SUMO-like domains (SLDs) on EBNA3 binding. (A) 293T cells were co-transfected with vector control, Xpress tagged full length WDR48 (FL) or a deletion mutant lacking the SLD2 domain (WDR48 1–634) and flag tagged EBNA3A 1–944 (F-E3A), EBNA3B 394–938 (F-E3B) or EBNA3C 365–545 (F-E3C). Cell lysates were immunoprecipitated with Flag agarose, separated by SDS PAGE, and probed with WDR48 or Flag antibody. (B) Immunoprecipitation assay to determine effect of deleting SLD1/2 on WDR48 binding to EBNA3A. Assays were performed as describe above with co-transfection of EBNA3A WT and either full length WDR48 (FL), WDR48 1–535, or WDR48 1–430 (which lacks both SLD1 and SLD2).
    Figure Legend Snippet: WDR48 SLD2 mediates binding to EBNA3B and EBNA3C, but is not required for EBNA3A binding. Immunoprecipitation assays were performed to assess effect of deleting the WDR48 SUMO-like domains (SLDs) on EBNA3 binding. (A) 293T cells were co-transfected with vector control, Xpress tagged full length WDR48 (FL) or a deletion mutant lacking the SLD2 domain (WDR48 1–634) and flag tagged EBNA3A 1–944 (F-E3A), EBNA3B 394–938 (F-E3B) or EBNA3C 365–545 (F-E3C). Cell lysates were immunoprecipitated with Flag agarose, separated by SDS PAGE, and probed with WDR48 or Flag antibody. (B) Immunoprecipitation assay to determine effect of deleting SLD1/2 on WDR48 binding to EBNA3A. Assays were performed as describe above with co-transfection of EBNA3A WT and either full length WDR48 (FL), WDR48 1–535, or WDR48 1–430 (which lacks both SLD1 and SLD2).

    Techniques Used: Binding Assay, Immunoprecipitation, Transfection, Plasmid Preparation, Mutagenesis, SDS Page, Cotransfection

    26) Product Images from "Human CalDAG-GEFI gene (RASGRP2) mutation affects platelet function and causes severe bleeding"

    Article Title: Human CalDAG-GEFI gene (RASGRP2) mutation affects platelet function and causes severe bleeding

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20130477

    RASGRP2 gene mutation screening in family members and CalDAG-GEFI expression in platelets of the patients. (a) CalDAG-GEFI primary structure showing the G248W substitution and the different structural domains: ras exchange motif (REM), catalytic domain (CDC25), calcium-binding EF hands (EF), and diacylglycerol-binding (C1). (b) Multiple alignment of genomic DNA sequence (Chr11: 64,494,383-64,512,928; National Center for Biotechnology Information Build 36) surrounding the putative disease-causing mutation (in bold). K is the IUPAC-IUB ambiguity code for G or T. (c) Family pedigree. Whole-exome sequencing was performed in #01, #02, #04, #05, and #06. Direct capillary sequencing confirmed the complete segregation of the p.G248W mutation. (d) Relative CalDAG-GEFI mRNA expression levels from homozygous (HOM) and healthy (Ctl) platelets. The relative amounts of RASGRP2 mRNA were normalized to GAPDH mRNA levels. Data are mean ± SEM (Student’s t test). (e) Representative Western blot for CalDAG-GEFI in platelet lysates from two homozygous (HOM) and two healthy subjects (Ctl). GAPDH expression was used as equal loading and electrophoretic transfer control.
    Figure Legend Snippet: RASGRP2 gene mutation screening in family members and CalDAG-GEFI expression in platelets of the patients. (a) CalDAG-GEFI primary structure showing the G248W substitution and the different structural domains: ras exchange motif (REM), catalytic domain (CDC25), calcium-binding EF hands (EF), and diacylglycerol-binding (C1). (b) Multiple alignment of genomic DNA sequence (Chr11: 64,494,383-64,512,928; National Center for Biotechnology Information Build 36) surrounding the putative disease-causing mutation (in bold). K is the IUPAC-IUB ambiguity code for G or T. (c) Family pedigree. Whole-exome sequencing was performed in #01, #02, #04, #05, and #06. Direct capillary sequencing confirmed the complete segregation of the p.G248W mutation. (d) Relative CalDAG-GEFI mRNA expression levels from homozygous (HOM) and healthy (Ctl) platelets. The relative amounts of RASGRP2 mRNA were normalized to GAPDH mRNA levels. Data are mean ± SEM (Student’s t test). (e) Representative Western blot for CalDAG-GEFI in platelet lysates from two homozygous (HOM) and two healthy subjects (Ctl). GAPDH expression was used as equal loading and electrophoretic transfer control.

    Techniques Used: Mutagenesis, Expressing, Binding Assay, Sequencing, CTL Assay, Western Blot

    27) Product Images from "Synthetic lethality of TNK2 inhibition in PTPN11-mutant leukemia"

    Article Title: Synthetic lethality of TNK2 inhibition in PTPN11-mutant leukemia

    Journal: Science signaling

    doi: 10.1126/scisignal.aao5617

    Working model: Synthetic lethality of TNK2 inhibition in PTPN11-mutant leukemia.
    Figure Legend Snippet: Working model: Synthetic lethality of TNK2 inhibition in PTPN11-mutant leukemia.

    Techniques Used: Inhibition, Mutagenesis

    TNK2 increases signaling through PTPN11/RAS/MAPK in cells overexpressing mutant PTPN11.
    Figure Legend Snippet: TNK2 increases signaling through PTPN11/RAS/MAPK in cells overexpressing mutant PTPN11.

    Techniques Used: Mutagenesis

    A primary patient sample containing a PTPN11 mutation demonstrates dasatinib sensitivity and over-reliance on TNK2.
    Figure Legend Snippet: A primary patient sample containing a PTPN11 mutation demonstrates dasatinib sensitivity and over-reliance on TNK2.

    Techniques Used: Mutagenesis

    Inhibition of TNK2 reduces signaling through PTPN11/RAS/MAPK.
    Figure Legend Snippet: Inhibition of TNK2 reduces signaling through PTPN11/RAS/MAPK.

    Techniques Used: Inhibition

    Functional assays show increased transformation potential and sensitivity to TNK2 inhibition.
    Figure Legend Snippet: Functional assays show increased transformation potential and sensitivity to TNK2 inhibition.

    Techniques Used: Functional Assay, Transformation Assay, Inhibition

    28) Product Images from "Cardiac-Myocyte-Specific Excision of the Vinculin Gene Disrupts Cellular Junctions, Causing Sudden Death or Dilated Cardiomyopathy ▿"

    Article Title: Cardiac-Myocyte-Specific Excision of the Vinculin Gene Disrupts Cellular Junctions, Causing Sudden Death or Dilated Cardiomyopathy ▿

    Journal:

    doi: 10.1128/MCB.00728-07

    Cardiac myocytes null for vinculin (Vin) show lack of cadherin (Cad) expression and abnormal distribution of connexin 43. Tissue from physiologically normal cVclKO and control hearts was obtained at 8 weeks of age and examined by deconvolution microscopy.
    Figure Legend Snippet: Cardiac myocytes null for vinculin (Vin) show lack of cadherin (Cad) expression and abnormal distribution of connexin 43. Tissue from physiologically normal cVclKO and control hearts was obtained at 8 weeks of age and examined by deconvolution microscopy.

    Techniques Used: Expressing, Microscopy

    29) Product Images from "Mitochondrial control of cell death induced by hyperosmotic stress"

    Article Title: Mitochondrial control of cell death induced by hyperosmotic stress

    Journal: Apoptosis

    doi: 10.1007/s10495-006-0328-x

    Bcl-2 targeted to mitochondria protects Rat-1 fibroblasts from hyperosmotic stress-induced apoptosis . (a–e) Rat-1 fibroblasts were transfected with an empty control vector (CMV) or with a plasmid expressing Bcl-2 in its wild-type configuration (WT) or fused to peptides targeting it to mitochondria (Acta) or to endoplasmic reticulum (Cb5). (a) Rat-1 cells transfected with the abovementioned constructs were submitted to immunofluorescence staining with antibodies specific for Bcl-2 and the 60 kDa heat-shock protein (HSP-60, which colocalizes with mitochondria), to ensure the correct localization of the Bcl-2 fusion proteins. (b) Proteinswere extracted from Rat-1 fibroblasts transfected with the abovementioned plasmids, separated according to molecular weight on mono-dimensional SDS-PAGE, then analyzed by immunoblotting with antibodies specific for Bcl-2 and GAPDH (loading control). (c–e) Transfected Rat-1 fibroblasts were treated with 300 mM sorbitol for the indicated time then subjected to protein extraction for subsequent immunoblot (c) or to cytofluorometric analysis of mitochondrial membrane potential (DiOC 6 (3) staining) and viability (PI staining) (d–e). (c) After the treatment with 300 mM sorbitol for the indicated time, proteins were extracted, run on mono-dimensional SDS-PAGE, then analyzed by immunoblotting with antibodies specific for total caspase-3 and GAPDH (loading control). Caspase-3 activation corresponds to the progressive accumulation of the 17 kDa active caspase-3 fragment in parallel with a decrease of the pro-caspase-3 band (approx. 35 kDa). Only when Bcl-2 is targeted to mitochondria, sorbitol-induced caspase-3 activation is efficiently prevented. (d) Representative dot plots obtained after 6 h of treatment with 0 (control) or 300 mM sorbitol. X-axis, DiOC 6 (3) staining; Y-axis, PI staining. (e) Kinetic response of Rat-1 fibroblasts to sorbitol treatment. White columns depict the percentage of cells which have dissipated the mitochondrial membrane potential but are still viable (DiOC 6 (3) low ). The percentage of cells which have lost the integrity of plasma membrane (PI + ) is illustrated by black columns. Data are mean of triplicate experiments ± SEM. For further details please see the section “Materials and methods”
    Figure Legend Snippet: Bcl-2 targeted to mitochondria protects Rat-1 fibroblasts from hyperosmotic stress-induced apoptosis . (a–e) Rat-1 fibroblasts were transfected with an empty control vector (CMV) or with a plasmid expressing Bcl-2 in its wild-type configuration (WT) or fused to peptides targeting it to mitochondria (Acta) or to endoplasmic reticulum (Cb5). (a) Rat-1 cells transfected with the abovementioned constructs were submitted to immunofluorescence staining with antibodies specific for Bcl-2 and the 60 kDa heat-shock protein (HSP-60, which colocalizes with mitochondria), to ensure the correct localization of the Bcl-2 fusion proteins. (b) Proteinswere extracted from Rat-1 fibroblasts transfected with the abovementioned plasmids, separated according to molecular weight on mono-dimensional SDS-PAGE, then analyzed by immunoblotting with antibodies specific for Bcl-2 and GAPDH (loading control). (c–e) Transfected Rat-1 fibroblasts were treated with 300 mM sorbitol for the indicated time then subjected to protein extraction for subsequent immunoblot (c) or to cytofluorometric analysis of mitochondrial membrane potential (DiOC 6 (3) staining) and viability (PI staining) (d–e). (c) After the treatment with 300 mM sorbitol for the indicated time, proteins were extracted, run on mono-dimensional SDS-PAGE, then analyzed by immunoblotting with antibodies specific for total caspase-3 and GAPDH (loading control). Caspase-3 activation corresponds to the progressive accumulation of the 17 kDa active caspase-3 fragment in parallel with a decrease of the pro-caspase-3 band (approx. 35 kDa). Only when Bcl-2 is targeted to mitochondria, sorbitol-induced caspase-3 activation is efficiently prevented. (d) Representative dot plots obtained after 6 h of treatment with 0 (control) or 300 mM sorbitol. X-axis, DiOC 6 (3) staining; Y-axis, PI staining. (e) Kinetic response of Rat-1 fibroblasts to sorbitol treatment. White columns depict the percentage of cells which have dissipated the mitochondrial membrane potential but are still viable (DiOC 6 (3) low ). The percentage of cells which have lost the integrity of plasma membrane (PI + ) is illustrated by black columns. Data are mean of triplicate experiments ± SEM. For further details please see the section “Materials and methods”

    Techniques Used: Transfection, Plasmid Preparation, Expressing, Construct, Immunofluorescence, Staining, Molecular Weight, SDS Page, Protein Extraction, Activation Assay

    Involvement of the Bcl-2 protein family in hyperosmotic stressinduced death of A549 cells . (a) A549 cells were treated with the indicated dose of sorbitol for 6 h, followed by the cytofluorometric assessment of mitochondrial membrane potential (DiOC 6 (3) staining) and viability (PI staining). White columns illustrate the percentage of cells with a low mitochondrial membrane potential but still viable (DiOC 6 (3) low ). Black columns indicate the percentage of cells with disrupted plasma membrane (PI + ). Data are mean of duplicate experiments ± SEM. (b, c) A549 cells were transfected with empty liposomes (Mock) or with siRNAs targeting Bax, Bak, Bcl-2, Bcl-X L , p53, the voltage-dependent anion channel 1 (VDAC1) or an irrelevant “unrelated” control (UNR). (b) To check for the effects of siRNAs, total proteins were purified from transfected A549 cells at different time points (12, 24 or 48 h), separated according to molecular weight on mono-dimensional SDS-PAGE, and finally analyzed by immunoblotting with the indicated antibodies. Antibodies specific for Mcl-1 and Bcl-X L or for GAPDH were employed as loading controls. (c) A549 cells transfected for 48 h with the indicated siRNAs were treated with 600 mM sorbitol for additional 6 h, then analyzed at FACS for mitochondrial membrane potential (DiOC 6 (3) staining) and viability (PI staining). White columns depict the percentage of cells that have dissipated the mitochondrial membrane potential but are still viable (DiOC 6 (3) low ). The percentage of cells with disrupted plasma membrane (PI + ) is illustrated by black columns. Data are mean of duplicate experiments ± SEM. Dashed lines indicate the range of statistical insignificance from control cells ( p > 0.05, ± 3 SEM). For additional details please see also “Materials and methods”
    Figure Legend Snippet: Involvement of the Bcl-2 protein family in hyperosmotic stressinduced death of A549 cells . (a) A549 cells were treated with the indicated dose of sorbitol for 6 h, followed by the cytofluorometric assessment of mitochondrial membrane potential (DiOC 6 (3) staining) and viability (PI staining). White columns illustrate the percentage of cells with a low mitochondrial membrane potential but still viable (DiOC 6 (3) low ). Black columns indicate the percentage of cells with disrupted plasma membrane (PI + ). Data are mean of duplicate experiments ± SEM. (b, c) A549 cells were transfected with empty liposomes (Mock) or with siRNAs targeting Bax, Bak, Bcl-2, Bcl-X L , p53, the voltage-dependent anion channel 1 (VDAC1) or an irrelevant “unrelated” control (UNR). (b) To check for the effects of siRNAs, total proteins were purified from transfected A549 cells at different time points (12, 24 or 48 h), separated according to molecular weight on mono-dimensional SDS-PAGE, and finally analyzed by immunoblotting with the indicated antibodies. Antibodies specific for Mcl-1 and Bcl-X L or for GAPDH were employed as loading controls. (c) A549 cells transfected for 48 h with the indicated siRNAs were treated with 600 mM sorbitol for additional 6 h, then analyzed at FACS for mitochondrial membrane potential (DiOC 6 (3) staining) and viability (PI staining). White columns depict the percentage of cells that have dissipated the mitochondrial membrane potential but are still viable (DiOC 6 (3) low ). The percentage of cells with disrupted plasma membrane (PI + ) is illustrated by black columns. Data are mean of duplicate experiments ± SEM. Dashed lines indicate the range of statistical insignificance from control cells ( p > 0.05, ± 3 SEM). For additional details please see also “Materials and methods”

    Techniques Used: Staining, Transfection, Purification, Molecular Weight, SDS Page, FACS

    30) Product Images from "The EBNA3 Family of Epstein-Barr Virus Nuclear Proteins Associates with the USP46/USP12 Deubiquitination Complexes to Regulate Lymphoblastoid Cell Line Growth"

    Article Title: The EBNA3 Family of Epstein-Barr Virus Nuclear Proteins Associates with the USP46/USP12 Deubiquitination Complexes to Regulate Lymphoblastoid Cell Line Growth

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004822

    Inability to derive USP46 null LCLs using CRISPR/ Cas9 mediated gene editing. (A) Western blot for USP46 in 721 LCLs cells transfected with a plasmid expressing either of two guide RNAs targeting different USP46 exons (CRISPR 1 or CRISPR 2) and a hygromycin resistance gene. Prior to harvesting, cells were subjected to hygromycin selection for one month (resulting in 6 clones for CRISPR 1 and 8 clones CRISPR 2). Untransfected 721 cells are also shown (WT). As a loading control, lysates were probed for beta actin (bottom panel). (B) Western blots of 293T cells that were transfected same CRISPR plasmids as in panel A and also subjected to one month of hygromycin selection. Hygromycin resistance cells were harvested and blotted for USP46 (specific band is indicated by arrowhead). Untransfected 293T cells are also shown (WT).
    Figure Legend Snippet: Inability to derive USP46 null LCLs using CRISPR/ Cas9 mediated gene editing. (A) Western blot for USP46 in 721 LCLs cells transfected with a plasmid expressing either of two guide RNAs targeting different USP46 exons (CRISPR 1 or CRISPR 2) and a hygromycin resistance gene. Prior to harvesting, cells were subjected to hygromycin selection for one month (resulting in 6 clones for CRISPR 1 and 8 clones CRISPR 2). Untransfected 721 cells are also shown (WT). As a loading control, lysates were probed for beta actin (bottom panel). (B) Western blots of 293T cells that were transfected same CRISPR plasmids as in panel A and also subjected to one month of hygromycin selection. Hygromycin resistance cells were harvested and blotted for USP46 (specific band is indicated by arrowhead). Untransfected 293T cells are also shown (WT).

    Techniques Used: CRISPR, Western Blot, Transfection, Plasmid Preparation, Expressing, Selection, Clone Assay

    31) Product Images from "Manipulating Kv4.2 identifies a specific component of hippocampal pyramidal neuron A-current that depends upon Kv4.2 expression"

    Article Title: Manipulating Kv4.2 identifies a specific component of hippocampal pyramidal neuron A-current that depends upon Kv4.2 expression

    Journal:

    doi: 10.1111/j.1471-4159.2006.04185.x

    Increasing the expression of Kv4.2 selectively increases I SA . (a) Representative I SA current traces from neuron expressing empty pSuperRed (Control) or pCMV-Kv4.2. I SA component of current was recorded as described in . (b) Summary data shows
    Figure Legend Snippet: Increasing the expression of Kv4.2 selectively increases I SA . (a) Representative I SA current traces from neuron expressing empty pSuperRed (Control) or pCMV-Kv4.2. I SA component of current was recorded as described in . (b) Summary data shows

    Techniques Used: Expressing

    Expression of a dominant-negative Kv4 construct suppresses I SA to a similar extent as Kv4.2 RNA interference. (a) Summary graph comparing the suppression of I SA by Kv4.2-RNAi ( n = 7) with the suppression by Kv4DN-EGFP ( n = 8). Both approaches suppress
    Figure Legend Snippet: Expression of a dominant-negative Kv4 construct suppresses I SA to a similar extent as Kv4.2 RNA interference. (a) Summary graph comparing the suppression of I SA by Kv4.2-RNAi ( n = 7) with the suppression by Kv4DN-EGFP ( n = 8). Both approaches suppress

    Techniques Used: Expressing, Dominant Negative Mutation, Construct

    Sindbis vectors express Kv4.2 and Kv4 dominant-negative constructs in CA1 pyramidal neurons in organotypic culture. (a) High-powered view of CA1 pyramidal neurons infected with Sindbis-Kv4.2 that co-expresses EGFP. Entire cell fills evenly with green
    Figure Legend Snippet: Sindbis vectors express Kv4.2 and Kv4 dominant-negative constructs in CA1 pyramidal neurons in organotypic culture. (a) High-powered view of CA1 pyramidal neurons infected with Sindbis-Kv4.2 that co-expresses EGFP. Entire cell fills evenly with green

    Techniques Used: Dominant Negative Mutation, Construct, Infection

    Kv4.2 RNA interference selectively suppresses I SA . (a) Representative I SA current traces from neuron expressing pSuperRed (Control) or pSuperRed-Kv4.2i (Kv4.2-RNAi). I SA component of current was recorded as described in . (b) Summary data shows
    Figure Legend Snippet: Kv4.2 RNA interference selectively suppresses I SA . (a) Representative I SA current traces from neuron expressing pSuperRed (Control) or pSuperRed-Kv4.2i (Kv4.2-RNAi). I SA component of current was recorded as described in . (b) Summary data shows

    Techniques Used: Expressing

    pSuperRed-Kv4.2i selectively suppresses the expression of Kv4.2. (a) pSuperRed expressed DsRed2 from a CMV promoter and shRNAs from the H1 Pol III promoter. ShRNA sequences are directionally inserted into the Bgl II- Hind III sites. (b) pSuperRed clearly
    Figure Legend Snippet: pSuperRed-Kv4.2i selectively suppresses the expression of Kv4.2. (a) pSuperRed expressed DsRed2 from a CMV promoter and shRNAs from the H1 Pol III promoter. ShRNA sequences are directionally inserted into the Bgl II- Hind III sites. (b) pSuperRed clearly

    Techniques Used: Expressing, shRNA

    Modulating I SA amplitude selectively modulates the amplitude of the fast component for recovery from inactivation. (a) Representative traces during recovery from inactivation for neurons transfected with Kv4.2, pSuperRed (control) or pSuperRed-Kv4.2i
    Figure Legend Snippet: Modulating I SA amplitude selectively modulates the amplitude of the fast component for recovery from inactivation. (a) Representative traces during recovery from inactivation for neurons transfected with Kv4.2, pSuperRed (control) or pSuperRed-Kv4.2i

    Techniques Used: Transfection

    32) Product Images from "Cell-based chemical fingerprinting identifies telomeres and lamin A as modifiers of DNA damage response in cancer cells"

    Article Title: Cell-based chemical fingerprinting identifies telomeres and lamin A as modifiers of DNA damage response in cancer cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-33139-x

    Lamin A prevents MST-312-induced DNA double strand breaks. ( A ) Western blot analysis of lamin A/C in the JFCR39 cancer cell line panel. Coomassie brilliant blue gel staining is shown to confirm equal loading. Three different blots or gels were derived from the same experiment and were processed in parallel. Each blot/gel contains NCI-H23 cells as a calibration standard. Full-length blots/gels were presented in Supplementary Fig. S5 . ( B ) Correlation between MST-312 sensitivity and lamin A protein expression level in JFCR39. Representative cell lines are shown in orange and blue . GI 50 values were determined by the cell growth inhibition curve of 48-h treated cells in Fig. 1D . ( C ) Indirect immunofluorescence staining with anti-lamin A/C antibody ( green ). MST-312-sensitive [NCI-H522 (short telomeres) and DMS114 cells (super long telomeres)] and MST-312-resistant cells (HT-29, A549) are shown. DNA was counterstained with DAPI ( blue ). ( D ) Ectopic expression of lamin A in NCI-H522 cells. Cells were infected with retrovirus for expression of lamin A (LMNA) exogene and selected by hygromycin. The resulting cells, NCI-H522/mock and NCI-H522/LMNA, were subjected to western blot analysis. α-tubulin served as loading control. Full-length blots were presented in Supplementary Fig. S5 . ( E ) Cells in ( D ) were treated with 5 μM MST-312 for 48 h and further incubated with colcemid. Metaphase spreads of chromosomes were analysed for DNA double strand breaks. Error bar indicates standard deviation. Statistical significance was evaluated by Tukey-Kramer test.
    Figure Legend Snippet: Lamin A prevents MST-312-induced DNA double strand breaks. ( A ) Western blot analysis of lamin A/C in the JFCR39 cancer cell line panel. Coomassie brilliant blue gel staining is shown to confirm equal loading. Three different blots or gels were derived from the same experiment and were processed in parallel. Each blot/gel contains NCI-H23 cells as a calibration standard. Full-length blots/gels were presented in Supplementary Fig. S5 . ( B ) Correlation between MST-312 sensitivity and lamin A protein expression level in JFCR39. Representative cell lines are shown in orange and blue . GI 50 values were determined by the cell growth inhibition curve of 48-h treated cells in Fig. 1D . ( C ) Indirect immunofluorescence staining with anti-lamin A/C antibody ( green ). MST-312-sensitive [NCI-H522 (short telomeres) and DMS114 cells (super long telomeres)] and MST-312-resistant cells (HT-29, A549) are shown. DNA was counterstained with DAPI ( blue ). ( D ) Ectopic expression of lamin A in NCI-H522 cells. Cells were infected with retrovirus for expression of lamin A (LMNA) exogene and selected by hygromycin. The resulting cells, NCI-H522/mock and NCI-H522/LMNA, were subjected to western blot analysis. α-tubulin served as loading control. Full-length blots were presented in Supplementary Fig. S5 . ( E ) Cells in ( D ) were treated with 5 μM MST-312 for 48 h and further incubated with colcemid. Metaphase spreads of chromosomes were analysed for DNA double strand breaks. Error bar indicates standard deviation. Statistical significance was evaluated by Tukey-Kramer test.

    Techniques Used: Microscale Thermophoresis, Western Blot, Staining, Derivative Assay, Expressing, Inhibition, Immunofluorescence, Infection, Incubation, Standard Deviation

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    Luciferase:

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    Blocking Assay:

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    SYBR Green Assay:

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    Incubation:

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    Expressing:

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    Bradford Assay:

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    Western Blot:

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    Countercurrent Chromatography:

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    Transfection:

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    Protease Inhibitor:

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    Article Snippet: For immunoprecipitation (IP), HEK 293T cells were scraped using a rubber policeman in cold PBS 36 h after transfection with EGFP-mDia2 expression vector together with mCherry-importin β expression vector, and lysed in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 10 mM EDTA, 1% Triton X-100, pH 8.0) containing protease inhibitor cocktail (Roche) and phosphatase inhibitors. .. Antibodies used for immunoblotting and IP were: monoclonal mouse anti-α-tubulin (Sigma); monoclonal mouse anti-importin β (Abcam); polyclonal rabbit anti-GFP (Medical & Biological Laboratory); HRP-conjugated anti-mouse or anti-rabbit secondary antibodies (Bio-Rad).

    Quantitative RT-PCR:

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    Article Snippet: Quantitative RT-PCR was performed using a real-time detection system with Power SYBR green PCR Master Mix (Applied Biosystems, Foster City, CA) and specific primers for rat (r-) or human (h-) Rho family GTPases: r-Cdc42 (5′-TTC CCG TCG GAG TAT GTA CC-3′ and 5′-CAG GCA CCC ACT TTT CTT TC-3′), r-Rac1 (5′-AGT TAC ACG ACC AAT GCG TTC-3′ and 5′-AAT GAT GCA GGA CTC ACA AGG-3′), r-RhoA (5′-TGT GGC AGA TAT TGA AGT GGA C-3′ and 5′-CTT CTG GAG TCC ATT TTT CTG G-3′), r-RhoG (5′-ACA ACT AAT GCC TTC CCC AAG-3′ and 5′-AAC AGA TGA CGA AGA CGT TGG-3′), h-Cdc42(5′-CCT TTC TTG CTT GTT GGG ACT C-3′ and 5′-CTC CAC ATA CTT GAC AGC CTT C-3′), h-Rac1(5′-GTC CCA ACA CTC CCA TCA TCC-3′ and 5′-ACA GCA CCA ATC TCC TTA GCC-3′), h-RhoA (5′-GCA GGT AGA GTT GGC TTT GTG-3′ and 5′-GAC TTC TGG GGT CCA CTT TTC-3′), and h-RhoG (5′-TGC CTG CTC ATC TGC TAC AC-3′ and 5′-ACG AAA ACG TTG GTC TGA GG-3′). mRNA levels were normalized to GTPase mRNA in untransfected cells. .. Protein depletion by RNAi was normalized to endogenous levels of tubulin using mouse monoclonal anti-α-tubulin antibody (clone DM1A; Sigma, St. Louis, MO).

    Cell Culture:

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    Inhibition:

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    Article Snippet: Rabbit anti-actin polyclonal Ab against a synthetic peptide from the NH2 -terminal region (A5060, Sigma), mouse anti-myosin mAb against the myosin heavy chain polypeptide of human uterus smooth muscle extract (M7786, Sigma), mouse anti-α-tubulin mAb against the COOH-terminal region of α-tubulin isoform of sea urchin sperm axonemes (T5168, Sigma), mouse anti-β-tubulin mAb against purified rat brain tubulin (T4026, Sigma), and mouse anti-γ-tubulin mAb against a synthetic peptide from the NH2 -terminal region (T6557, Sigma) were used. .. This Ab was used to detect the parasites in cell invasion inhibition assay, as described below.

    Protein Concentration:

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    Polymerase Chain Reaction:

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    Article Snippet: Quantitative RT-PCR was performed using a real-time detection system with Power SYBR green PCR Master Mix (Applied Biosystems, Foster City, CA) and specific primers for rat (r-) or human (h-) Rho family GTPases: r-Cdc42 (5′-TTC CCG TCG GAG TAT GTA CC-3′ and 5′-CAG GCA CCC ACT TTT CTT TC-3′), r-Rac1 (5′-AGT TAC ACG ACC AAT GCG TTC-3′ and 5′-AAT GAT GCA GGA CTC ACA AGG-3′), r-RhoA (5′-TGT GGC AGA TAT TGA AGT GGA C-3′ and 5′-CTT CTG GAG TCC ATT TTT CTG G-3′), r-RhoG (5′-ACA ACT AAT GCC TTC CCC AAG-3′ and 5′-AAC AGA TGA CGA AGA CGT TGG-3′), h-Cdc42(5′-CCT TTC TTG CTT GTT GGG ACT C-3′ and 5′-CTC CAC ATA CTT GAC AGC CTT C-3′), h-Rac1(5′-GTC CCA ACA CTC CCA TCA TCC-3′ and 5′-ACA GCA CCA ATC TCC TTA GCC-3′), h-RhoA (5′-GCA GGT AGA GTT GGC TTT GTG-3′ and 5′-GAC TTC TGG GGT CCA CTT TTC-3′), and h-RhoG (5′-TGC CTG CTC ATC TGC TAC AC-3′ and 5′-ACG AAA ACG TTG GTC TGA GG-3′). mRNA levels were normalized to GTPase mRNA in untransfected cells. .. Protein depletion by RNAi was normalized to endogenous levels of tubulin using mouse monoclonal anti-α-tubulin antibody (clone DM1A; Sigma, St. Louis, MO).

    Affinity Purification:

    Article Title: Antagonism between C/EBP? and FOG in eosinophil lineage commitment of multipotent hematopoietic progenitors
    Article Snippet: .. Primary antibodies were: rabbit anti-chicken C/EBPβ (formerly anti-NF-M; ; kindly provided by Dr. A. Leutz, Max Delbruck Center for Molecular Medicine, Berlin, Germany) used at a 1:1000 dilution; affinity-purified rabbit anti-Mim-1 antibody ( ) (1:300 dilution), mouse anti-α-tubulin monoclonal antibody (clone DM 1A, Sigma) (1:300 dilution), and mouse anti-HA epitope monoclonal antibody (clone 12CA5; Roche) (1:5000 dilution). .. Secondary antibodies were horseradish peroxidase-conjugated anti-rabbit immunoglobulin and anti-mouse immunoglobulin (Amersham-Pharmacia) as appropriate, both 1:5000 in TBS-T. Immunodetection was performed by enhanced chemiluminescence (ECL, Amersham-Pharmacia) as recommended by the manufacturer.

    Immunofluorescence:

    Article Title: Cytotoxic Effects of Tropodithietic Acid on Mammalian Clonal Cell Lines of Neuronal and Glial Origin
    Article Snippet: Paragraph title: 3.5. Indirect Immunofluorescence ... Cells were incubated overnight at 4 °C with the following antibodies (the working dilutions are given in brackets): mouse mAb anti-α-tubulin (1:250) (Sigma, Munich, Germany).

    Article Title: Disease-associated mutations in TUBA1A result in a spectrum of defects in the tubulin folding and heterodimer assembly pathway
    Article Snippet: .. The efficiency of co-transfection in imaged cells was verified following confocal data acquisition by immunofluorescence using an anti-FLAG (rabbit polyclonal; Sigma-Aldrich Inc., St Louis, MO, USA) and either an anti-α-tubulin (mouse monoclonal DM1A; Sigma-Aldrich Inc., St Louis, MO, USA) antibody or an anti-βIII-tubulin (Tuj1) (mouse monoclonal Tuj1; Covance, Princeton, NJ, USA) antibody. ..

    Immunodetection:

    Article Title: Antagonism between C/EBP? and FOG in eosinophil lineage commitment of multipotent hematopoietic progenitors
    Article Snippet: Primary antibodies were: rabbit anti-chicken C/EBPβ (formerly anti-NF-M; ; kindly provided by Dr. A. Leutz, Max Delbruck Center for Molecular Medicine, Berlin, Germany) used at a 1:1000 dilution; affinity-purified rabbit anti-Mim-1 antibody ( ) (1:300 dilution), mouse anti-α-tubulin monoclonal antibody (clone DM 1A, Sigma) (1:300 dilution), and mouse anti-HA epitope monoclonal antibody (clone 12CA5; Roche) (1:5000 dilution). .. Secondary antibodies were horseradish peroxidase-conjugated anti-rabbit immunoglobulin and anti-mouse immunoglobulin (Amersham-Pharmacia) as appropriate, both 1:5000 in TBS-T. Immunodetection was performed by enhanced chemiluminescence (ECL, Amersham-Pharmacia) as recommended by the manufacturer.

    Labeling:

    Article Title: A novel polymer of tubulin forms the conoid of Toxoplasma gondii
    Article Snippet: .. After incubating in blocking buffer (5% fat-free dry milk in PBS with 0.02% Tween-20), blots were labeled with monoclonal mouse anti-GFP (Clontech Laboratories, Inc.) or monoclonal mouse anti–α-tubulin (Sigma-Aldrich; B5-1–2) diluted 1:1,000 in blocking buffer. .. Bound antibody was detected with goat anti–mouse IgG conjugated to horseradish peroxidase (1:3,000 in blocking buffer; Bio-Rad Laboratories), using the Supersignal West Pico Kit (Pierce Chemical Co.) according to the manufacturer's protocol.

    Article Title: Cytotoxic Effects of Tropodithietic Acid on Mammalian Clonal Cell Lines of Neuronal and Glial Origin
    Article Snippet: Cells were incubated overnight at 4 °C with the following antibodies (the working dilutions are given in brackets): mouse mAb anti-α-tubulin (1:250) (Sigma, Munich, Germany). .. Actin Staining was performed with Phalloidin labeled with FITC (Phalloidin Green, 1 µM) from Sigma (Munich, Germany) and mouse mAb anti-HSP60 (1:1000) from Enzo Life Sciences.

    Purification:

    Article Title: Abnormal proplatelet formation and emperipolesis in cultured human megakaryocytes from gray platelet syndrome patients
    Article Snippet: Type I collagen was purified as described previously . .. The following antibodies were used: monoclonal anti-CD61 (clone SZ21) (Immunotech, Marseille, France); goat monoclonal anti-CD61 (clone C-20) and mouse monoclonal anti-P-Selectin (clone 1E3) (Santa Cruz Biotechnology, California, USA); mouse monoclonal anti-α-tubulin (clone DM1A) and mouse monoclonal anti-human P-Selectin-allophycocyanin (APC) (clone clone AK4) (Sigma Aldrich, Milan, Italy); rabbit polyclonal anti-von Willebrand Factor (Dako, Milan, Italy); mouse monoclonal anti-Thrombospondin (clone A6.1), mouse monoclonal anti-CD61-fluorescein isothiocyanate (FITC) (clone PM6/13) and mouse monoclonal anti-CD42b-phycoerythrin (PE) (clone HIP1) (Abcam, Cambridge, UK); anti-CD61-FITC and anti-CD42b-PE (BD Biosciences, San José, CA, USA); Alexa Fluor-conjugated antibodies (Invitrogen, Milan, Italy).

    Article Title: Elongation Factor-1α Is a Novel Protein Associated with Host Cell Invasion and a Potential Protective Antigen of Cryptosporidium parvum *
    Article Snippet: .. Rabbit anti-actin polyclonal Ab against a synthetic peptide from the NH2 -terminal region (A5060, Sigma), mouse anti-myosin mAb against the myosin heavy chain polypeptide of human uterus smooth muscle extract (M7786, Sigma), mouse anti-α-tubulin mAb against the COOH-terminal region of α-tubulin isoform of sea urchin sperm axonemes (T5168, Sigma), mouse anti-β-tubulin mAb against purified rat brain tubulin (T4026, Sigma), and mouse anti-γ-tubulin mAb against a synthetic peptide from the NH2 -terminal region (T6557, Sigma) were used. ..

    Article Title: Kinesin-1 Regulates Microtubule Dynamics via a c-Jun N-terminal Kinase-dependent Mechanism *
    Article Snippet: Purified monoclonal mouse anti-kinesin antibody (clone Suk-4) was from Covance (Princeton, NJ). .. Monoclonal mouse anti-α-tubulin (clone DM1A) anti-mouse IgG fluorescein isothiocyanate and TRITC conjugates were from Sigma.

    Immunostaining:

    Article Title: Direct fluorescent-dye labeling of α-tubulin in mammalian cells for live cell and superresolution imaging
    Article Snippet: .. Immunostaining Cells were fixed with 4% paraformaldehyde (PFA), permeabilized with 0.5% Triton X-100 (in phosphate-buffered saline [PBS]) for 10 min, and blocked with 10% FBS (in PBS) for 15 min. All cells were stained with mouse monoclonal anti-α-tubulin antibodies (1:1000, DM1A; Sigma-Aldrich), rabbit polyclonal anti-HA antibodies (1:500; Applied Biological Materials, Richmond, Canada), or monoclonal anti-acetylated tubulin antibodies (1:1000, 6-11B-1; Sigma-Aldrich). .. Cells were then subjected to a secondary antibody staining using Alexa Fluor 594 or Alexa Fluor 488 anti-mouse or anti-rabbit secondary antibodies (Life Technologies).

    Cotransfection:

    Article Title: Disease-associated mutations in TUBA1A result in a spectrum of defects in the tubulin folding and heterodimer assembly pathway
    Article Snippet: .. The efficiency of co-transfection in imaged cells was verified following confocal data acquisition by immunofluorescence using an anti-FLAG (rabbit polyclonal; Sigma-Aldrich Inc., St Louis, MO, USA) and either an anti-α-tubulin (mouse monoclonal DM1A; Sigma-Aldrich Inc., St Louis, MO, USA) antibody or an anti-βIII-tubulin (Tuj1) (mouse monoclonal Tuj1; Covance, Princeton, NJ, USA) antibody. ..

    Lysis:

    Article Title: Novel localization of formin mDia2: importin β-mediated delivery to and retention at the cytoplasmic side of the nuclear envelope
    Article Snippet: The IP assay was made up of cell lysate containing 2 mg proteins, 4-5 µg GFP antibody (Medical & Biological Laboratory), 20 µl protein G sepharose beads (GE Healthcare) and topped up with lysis buffer to a final volume of 500-600 µl. .. Antibodies used for immunoblotting and IP were: monoclonal mouse anti-α-tubulin (Sigma); monoclonal mouse anti-importin β (Abcam); polyclonal rabbit anti-GFP (Medical & Biological Laboratory); HRP-conjugated anti-mouse or anti-rabbit secondary antibodies (Bio-Rad).

    Article Title: Comparative study on the effect of human BST-2/Tetherin on HIV-1 release in cells of various species
    Article Snippet: Briefly, the cells were lysed with lysis buffer (1% NP-40, 50 mM Tris-HCl [pH7.5], 150 mM NaCl, 1 mM EDTA, 1 mM Na3 VO4 , and 1 mM PMSF). .. For detection, the mouse anti-hBST-2 monoclonal antibody, a mouse anti-Tubulin monoclonal antibody (clone DM1A; Sigma), and an HRP-conjugated horse anti-mouse IgG antibody (Cell Signalling) were used.

    Activated Clotting Time Assay:

    Article Title: Salmonella enterica Serovar Typhimurium Invades Fibroblasts by Multiple Routes Differing from the Entry into Epithelial Cells ▿
    Article Snippet: Quantitative RT-PCR was performed using a real-time detection system with Power SYBR green PCR Master Mix (Applied Biosystems, Foster City, CA) and specific primers for rat (r-) or human (h-) Rho family GTPases: r-Cdc42 (5′-TTC CCG TCG GAG TAT GTA CC-3′ and 5′-CAG GCA CCC ACT TTT CTT TC-3′), r-Rac1 (5′-AGT TAC ACG ACC AAT GCG TTC-3′ and 5′-AAT GAT GCA GGA CTC ACA AGG-3′), r-RhoA (5′-TGT GGC AGA TAT TGA AGT GGA C-3′ and 5′-CTT CTG GAG TCC ATT TTT CTG G-3′), r-RhoG (5′-ACA ACT AAT GCC TTC CCC AAG-3′ and 5′-AAC AGA TGA CGA AGA CGT TGG-3′), h-Cdc42(5′-CCT TTC TTG CTT GTT GGG ACT C-3′ and 5′-CTC CAC ATA CTT GAC AGC CTT C-3′), h-Rac1(5′-GTC CCA ACA CTC CCA TCA TCC-3′ and 5′-ACA GCA CCA ATC TCC TTA GCC-3′), h-RhoA (5′-GCA GGT AGA GTT GGC TTT GTG-3′ and 5′-GAC TTC TGG GGT CCA CTT TTC-3′), and h-RhoG (5′-TGC CTG CTC ATC TGC TAC AC-3′ and 5′-ACG AAA ACG TTG GTC TGA GG-3′). mRNA levels were normalized to GTPase mRNA in untransfected cells. .. Protein depletion by RNAi was normalized to endogenous levels of tubulin using mouse monoclonal anti-α-tubulin antibody (clone DM1A; Sigma, St. Louis, MO).

    SDS Page:

    Article Title: Antagonism between C/EBP? and FOG in eosinophil lineage commitment of multipotent hematopoietic progenitors
    Article Snippet: Cells were lysed as described , the debris separated by centrifugation at 4°C, the extracts fractionated by SDS-PAGE, and blotted onto a PVDF membrane (Immobilon-P, Millipore). .. Primary antibodies were: rabbit anti-chicken C/EBPβ (formerly anti-NF-M; ; kindly provided by Dr. A. Leutz, Max Delbruck Center for Molecular Medicine, Berlin, Germany) used at a 1:1000 dilution; affinity-purified rabbit anti-Mim-1 antibody ( ) (1:300 dilution), mouse anti-α-tubulin monoclonal antibody (clone DM 1A, Sigma) (1:300 dilution), and mouse anti-HA epitope monoclonal antibody (clone 12CA5; Roche) (1:5000 dilution).

    Article Title: Oatp1 Enhances Bioluminescence by Acting as a Plasma Membrane Transporter for d-luciferin
    Article Snippet: Extracts were run on an SDS PAGE gel (NuPAGE, Invitrogen), transferred to a nitrocellulose membrane and luciferase-YFP detected using polyclonal goat anti-luciferase antibody (g475A, Promega), at 1 in 25,000 dilution with incubation for 1 h at room temperature. .. For α-tubulin staining a monoclonal mouse anti-α-tubulin (T1799, Sigma-Aldrich) was used at 1 in 1,000 dilution with overnight incubation at 4 °C.

    Article Title: Comparative study on the effect of human BST-2/Tetherin on HIV-1 release in cells of various species
    Article Snippet: The lysates were separated by SDS-PAGE and transferred to Immobilon transfer membrane (Millipore). .. For detection, the mouse anti-hBST-2 monoclonal antibody, a mouse anti-Tubulin monoclonal antibody (clone DM1A; Sigma), and an HRP-conjugated horse anti-mouse IgG antibody (Cell Signalling) were used.

    Plasmid Preparation:

    Article Title: Cytotoxic Effects of Tropodithietic Acid on Mammalian Clonal Cell Lines of Neuronal and Glial Origin
    Article Snippet: Cells were incubated overnight at 4 °C with the following antibodies (the working dilutions are given in brackets): mouse mAb anti-α-tubulin (1:250) (Sigma, Munich, Germany). .. Nuclei were stained by 4,6-diamidino-2-phenylindole (DAPI) (1.5 mg/mL) included in the mounting medium (Vectashield; Vector Laboratories, Burlingame, CA, USA).

    Article Title: Novel localization of formin mDia2: importin β-mediated delivery to and retention at the cytoplasmic side of the nuclear envelope
    Article Snippet: For immunoprecipitation (IP), HEK 293T cells were scraped using a rubber policeman in cold PBS 36 h after transfection with EGFP-mDia2 expression vector together with mCherry-importin β expression vector, and lysed in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 10 mM EDTA, 1% Triton X-100, pH 8.0) containing protease inhibitor cocktail (Roche) and phosphatase inhibitors. .. Antibodies used for immunoblotting and IP were: monoclonal mouse anti-α-tubulin (Sigma); monoclonal mouse anti-importin β (Abcam); polyclonal rabbit anti-GFP (Medical & Biological Laboratory); HRP-conjugated anti-mouse or anti-rabbit secondary antibodies (Bio-Rad).

    Article Title: Kinesin-1 Regulates Microtubule Dynamics via a c-Jun N-terminal Kinase-dependent Mechanism *
    Article Snippet: Monoclonal mouse anti-α-tubulin (clone DM1A) anti-mouse IgG fluorescein isothiocyanate and TRITC conjugates were from Sigma. .. GFP-tagged tubulin Living ColorsTM vector was from Clontech Laboratories.

    SDS-Gel:

    Article Title: A novel polymer of tubulin forms the conoid of Toxoplasma gondii
    Article Snippet: Western blotting Parasites were harvested, washed with PBS, lysed with 0.5% NP40, 2 mM EDTA in PBS, immediately put in SDS gel loading buffer, and heated at 70°C for 20 min. .. After incubating in blocking buffer (5% fat-free dry milk in PBS with 0.02% Tween-20), blots were labeled with monoclonal mouse anti-GFP (Clontech Laboratories, Inc.) or monoclonal mouse anti–α-tubulin (Sigma-Aldrich; B5-1–2) diluted 1:1,000 in blocking buffer.

    Immunoprecipitation:

    Article Title: Novel localization of formin mDia2: importin β-mediated delivery to and retention at the cytoplasmic side of the nuclear envelope
    Article Snippet: Paragraph title: Immunoblotting and immunoprecipitation assay ... Antibodies used for immunoblotting and IP were: monoclonal mouse anti-α-tubulin (Sigma); monoclonal mouse anti-importin β (Abcam); polyclonal rabbit anti-GFP (Medical & Biological Laboratory); HRP-conjugated anti-mouse or anti-rabbit secondary antibodies (Bio-Rad).

    Construct:

    Article Title: Kinesin-1 Regulates Microtubule Dynamics via a c-Jun N-terminal Kinase-dependent Mechanism *
    Article Snippet: Monoclonal mouse anti-α-tubulin (clone DM1A) anti-mouse IgG fluorescein isothiocyanate and TRITC conjugates were from Sigma. .. The YFP-CLIP-170 and mCherry-CLIP-170 were similar to the GFP-CLIP-170 construct described previously ( ).

    CTG Assay:

    Article Title: Salmonella enterica Serovar Typhimurium Invades Fibroblasts by Multiple Routes Differing from the Entry into Epithelial Cells ▿
    Article Snippet: Quantitative RT-PCR was performed using a real-time detection system with Power SYBR green PCR Master Mix (Applied Biosystems, Foster City, CA) and specific primers for rat (r-) or human (h-) Rho family GTPases: r-Cdc42 (5′-TTC CCG TCG GAG TAT GTA CC-3′ and 5′-CAG GCA CCC ACT TTT CTT TC-3′), r-Rac1 (5′-AGT TAC ACG ACC AAT GCG TTC-3′ and 5′-AAT GAT GCA GGA CTC ACA AGG-3′), r-RhoA (5′-TGT GGC AGA TAT TGA AGT GGA C-3′ and 5′-CTT CTG GAG TCC ATT TTT CTG G-3′), r-RhoG (5′-ACA ACT AAT GCC TTC CCC AAG-3′ and 5′-AAC AGA TGA CGA AGA CGT TGG-3′), h-Cdc42(5′-CCT TTC TTG CTT GTT GGG ACT C-3′ and 5′-CTC CAC ATA CTT GAC AGC CTT C-3′), h-Rac1(5′-GTC CCA ACA CTC CCA TCA TCC-3′ and 5′-ACA GCA CCA ATC TCC TTA GCC-3′), h-RhoA (5′-GCA GGT AGA GTT GGC TTT GTG-3′ and 5′-GAC TTC TGG GGT CCA CTT TTC-3′), and h-RhoG (5′-TGC CTG CTC ATC TGC TAC AC-3′ and 5′-ACG AAA ACG TTG GTC TGA GG-3′). mRNA levels were normalized to GTPase mRNA in untransfected cells. .. Protein depletion by RNAi was normalized to endogenous levels of tubulin using mouse monoclonal anti-α-tubulin antibody (clone DM1A; Sigma, St. Louis, MO).

    Staining:

    Article Title: Cytotoxic Effects of Tropodithietic Acid on Mammalian Clonal Cell Lines of Neuronal and Glial Origin
    Article Snippet: Cells were incubated overnight at 4 °C with the following antibodies (the working dilutions are given in brackets): mouse mAb anti-α-tubulin (1:250) (Sigma, Munich, Germany). .. Actin Staining was performed with Phalloidin labeled with FITC (Phalloidin Green, 1 µM) from Sigma (Munich, Germany) and mouse mAb anti-HSP60 (1:1000) from Enzo Life Sciences.

    Article Title: Oatp1 Enhances Bioluminescence by Acting as a Plasma Membrane Transporter for d-luciferin
    Article Snippet: .. For α-tubulin staining a monoclonal mouse anti-α-tubulin (T1799, Sigma-Aldrich) was used at 1 in 1,000 dilution with overnight incubation at 4 °C. .. For mStrawberry staining, rabbit polyclonal anti-RFP (ab34771, AbCam) was used at 1 in 10,000 dilution with overnight incubation at 4 °C.

    Article Title: Direct fluorescent-dye labeling of α-tubulin in mammalian cells for live cell and superresolution imaging
    Article Snippet: .. Immunostaining Cells were fixed with 4% paraformaldehyde (PFA), permeabilized with 0.5% Triton X-100 (in phosphate-buffered saline [PBS]) for 10 min, and blocked with 10% FBS (in PBS) for 15 min. All cells were stained with mouse monoclonal anti-α-tubulin antibodies (1:1000, DM1A; Sigma-Aldrich), rabbit polyclonal anti-HA antibodies (1:500; Applied Biological Materials, Richmond, Canada), or monoclonal anti-acetylated tubulin antibodies (1:1000, 6-11B-1; Sigma-Aldrich). .. Cells were then subjected to a secondary antibody staining using Alexa Fluor 594 or Alexa Fluor 488 anti-mouse or anti-rabbit secondary antibodies (Life Technologies).

    other:

    Article Title: A virus responds instantly to the presence of the vector on the host and forms transmission morphs
    Article Snippet: Antisera The following antibodies or antisera were used: rabbit anti-P2 , anti-P3 , anti-P6 , monoclonal mouse anti-α-tubulin DM1A ( http://www.sigmaaldrich.com ; ), and rabbit anti-P4 ( http://plant.neogeneurope.com ).

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    Millipore mouse anti glyceraldehyde 3 phosphate dehydrogenase gapdh monoclonal antibody
    Protein quantity and quality of buffered ethanol 70% (BE70) fixative. (A) Amount of protein extracted from each condition was measured using the bicinchoninic acid (BCA) Protein Assay Kit. The protein extraction yield was expressed as the mean of three replicated samples (mean ± SD). (B) Protein integrity of different fixative solutions was assessed by western blotting. Proteins extracted from different fixative solutions were separated by 4% to 12% reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), electroblotted to nitrocellulose membrane, and probed with anti-aquaporin 1 (AQP1; 1:1000). (C) Western blotting by anti-glyceraldehyde 3-phosphate dehydrogenase <t>(GAPDH)</t> antibody. Relative GAPDH signal of each entity was normalized to neutral-buffered formalin (NBF). Abbreviations: E, 70% ethanol.
    Mouse Anti Glyceraldehyde 3 Phosphate Dehydrogenase Gapdh Monoclonal Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti glyceraldehyde 3 phosphate dehydrogenase gapdh monoclonal antibody/product/Millipore
    Average 92 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    mouse anti glyceraldehyde 3 phosphate dehydrogenase gapdh monoclonal antibody - by Bioz Stars, 2020-04
    92/100 stars
      Buy from Supplier

    93
    Millipore mouse anti glyceraldehyde 3 phosphate dehydrogenase
    Protein quantity and quality of buffered ethanol 70% (BE70) fixative. (A) Amount of protein extracted from each condition was measured using the bicinchoninic acid (BCA) Protein Assay Kit. The protein extraction yield was expressed as the mean of three replicated samples (mean ± SD). (B) Protein integrity of different fixative solutions was assessed by western blotting. Proteins extracted from different fixative solutions were separated by 4% to 12% reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), electroblotted to nitrocellulose membrane, and probed with anti-aquaporin 1 (AQP1; 1:1000). (C) Western blotting by anti-glyceraldehyde 3-phosphate dehydrogenase <t>(GAPDH)</t> antibody. Relative GAPDH signal of each entity was normalized to neutral-buffered formalin (NBF). Abbreviations: E, 70% ethanol.
    Mouse Anti Glyceraldehyde 3 Phosphate Dehydrogenase, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti glyceraldehyde 3 phosphate dehydrogenase/product/Millipore
    Average 93 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    mouse anti glyceraldehyde 3 phosphate dehydrogenase - by Bioz Stars, 2020-04
    93/100 stars
      Buy from Supplier

    93
    Millipore anti glyceraldehyde 3 phosphate dehydrogenase gapdh antibody
    Protein quantity and quality of buffered ethanol 70% (BE70) fixative. (A) Amount of protein extracted from each condition was measured using the bicinchoninic acid (BCA) Protein Assay Kit. The protein extraction yield was expressed as the mean of three replicated samples (mean ± SD). (B) Protein integrity of different fixative solutions was assessed by western blotting. Proteins extracted from different fixative solutions were separated by 4% to 12% reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), electroblotted to nitrocellulose membrane, and probed with anti-aquaporin 1 (AQP1; 1:1000). (C) Western blotting by anti-glyceraldehyde 3-phosphate dehydrogenase <t>(GAPDH)</t> antibody. Relative GAPDH signal of each entity was normalized to neutral-buffered formalin (NBF). Abbreviations: E, 70% ethanol.
    Anti Glyceraldehyde 3 Phosphate Dehydrogenase Gapdh Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti glyceraldehyde 3 phosphate dehydrogenase gapdh antibody/product/Millipore
    Average 93 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    anti glyceraldehyde 3 phosphate dehydrogenase gapdh antibody - by Bioz Stars, 2020-04
    93/100 stars
      Buy from Supplier

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    Protein quantity and quality of buffered ethanol 70% (BE70) fixative. (A) Amount of protein extracted from each condition was measured using the bicinchoninic acid (BCA) Protein Assay Kit. The protein extraction yield was expressed as the mean of three replicated samples (mean ± SD). (B) Protein integrity of different fixative solutions was assessed by western blotting. Proteins extracted from different fixative solutions were separated by 4% to 12% reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), electroblotted to nitrocellulose membrane, and probed with anti-aquaporin 1 (AQP1; 1:1000). (C) Western blotting by anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody. Relative GAPDH signal of each entity was normalized to neutral-buffered formalin (NBF). Abbreviations: E, 70% ethanol.

    Journal: Journal of Histochemistry and Cytochemistry

    Article Title: A Buffered Alcohol-Based Fixative for Histomorphologic and Molecular Applications

    doi: 10.1369/0022155416649579

    Figure Lengend Snippet: Protein quantity and quality of buffered ethanol 70% (BE70) fixative. (A) Amount of protein extracted from each condition was measured using the bicinchoninic acid (BCA) Protein Assay Kit. The protein extraction yield was expressed as the mean of three replicated samples (mean ± SD). (B) Protein integrity of different fixative solutions was assessed by western blotting. Proteins extracted from different fixative solutions were separated by 4% to 12% reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), electroblotted to nitrocellulose membrane, and probed with anti-aquaporin 1 (AQP1; 1:1000). (C) Western blotting by anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody. Relative GAPDH signal of each entity was normalized to neutral-buffered formalin (NBF). Abbreviations: E, 70% ethanol.

    Article Snippet: The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with Tween (TBST; 50 mM Tris, pH 7.5, 150 mM NaCl, and 0.05% Tween-20) for 1 hr, washed, and incubated overnight at 4C in TBST with rabbit anti-AQP1 polyclonal antibody (Cat. No. sc-20810; dilution 1:100; Santa Cruz Biotechnology, Dallas, TA) and mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) monoclonal antibody (clone 6C5; dilution 1:3000; Calbiochem, Gibbstown, NJ).

    Techniques: BIA-KA, Protein Extraction, Western Blot, Polyacrylamide Gel Electrophoresis, SDS Page