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    Name:
    Protein A Magnetic Beads
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    Protein A Magnetic Beads 1 ml
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    s1425s
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    1 ml
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    Magnetic Separation Equipment
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    New England Biolabs rna
    Protein A Magnetic Beads
    Protein A Magnetic Beads 1 ml
    https://www.bioz.com/result/rna/product/New England Biolabs
    Average 97 stars, based on 331 article reviews
    Price from $9.99 to $1999.99
    rna - by Bioz Stars, 2021-02
    97/100 stars

    Images

    1) Product Images from "Robust transcriptome-wide discovery of RNA binding protein binding sites with enhanced CLIP (eCLIP)"

    Article Title: Robust transcriptome-wide discovery of RNA binding protein binding sites with enhanced CLIP (eCLIP)

    Journal: Nature methods

    doi: 10.1038/nmeth.3810

    Improved identification of RNA binding protein (RBP) targets by enhanced C ross L inking and I mmuno P recipitation followed by high-throughput sequencing (eCLIP-seq) (a) RBP-RNA interactions are stabilized with UV crosslinking, followed by limited RNase I digestion, immunoprecipitation of RBP-RNA complexes with a specific antibody of interest, and stringent washes. After dephosphorylation of RNA fragments, an “inline barcoded” RNA adapter is ligated to the 3′ end. After protein gel electrophoresis and nitrocellulose membrane transfer, a region 75 kDa (~220 nt of RNA) above the protein size is excised and proteinase K treated to isolate RNA. RNA is further prepared into paired-end high-throughput sequencing libraries, where read 1 begins with the inline barcode and read 2 begins with a random-mer sequence (added during the 3′ DNA adapter ligation) followed by sequence corresponding to the 5′ end of the original RNA fragment (which often marks reverse transcriptase termination at the crosslink site (red X)). (b) Bars indicate the number of reads remaining after processing steps. PCR duplicate reads that map to the same genomic position and have the same random-mer as previously considered reads are discarded, leaving only “Usable reads”. (c) Varying numbers of uniquely mapped reads were randomly sampled from RBFOX2 iCLIP and eCLIP experiments and PCR duplicate removal was performed. Points indicate the mean of 100 downsampling experiments (for all, s.e.m. is less than 0.1% of mean value). (d) RBFOX2 read density in reads per million usable (RPM). Shown are iCLIP, two biological replicates for eCLIP with paired size-matched input (SMInput) and IgG-only controls. CLIPper-identified clusters indicated as boxes below, with dark colored boxes indicating binding sites enriched above SMInput.
    Figure Legend Snippet: Improved identification of RNA binding protein (RBP) targets by enhanced C ross L inking and I mmuno P recipitation followed by high-throughput sequencing (eCLIP-seq) (a) RBP-RNA interactions are stabilized with UV crosslinking, followed by limited RNase I digestion, immunoprecipitation of RBP-RNA complexes with a specific antibody of interest, and stringent washes. After dephosphorylation of RNA fragments, an “inline barcoded” RNA adapter is ligated to the 3′ end. After protein gel electrophoresis and nitrocellulose membrane transfer, a region 75 kDa (~220 nt of RNA) above the protein size is excised and proteinase K treated to isolate RNA. RNA is further prepared into paired-end high-throughput sequencing libraries, where read 1 begins with the inline barcode and read 2 begins with a random-mer sequence (added during the 3′ DNA adapter ligation) followed by sequence corresponding to the 5′ end of the original RNA fragment (which often marks reverse transcriptase termination at the crosslink site (red X)). (b) Bars indicate the number of reads remaining after processing steps. PCR duplicate reads that map to the same genomic position and have the same random-mer as previously considered reads are discarded, leaving only “Usable reads”. (c) Varying numbers of uniquely mapped reads were randomly sampled from RBFOX2 iCLIP and eCLIP experiments and PCR duplicate removal was performed. Points indicate the mean of 100 downsampling experiments (for all, s.e.m. is less than 0.1% of mean value). (d) RBFOX2 read density in reads per million usable (RPM). Shown are iCLIP, two biological replicates for eCLIP with paired size-matched input (SMInput) and IgG-only controls. CLIPper-identified clusters indicated as boxes below, with dark colored boxes indicating binding sites enriched above SMInput.

    Techniques Used: RNA Binding Assay, Next-Generation Sequencing, Immunoprecipitation, De-Phosphorylation Assay, Nucleic Acid Electrophoresis, Sequencing, Ligation, Polymerase Chain Reaction, Binding Assay

    2) Product Images from "BRCA1 modulates the autophosphorylation status of DNA-PKcs in S phase of the cell cycle"

    Article Title: BRCA1 modulates the autophosphorylation status of DNA-PKcs in S phase of the cell cycle

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku824

    DNA-PKcs interacts with the tandem BRCT domain of BRCA1.  (A)  FLAG-tagged fragments of BRCA1 were transiently expressed in HeLa cells and subsequently IPed using an antiserum specific for the FLAG-tag. The immunoprecipitates were analyzed by western blot analysis using anti-DNA-PKcs or anti-FLAG antibodies.  (B)  DNA-PKcs was IP from HeLa cells, washed three times and the IP DNA-PKcs which was still bound to the protein A-sepharose was incubated with purified GST or GST-tagged tandem BRCT domain of BRCA1 or MDC1. The pull-downs were then washed and western blot analysis was performed using antibodies against GST or DNA-PKcs.  (C)  Representation of BRCA1 fragments used in (D and E) and their ability to interact with DNA-PKcs.
    Figure Legend Snippet: DNA-PKcs interacts with the tandem BRCT domain of BRCA1. (A) FLAG-tagged fragments of BRCA1 were transiently expressed in HeLa cells and subsequently IPed using an antiserum specific for the FLAG-tag. The immunoprecipitates were analyzed by western blot analysis using anti-DNA-PKcs or anti-FLAG antibodies. (B) DNA-PKcs was IP from HeLa cells, washed three times and the IP DNA-PKcs which was still bound to the protein A-sepharose was incubated with purified GST or GST-tagged tandem BRCT domain of BRCA1 or MDC1. The pull-downs were then washed and western blot analysis was performed using antibodies against GST or DNA-PKcs. (C) Representation of BRCA1 fragments used in (D and E) and their ability to interact with DNA-PKcs.

    Techniques Used: FLAG-tag, Western Blot, Incubation, Purification

    The interaction between DNA-PKcs and the tandem BRCT domain of BRCA1 is phospho-independent.  (A)  Exponentially HeLa cells were mock or irradiated with 20 Gy and allowed to recover for 1 h. DNA-PKcs was IPed, washed three times and the IP DNA-PKcs which was still bound to the protein A-sepharose was incubated with GST or GST-tagged BRCA1 tandem BRCT domain (BRCT) protein fragment in the presence or absence of lambda phosphatase (PPase). The pull-downs were then washed and western blot analysis was performed using antibodies against GST or DNA-PKcs. Antibodies against phosphorylated serine 2056 were used to show IR-induced phosphorylation of DNA-PKcs.  (B)  Purified DNA-PKcs was incubated with GST or GST-tagged protein fragments of BRCA1 encoding the RING finger domain (RING) or the tandem BRCT domain (BRCT) in the presence or absence of lambda phosphatase (PPase). The pull-downs were then washed and western blot analysis was performed using antibodies against GST or DNA-PKcs.
    Figure Legend Snippet: The interaction between DNA-PKcs and the tandem BRCT domain of BRCA1 is phospho-independent. (A) Exponentially HeLa cells were mock or irradiated with 20 Gy and allowed to recover for 1 h. DNA-PKcs was IPed, washed three times and the IP DNA-PKcs which was still bound to the protein A-sepharose was incubated with GST or GST-tagged BRCA1 tandem BRCT domain (BRCT) protein fragment in the presence or absence of lambda phosphatase (PPase). The pull-downs were then washed and western blot analysis was performed using antibodies against GST or DNA-PKcs. Antibodies against phosphorylated serine 2056 were used to show IR-induced phosphorylation of DNA-PKcs. (B) Purified DNA-PKcs was incubated with GST or GST-tagged protein fragments of BRCA1 encoding the RING finger domain (RING) or the tandem BRCT domain (BRCT) in the presence or absence of lambda phosphatase (PPase). The pull-downs were then washed and western blot analysis was performed using antibodies against GST or DNA-PKcs.

    Techniques Used: Irradiation, Incubation, Western Blot, Purification

    BRCA1 preferentially interacts with the N-terminal region of DNA-PKcs near the 2056 cluster.  (A)  FLAG-tagged N-terminal fragment (N-PKcs) and C-terminal fragment (C-PKcs) of DNA-PKcs were expressed and IPed from Sf9 cells using FLAG antibody, washed three times and the IP DNA-PKcs fragments still bound to the protein A-sepharose were incubated with GST or BRCT. Western blot analysis was then performed using antibodies against GST or FLAG.  (B)  GST-tagged fragments of DNA-PKcs encoding the amino acids indicated in the figure were purified from bacteria and left bound on the glutathione-agarose. These fragments were then incubated with purified His-tagged BRCA1 tandem BRCT domain protein fragment (BRCT). Following the incubation, the GST pull-downs were washed and western blot analysis was performed using anti-His and GST antibodies.
    Figure Legend Snippet: BRCA1 preferentially interacts with the N-terminal region of DNA-PKcs near the 2056 cluster. (A) FLAG-tagged N-terminal fragment (N-PKcs) and C-terminal fragment (C-PKcs) of DNA-PKcs were expressed and IPed from Sf9 cells using FLAG antibody, washed three times and the IP DNA-PKcs fragments still bound to the protein A-sepharose were incubated with GST or BRCT. Western blot analysis was then performed using antibodies against GST or FLAG. (B) GST-tagged fragments of DNA-PKcs encoding the amino acids indicated in the figure were purified from bacteria and left bound on the glutathione-agarose. These fragments were then incubated with purified His-tagged BRCA1 tandem BRCT domain protein fragment (BRCT). Following the incubation, the GST pull-downs were washed and western blot analysis was performed using anti-His and GST antibodies.

    Techniques Used: Incubation, Western Blot, Purification

    3) Product Images from "Phosphorylation of the RNA Polymerase II Carboxyl-Terminal Domain by CDK9 Is Directly Responsible for Human Immunodeficiency Virus Type 1 Tat-Activated Transcriptional Elongation"

    Article Title: Phosphorylation of the RNA Polymerase II Carboxyl-Terminal Domain by CDK9 Is Directly Responsible for Human Immunodeficiency Virus Type 1 Tat-Activated Transcriptional Elongation

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.22.13.4622-4637.2002

    Phosphorylation of the CTD is required for efficient transcription through terminator sequences. (A) Structure of templates. The pW1 template carries a terminator formed by an RNA stem-loop, followed by nine uridines (τ). pH3.3 contains two tandem arrest sequences (τ′ 1a and τ′ 1b ) that induce DNA bending. In pΔTerm, the terminator sequence was replaced by the original HIV sequence. (B) Transcription reactions were performed with three different immobilized DNA templates (pW1, pH3.3, or pΔTerm). Reactions contained 100 ng of LacR and were performed in the absence (−) or presence (+) of 20 ng of Tat. After labeling for 20 min with [α- 32 P]UTP (−PP1), the immobilized templates were purified and treated with RNase H in the presence of the RHX1 and RHLAC oligonucleotides to remove the labeled RNA transcripts from transcription complexes that had read through the LacR site. The arrested complexes were then dephosphorylated by PP1 treatment (+PP1). After the complexes were washed with TMZ buffer, the dephosphorylated complexes were chased by the addition of 250 μM ATP, GTP, and CTP; 5 μM UTP; and 25 mM IPTG in the absence (−) or presence (+) of 100 μM DRB. Positions of transcripts at the runoffs (ρ), terminator (τ), and lac repressor (LacR) in pW1 template are indicated. τ′ 1a and τ′ 1b indicate the stop sites from the pH3.3 template. (C) Immunoblot. Samples of the reactions shown in panel B were immunoblotted with the N-20 antibody against RNA Pol II and the anti-Spt5 antibody. Tat-dependent hyperphosphorylation of RNA polymerase CTD and Spt5 was observed during the chase of dephosphorylated complexes assembled on each template.
    Figure Legend Snippet: Phosphorylation of the CTD is required for efficient transcription through terminator sequences. (A) Structure of templates. The pW1 template carries a terminator formed by an RNA stem-loop, followed by nine uridines (τ). pH3.3 contains two tandem arrest sequences (τ′ 1a and τ′ 1b ) that induce DNA bending. In pΔTerm, the terminator sequence was replaced by the original HIV sequence. (B) Transcription reactions were performed with three different immobilized DNA templates (pW1, pH3.3, or pΔTerm). Reactions contained 100 ng of LacR and were performed in the absence (−) or presence (+) of 20 ng of Tat. After labeling for 20 min with [α- 32 P]UTP (−PP1), the immobilized templates were purified and treated with RNase H in the presence of the RHX1 and RHLAC oligonucleotides to remove the labeled RNA transcripts from transcription complexes that had read through the LacR site. The arrested complexes were then dephosphorylated by PP1 treatment (+PP1). After the complexes were washed with TMZ buffer, the dephosphorylated complexes were chased by the addition of 250 μM ATP, GTP, and CTP; 5 μM UTP; and 25 mM IPTG in the absence (−) or presence (+) of 100 μM DRB. Positions of transcripts at the runoffs (ρ), terminator (τ), and lac repressor (LacR) in pW1 template are indicated. τ′ 1a and τ′ 1b indicate the stop sites from the pH3.3 template. (C) Immunoblot. Samples of the reactions shown in panel B were immunoblotted with the N-20 antibody against RNA Pol II and the anti-Spt5 antibody. Tat-dependent hyperphosphorylation of RNA polymerase CTD and Spt5 was observed during the chase of dephosphorylated complexes assembled on each template.

    Techniques Used: Sequencing, Labeling, Purification

    Tat and TAR stimulate hyperphosphorylation of CTD in transcription elongation complexes. (A) Rephosphorylation by CDK9. Elongation complexes assembled on the pW1 templates were dephosphorylated by PP1 treatment (Pol II a ). Chase of the dephosphorylated complexes from the LacR site in the presence of all four nucleotides and IPTG permits rephosphorylation of the RNA polymerase CTD (Pol II o *) in the presence (+) of 20 ng of Tat but not in the absence (−) of Tat. (B) Inhibition of CDK9 by DRB. Between 0 and 10 μM DRB was included in the chase reactions containing dephosphorylated elongation complexes. Transcription was performed on the pW1 DNA templates in the absence (−) and presence (+) of 20 ng of Tat. (C) Activation of CDK9 by Tat and TAR. Elongation complexes were assembled on templates carrying wild-type TAR (WT) or mutant TAR elements in the Tat-binding site (mGC) or in the CycT1-binding site (mLG) in the absence (−) or presence (+) of 20 ng of Tat. After dephosphorylation of RNA polymerase CTD, the complexes were chased as described above.
    Figure Legend Snippet: Tat and TAR stimulate hyperphosphorylation of CTD in transcription elongation complexes. (A) Rephosphorylation by CDK9. Elongation complexes assembled on the pW1 templates were dephosphorylated by PP1 treatment (Pol II a ). Chase of the dephosphorylated complexes from the LacR site in the presence of all four nucleotides and IPTG permits rephosphorylation of the RNA polymerase CTD (Pol II o *) in the presence (+) of 20 ng of Tat but not in the absence (−) of Tat. (B) Inhibition of CDK9 by DRB. Between 0 and 10 μM DRB was included in the chase reactions containing dephosphorylated elongation complexes. Transcription was performed on the pW1 DNA templates in the absence (−) and presence (+) of 20 ng of Tat. (C) Activation of CDK9 by Tat and TAR. Elongation complexes were assembled on templates carrying wild-type TAR (WT) or mutant TAR elements in the Tat-binding site (mGC) or in the CycT1-binding site (mLG) in the absence (−) or presence (+) of 20 ng of Tat. After dephosphorylation of RNA polymerase CTD, the complexes were chased as described above.

    Techniques Used: Inhibition, Activation Assay, Mutagenesis, Binding Assay, De-Phosphorylation Assay

    Strategy used for analyzing transcription elongation complexes. (A) Structure of HIV-LTR template. DNA templates containing the lac operator (lacO) binding site for the lac repressor protein (LacR) and a terminator (τ) sequence were biotinylated and bound to streptavidin beads. (B) Elongation complexes were trapped by the lac repressor (LacR) after incubation of the immobilized templates with HeLa nuclear extract (NE) in the presence of nucleotide triphosphates and LacR and in the absence or presence of Tat. The CTD of the RNA polymerase was phosphorylated during the elongation reaction due to the activity of CDK7 and CDK9. (C) Elongation complexes arrested by LacR were treated with PP1 to remove phosphate groups from the CTD. (D) The phosphatase-treated complexes can resume transcription elongation after the addition of nucleotides and IPTG. During the chase reaction the CTD became phosphorylated by CDK9. The addition of DRB blocked the rephosphorylation of the CTD and induced pausing of the transcription complex at the terminator sequences.
    Figure Legend Snippet: Strategy used for analyzing transcription elongation complexes. (A) Structure of HIV-LTR template. DNA templates containing the lac operator (lacO) binding site for the lac repressor protein (LacR) and a terminator (τ) sequence were biotinylated and bound to streptavidin beads. (B) Elongation complexes were trapped by the lac repressor (LacR) after incubation of the immobilized templates with HeLa nuclear extract (NE) in the presence of nucleotide triphosphates and LacR and in the absence or presence of Tat. The CTD of the RNA polymerase was phosphorylated during the elongation reaction due to the activity of CDK7 and CDK9. (C) Elongation complexes arrested by LacR were treated with PP1 to remove phosphate groups from the CTD. (D) The phosphatase-treated complexes can resume transcription elongation after the addition of nucleotides and IPTG. During the chase reaction the CTD became phosphorylated by CDK9. The addition of DRB blocked the rephosphorylation of the CTD and induced pausing of the transcription complex at the terminator sequences.

    Techniques Used: Binding Assay, Sequencing, Incubation, Activity Assay

    CDK9 phosphorylates Ser5 and Ser2 of the CTD in elongation complexes. (A) Transcription reactions. Preinitiation complexes (PIC) were assembled on immobilized wild-type template (WT) by using hexokinase/glucose-treated HeLa nuclear extract in the presence of 50 μM dATP and in the absence (−) or presence (+) of 20 ng of Tat. Transcription complexes paused at the uridine residue at position 14 were obtained after elongation of the preinitiation complexes in the absence of ATP. Standard transcription reactions were performed in parallel with templates carrying either the wild-type TAR element (WT) or a mutation in the Tat-binding site (mGC). Protein composition and phosphorylation of RNA Pol II CTD were analyzed from different transcription complexes by immunoblotting with the N-20, H5, and H14 antibodies directed against RNA Pol II (RNAP) and antibodies against CDK9, CycT1, CDK7, and CycH. (B) Rephosphorylation reactions. Transcription elongation complexes arrested by LacR were dephosphorylated by PP1, washed with EBCD buffer containing 0.1% Sarkosyl, and chased in the presence of 25 mM IPTG and all four nucleotide triphosphates. The proteins were detected by immunoblotting with the N-20, 8WG16, H5, and H14 antibodies directed against RNA Pol II and antibodies against CDK9 and CycT1.
    Figure Legend Snippet: CDK9 phosphorylates Ser5 and Ser2 of the CTD in elongation complexes. (A) Transcription reactions. Preinitiation complexes (PIC) were assembled on immobilized wild-type template (WT) by using hexokinase/glucose-treated HeLa nuclear extract in the presence of 50 μM dATP and in the absence (−) or presence (+) of 20 ng of Tat. Transcription complexes paused at the uridine residue at position 14 were obtained after elongation of the preinitiation complexes in the absence of ATP. Standard transcription reactions were performed in parallel with templates carrying either the wild-type TAR element (WT) or a mutation in the Tat-binding site (mGC). Protein composition and phosphorylation of RNA Pol II CTD were analyzed from different transcription complexes by immunoblotting with the N-20, H5, and H14 antibodies directed against RNA Pol II (RNAP) and antibodies against CDK9, CycT1, CDK7, and CycH. (B) Rephosphorylation reactions. Transcription elongation complexes arrested by LacR were dephosphorylated by PP1, washed with EBCD buffer containing 0.1% Sarkosyl, and chased in the presence of 25 mM IPTG and all four nucleotide triphosphates. The proteins were detected by immunoblotting with the N-20, 8WG16, H5, and H14 antibodies directed against RNA Pol II and antibodies against CDK9 and CycT1.

    Techniques Used: Mutagenesis, Binding Assay

    4) Product Images from "Normal and Friedreich Ataxia Cells Express Different Isoforms of Frataxin with Complementary Roles in Iron-Sulfur Cluster Assembly *"

    Article Title: Normal and Friedreich Ataxia Cells Express Different Isoforms of Frataxin with Complementary Roles in Iron-Sulfur Cluster Assembly *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.145144

    Interactions of native FXN isoforms with native NFS1 and ISCU. Lymphoblastoid cell lysate was analyzed by Superdex 75 size exclusion chromatography. Fractions comprising the entire molecular mass fractionation range of the column were analyzed by Western blotting with ( A ) anti-NFS1 monoclonal antibody MyBioSource, ( B ) anti-FXN, or ( C ) polyclonal antibodies. The high and low molecular weight fractions were pooled ( HMW and LMW box , respectively), and immunoprecipitation was performed with PAC 2517 anti-FXN antibody immobilized on Protein A Magnetic beads, as described under “Experimental Procedures.” Aliquots of each pool (HMW or LMW) before immunoprecipitation ( Input , ∼5% of total volume), the flow-through fraction ( Not bound ; ∼5% of total volume), and the affinity-purified fraction ( Bound ; 100% of total volume) were analyzed by Western blotting ( WB ) with anti-NFS1 monoclonal antibody ( D ) or anti-FXN monoclonal antibody ( E ) or anti-Isu1 polyclonal antibody ( F ). FXN and ISCU were detected in individual HMW fractions ( B and C ) but not in the HMW fraction pool ( E and F , input and not Bound ) because ∼4 times less total protein was loaded on the gel in the latter analysis. For the control (MOCK), the Protein A Magnetic beads without antibody were incubated overnight with non-fractionated lymphoblastoid cell lysate, and Input , Not bound , and Bound proteins were analyzed as described above.
    Figure Legend Snippet: Interactions of native FXN isoforms with native NFS1 and ISCU. Lymphoblastoid cell lysate was analyzed by Superdex 75 size exclusion chromatography. Fractions comprising the entire molecular mass fractionation range of the column were analyzed by Western blotting with ( A ) anti-NFS1 monoclonal antibody MyBioSource, ( B ) anti-FXN, or ( C ) polyclonal antibodies. The high and low molecular weight fractions were pooled ( HMW and LMW box , respectively), and immunoprecipitation was performed with PAC 2517 anti-FXN antibody immobilized on Protein A Magnetic beads, as described under “Experimental Procedures.” Aliquots of each pool (HMW or LMW) before immunoprecipitation ( Input , ∼5% of total volume), the flow-through fraction ( Not bound ; ∼5% of total volume), and the affinity-purified fraction ( Bound ; 100% of total volume) were analyzed by Western blotting ( WB ) with anti-NFS1 monoclonal antibody ( D ) or anti-FXN monoclonal antibody ( E ) or anti-Isu1 polyclonal antibody ( F ). FXN and ISCU were detected in individual HMW fractions ( B and C ) but not in the HMW fraction pool ( E and F , input and not Bound ) because ∼4 times less total protein was loaded on the gel in the latter analysis. For the control (MOCK), the Protein A Magnetic beads without antibody were incubated overnight with non-fractionated lymphoblastoid cell lysate, and Input , Not bound , and Bound proteins were analyzed as described above.

    Techniques Used: Size-exclusion Chromatography, Fractionation, Western Blot, Molecular Weight, Immunoprecipitation, Magnetic Beads, Flow Cytometry, Affinity Purification, Incubation

    5) Product Images from "IFN-γ-independent immune markers of Mycobacterium tuberculosis exposure"

    Article Title: IFN-γ-independent immune markers of Mycobacterium tuberculosis exposure

    Journal: Nature Medicine

    doi: 10.1038/s41591-019-0441-3

    Stratification of ‘resisters’ and LTBI controls by IGRA, TST and BCG scar. a – c , IGRA results ( a ) are shown by the average of three sequential QFT-Gold readings minus the background (nil) stratified across RSTR ( n = 40) or LTBI ( n = 39). TST results, stratified across RSTR or LTBI, are reported in mM of induration measured in phase 1 ( b ) and phase 2 ( c ) of the clinical trial. Medians are depicted by lines with interquartile ranges. d , e , Plasma levels of IgG reactive to PPD ( d ) and ESAT6 and CFP10 ( e ) were quantified in RSTR ( n = 40) and LTBI ( n = 39) individuals using a customized multiplex Luminex in serial dilutions. AUCs were determined from MFIs generated with each dilution and plotted for each individual. Data points are stratified by BCG scar status. Statistical significance was calculated by Mann–Whitney U test and two-tailed P values are indicated.
    Figure Legend Snippet: Stratification of ‘resisters’ and LTBI controls by IGRA, TST and BCG scar. a – c , IGRA results ( a ) are shown by the average of three sequential QFT-Gold readings minus the background (nil) stratified across RSTR ( n = 40) or LTBI ( n = 39). TST results, stratified across RSTR or LTBI, are reported in mM of induration measured in phase 1 ( b ) and phase 2 ( c ) of the clinical trial. Medians are depicted by lines with interquartile ranges. d , e , Plasma levels of IgG reactive to PPD ( d ) and ESAT6 and CFP10 ( e ) were quantified in RSTR ( n = 40) and LTBI ( n = 39) individuals using a customized multiplex Luminex in serial dilutions. AUCs were determined from MFIs generated with each dilution and plotted for each individual. Data points are stratified by BCG scar status. Statistical significance was calculated by Mann–Whitney U test and two-tailed P values are indicated.

    Techniques Used: Multiplex Assay, Luminex, Generated, MANN-WHITNEY, Two Tailed Test

    Qualitatively distinct PPD-specific antibody responses in ‘resisters’ compared with LTBI individuals. a , Graphs depict the AUCs calculated from the ratio of live to total intracellular bacterial burden in primary human monocyte-derived macrophages after treatment with purified IgG at 0.1 mg ml –1 , 0.01 mg ml –1 and 0.001 mg ml –1 (left) from ‘resisters’ ( n = 40) and TST/IGRA-positive LTBI controls ( n = 39). Extension of analysis to additional donors was performed at a single concentration of purified IgG of 0.1 mg ml –1 due to sample availability (middle). Levels of secreted IL-1β from supernatants were measured by ELISA and are shown relative to no antibody treatment. Purified IgG from individuals in this study, with culture-confirmed pulmonary TB (ATB), is shown as a benchmark. Each line represents one healthy macrophage donor individual. For dot plots, lines are medians. The statistical significance was calculated using Wilcoxon’s matched-pairs signed rank, and two-tailed P values are shown. b , The calculated avidity against PPD from pooled plasma from ‘resisters’ ( n = 40), TST/IGRA-positive LTBI controls ( n = 39) and healthy, HIV-uninfected North Americans ( n = 10) are shown, with lines representing the fitted curves and dotted lines the 95% confidence intervals. Each plasma group was tested in triplicate, with associated calculated avidity represented in the dot plot. The statistical significance was calculated using the Student’s t -test, and a two-tailed P value is indicated. OD, optical density or absorbance. c – e , Plasma from ‘resisters’ ( n = 40) and LTBI controls ( n = 39) was assayed for the ability to mediate: PPD and ESAT6/CFP10-specific, antibody-dependent, monocyte-mediated cellular phagocytosis ( c ); PPD-specific, antibody-dependent neutrophil phagocytosis ( d ); and PPD-specific, NK cell activation by CD107a expression, macrophage inflammatory protein-1β and IFN-γ production ( e ). Data are representative of experiments performed in duplicate over three dilutions. Assays utilizing primary human neutrophils ( d ) and NK cells ( e ) were additionally performed utilizing three independent, healthy, HIV-negative donors. f , Affinity for FcγR2A(R), FcγR2A(H), FcγR3A(V) and FcγR3A(F) were determined using customized Luminex to PPD in ‘resisters’ ( n = 40) and LTBI individuals ( n = 39), using plasma diluted at 1:100. MFI is shown on the graph. The statistical significance was calculated using the Mann–Whitney U test, and P values are indicated. Dotted lines represent the median level detected in HIV-negative, healthy North American volunteers. g , Ratios of plasma levels of IgM, IgG and IgA1 reactive to PPD and LAM in ‘resisters’ ( n = 40) and TST/IGRA-positive LTBI controls ( n = 39) are depicted with medians and interquartile ranges. The statistical significance was calculated using the Mann–Whitney U test, and two-tailed P values are indicated. h , Ratios of plasma levels of IgG1, IgG2, IgG3 and IgG4 reactive to PPD in ‘resisters’ ( n = 40) and TST/IGRA-positive LTBI individuals ( n = 39) were measured by customized multiplex Luminex in serial dilutions. AUCs are depicted with medians and interquartile ranges. The statistical significance was calculated using the Mann–Whitney U test, and two-tailed P values are indicated. i , The relative distribution of glycoform substructures isolated from non-antigen-specific and PPD-specific IgG are depicted, with each column representing each individual. j , Principal component analysis demonstrates the overlapping profiles of ‘resisters’ ( n = 40) and TST/IGRA-positive LTBI individuals ( n = 39) in the dominant total glycans isolated from non-antigen-specific IgG compared with partially separating profiles from PPD-specific IgG. ADCP, antibody-dependent cellular phagocytosis; ADNP, antibody-dependent neutrophil phagocytosis; MIP, macrophage inflammatory protein.
    Figure Legend Snippet: Qualitatively distinct PPD-specific antibody responses in ‘resisters’ compared with LTBI individuals. a , Graphs depict the AUCs calculated from the ratio of live to total intracellular bacterial burden in primary human monocyte-derived macrophages after treatment with purified IgG at 0.1 mg ml –1 , 0.01 mg ml –1 and 0.001 mg ml –1 (left) from ‘resisters’ ( n = 40) and TST/IGRA-positive LTBI controls ( n = 39). Extension of analysis to additional donors was performed at a single concentration of purified IgG of 0.1 mg ml –1 due to sample availability (middle). Levels of secreted IL-1β from supernatants were measured by ELISA and are shown relative to no antibody treatment. Purified IgG from individuals in this study, with culture-confirmed pulmonary TB (ATB), is shown as a benchmark. Each line represents one healthy macrophage donor individual. For dot plots, lines are medians. The statistical significance was calculated using Wilcoxon’s matched-pairs signed rank, and two-tailed P values are shown. b , The calculated avidity against PPD from pooled plasma from ‘resisters’ ( n = 40), TST/IGRA-positive LTBI controls ( n = 39) and healthy, HIV-uninfected North Americans ( n = 10) are shown, with lines representing the fitted curves and dotted lines the 95% confidence intervals. Each plasma group was tested in triplicate, with associated calculated avidity represented in the dot plot. The statistical significance was calculated using the Student’s t -test, and a two-tailed P value is indicated. OD, optical density or absorbance. c – e , Plasma from ‘resisters’ ( n = 40) and LTBI controls ( n = 39) was assayed for the ability to mediate: PPD and ESAT6/CFP10-specific, antibody-dependent, monocyte-mediated cellular phagocytosis ( c ); PPD-specific, antibody-dependent neutrophil phagocytosis ( d ); and PPD-specific, NK cell activation by CD107a expression, macrophage inflammatory protein-1β and IFN-γ production ( e ). Data are representative of experiments performed in duplicate over three dilutions. Assays utilizing primary human neutrophils ( d ) and NK cells ( e ) were additionally performed utilizing three independent, healthy, HIV-negative donors. f , Affinity for FcγR2A(R), FcγR2A(H), FcγR3A(V) and FcγR3A(F) were determined using customized Luminex to PPD in ‘resisters’ ( n = 40) and LTBI individuals ( n = 39), using plasma diluted at 1:100. MFI is shown on the graph. The statistical significance was calculated using the Mann–Whitney U test, and P values are indicated. Dotted lines represent the median level detected in HIV-negative, healthy North American volunteers. g , Ratios of plasma levels of IgM, IgG and IgA1 reactive to PPD and LAM in ‘resisters’ ( n = 40) and TST/IGRA-positive LTBI controls ( n = 39) are depicted with medians and interquartile ranges. The statistical significance was calculated using the Mann–Whitney U test, and two-tailed P values are indicated. h , Ratios of plasma levels of IgG1, IgG2, IgG3 and IgG4 reactive to PPD in ‘resisters’ ( n = 40) and TST/IGRA-positive LTBI individuals ( n = 39) were measured by customized multiplex Luminex in serial dilutions. AUCs are depicted with medians and interquartile ranges. The statistical significance was calculated using the Mann–Whitney U test, and two-tailed P values are indicated. i , The relative distribution of glycoform substructures isolated from non-antigen-specific and PPD-specific IgG are depicted, with each column representing each individual. j , Principal component analysis demonstrates the overlapping profiles of ‘resisters’ ( n = 40) and TST/IGRA-positive LTBI individuals ( n = 39) in the dominant total glycans isolated from non-antigen-specific IgG compared with partially separating profiles from PPD-specific IgG. ADCP, antibody-dependent cellular phagocytosis; ADNP, antibody-dependent neutrophil phagocytosis; MIP, macrophage inflammatory protein.

    Techniques Used: Derivative Assay, Purification, Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Activation Assay, Expressing, Luminex, MANN-WHITNEY, Laser Capture Microdissection, Multiplex Assay, Isolation

    Detectable Mtb -specific humoral immunity in ‘resisters’. a – c , Plasma levels of IgM ( a ), IgG ( b ) and IgA1 ( c ) reactive to PPD, Ag85A, ESAT6 and CFP10, α-crystalline (HspX), GroES and LAM were quantified in ‘resisters’ ( n = 40) and LTBI individuals ( n = 39) with AUCs determined from MFIs generated using a customized Luminex assay, generated with three dilutions and plotted for each individual with medians and interquartile ranges depicted for each group. The statistical significance was calculated using the Mann–Whitney U test, and two-tailed P values are indicated. Dotted lines represent the median level detected in HIV-negative, healthy North American volunteers.
    Figure Legend Snippet: Detectable Mtb -specific humoral immunity in ‘resisters’. a – c , Plasma levels of IgM ( a ), IgG ( b ) and IgA1 ( c ) reactive to PPD, Ag85A, ESAT6 and CFP10, α-crystalline (HspX), GroES and LAM were quantified in ‘resisters’ ( n = 40) and LTBI individuals ( n = 39) with AUCs determined from MFIs generated using a customized Luminex assay, generated with three dilutions and plotted for each individual with medians and interquartile ranges depicted for each group. The statistical significance was calculated using the Mann–Whitney U test, and two-tailed P values are indicated. Dotted lines represent the median level detected in HIV-negative, healthy North American volunteers.

    Techniques Used: Laser Capture Microdissection, Generated, Luminex, MANN-WHITNEY, Two Tailed Test

    6) Product Images from "A Method for Selectively Enriching Microbial DNA from Contaminating Vertebrate Host DNA"

    Article Title: A Method for Selectively Enriching Microbial DNA from Contaminating Vertebrate Host DNA

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0076096

    Analysis of SOLiD 4 sequence data from human saliva and blood samples before and after enrichment with MBD-Fc protein A paramagnetic beads. (A) Unenriched control, enriched and, remaining after enrichment (bead bound) samples are indicated above the figure. The data reflect reads mapping to known oral microbes in HOMD [20] . (B) Reads mapping to the PhageSeed database of viral sequences also show strong enrichment across two replicates.
    Figure Legend Snippet: Analysis of SOLiD 4 sequence data from human saliva and blood samples before and after enrichment with MBD-Fc protein A paramagnetic beads. (A) Unenriched control, enriched and, remaining after enrichment (bead bound) samples are indicated above the figure. The data reflect reads mapping to known oral microbes in HOMD [20] . (B) Reads mapping to the PhageSeed database of viral sequences also show strong enrichment across two replicates.

    Techniques Used: Sequencing

    Analysis of Illumina sequence reads of a black molly ( Poecilia cf. sphenops ) whole fish DNA library before and after enrichment with MBD-Fc protein A paramagnetic beads. Shown is a concordance plot comparing relative abundances of microbial genera between the enriched and unenriched samples.
    Figure Legend Snippet: Analysis of Illumina sequence reads of a black molly ( Poecilia cf. sphenops ) whole fish DNA library before and after enrichment with MBD-Fc protein A paramagnetic beads. Shown is a concordance plot comparing relative abundances of microbial genera between the enriched and unenriched samples.

    Techniques Used: Sequencing, Fluorescence In Situ Hybridization

    7) Product Images from "Targeting of Tumor Necrosis Factor Receptor 1 to Low Density Plasma Membrane Domains in Human Endothelial Cells *"

    Article Title: Targeting of Tumor Necrosis Factor Receptor 1 to Low Density Plasma Membrane Domains in Human Endothelial Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.122853

    Expression and localization of transduced TNFR1 proteins. A , FLAG-TNFR1 surface protein expression in EAhy.926 transductants was measured by FACS analysis as described under “Experimental Procedures,” comparing pBabe.puro vector control, full-length FLAG-TNFR1 (L380) or three FLAG-tagged TNFR1 constructs with deletions in the putative CAV-1 binding domain (Δ236–239, Δ234–243, and Δ229–244). Indirect immunostaining was with polyclonal rabbit anti-FLAG IgG ( gray shading ) or with negative control nonspecific rabbit IgG ( black line ) followed by AlexaFluor 488-conjugated donkey anti-rabbit secondary antibody (Invitrogen). One of two experiments with similar results is shown. B , expression of transduced TNFR1 proteins was measured by immunoprecipitating FLAG-containing proteins (with rabbit anti-FLAG), followed by immunoblotting for TNFR1 (with mouse anti-TNFR1). Top , pooled low density membrane fractions positive for CAV-1 protein obtained from sucrose gradient ultracentrifugation of EAhy.926 protein lysates. Bottom , whole cell lysates. One of two experiments with similar results is shown. FL , full-length TNFR1; V , vector. C , EAhy.926 whole cell lysates of full-length and Δ229–244 FLAG-TNFR1 ( left ) as well as sucrose gradient separations ( middle and right ) were analyzed by Western blot with anti-FLAG-horseradish peroxidase and anti-CAV-1 primary antibodies. One of two experiments with similar results is shown. D , densitometric quantification comparing full-length and Δ229–244 TNFR1 localization with sucrose gradient light fractions of the experiment depicted in C . The graph shows the expression of FLAG-TNFR1 in fractions 3, 4, and 5 normalized to CAV-1 after correcting for different expression levels measured in whole cell lysates.
    Figure Legend Snippet: Expression and localization of transduced TNFR1 proteins. A , FLAG-TNFR1 surface protein expression in EAhy.926 transductants was measured by FACS analysis as described under “Experimental Procedures,” comparing pBabe.puro vector control, full-length FLAG-TNFR1 (L380) or three FLAG-tagged TNFR1 constructs with deletions in the putative CAV-1 binding domain (Δ236–239, Δ234–243, and Δ229–244). Indirect immunostaining was with polyclonal rabbit anti-FLAG IgG ( gray shading ) or with negative control nonspecific rabbit IgG ( black line ) followed by AlexaFluor 488-conjugated donkey anti-rabbit secondary antibody (Invitrogen). One of two experiments with similar results is shown. B , expression of transduced TNFR1 proteins was measured by immunoprecipitating FLAG-containing proteins (with rabbit anti-FLAG), followed by immunoblotting for TNFR1 (with mouse anti-TNFR1). Top , pooled low density membrane fractions positive for CAV-1 protein obtained from sucrose gradient ultracentrifugation of EAhy.926 protein lysates. Bottom , whole cell lysates. One of two experiments with similar results is shown. FL , full-length TNFR1; V , vector. C , EAhy.926 whole cell lysates of full-length and Δ229–244 FLAG-TNFR1 ( left ) as well as sucrose gradient separations ( middle and right ) were analyzed by Western blot with anti-FLAG-horseradish peroxidase and anti-CAV-1 primary antibodies. One of two experiments with similar results is shown. D , densitometric quantification comparing full-length and Δ229–244 TNFR1 localization with sucrose gradient light fractions of the experiment depicted in C . The graph shows the expression of FLAG-TNFR1 in fractions 3, 4, and 5 normalized to CAV-1 after correcting for different expression levels measured in whole cell lysates.

    Techniques Used: Expressing, FACS, Plasmid Preparation, Construct, Binding Assay, Immunostaining, Negative Control, Western Blot

    Pull-down of EA.hy926 proteins with biotinylated TNFR1 sequence-containing peptide. A , EAhy.926 lysate pull-downs with biotinylated TAT-TNFR1(229–244) wild type sequence-specific peptide ( WT ) or with biotinylated TAT-TNFR1(229–244SCR) scramble peptide ( Scr ) were separated by SDS-PAGE, and the gel was stained for protein as described under “Experimental Procedures.” Note the enrichment of several bands (marked by arrows ) in the pull down by the specific peptide compared with scrambled control. One of two independent experiments with similar results. B , pull-down of EAhy.926 lysates with TAT peptide, with TAT-TNFR1(229–244) peptide, or with TAT-TNFR1(229–244SCR) scramble peptide followed by immunoblotting. Note that the wild type-specific but not the scrambled TNFR1 sequence pulls down TNFR1, TRAF2, and CAV-1, suggesting interaction with a multiprotein complex. C , sucrose gradient fractionation was performed as described under “Experimental Procedures,” and samples from each fraction were immunoblotted both for CAV-1 and TNFR1. Three pools were generated as indicated for use in additional pull-down experiments. D , pull-down by TAT-TNFR1(229–244) from the pooled fractions generated in C . Note that the TNFR1 wild type sequence-containing peptide TAT-TNFR1(229–244) only pulled down CAV-1 from the light membrane fraction (pool 2) of the gradient. One of two independent experiments with similar results is shown. E , pull-down of lysates prepared from EA.hy926 cells pretreated with MβCD, a manipulation that disrupts low density membrane fractions by extraction of cholesterol. Note that the ability of TAT-TNFR1(229–244) to pull down interacting proteins, such as TNFR1 or CAV-1, was strongly inhibited by preincubation with MβCD. One of two independent experiments with similar results is shown. Ctr , cells without MβCD.
    Figure Legend Snippet: Pull-down of EA.hy926 proteins with biotinylated TNFR1 sequence-containing peptide. A , EAhy.926 lysate pull-downs with biotinylated TAT-TNFR1(229–244) wild type sequence-specific peptide ( WT ) or with biotinylated TAT-TNFR1(229–244SCR) scramble peptide ( Scr ) were separated by SDS-PAGE, and the gel was stained for protein as described under “Experimental Procedures.” Note the enrichment of several bands (marked by arrows ) in the pull down by the specific peptide compared with scrambled control. One of two independent experiments with similar results. B , pull-down of EAhy.926 lysates with TAT peptide, with TAT-TNFR1(229–244) peptide, or with TAT-TNFR1(229–244SCR) scramble peptide followed by immunoblotting. Note that the wild type-specific but not the scrambled TNFR1 sequence pulls down TNFR1, TRAF2, and CAV-1, suggesting interaction with a multiprotein complex. C , sucrose gradient fractionation was performed as described under “Experimental Procedures,” and samples from each fraction were immunoblotted both for CAV-1 and TNFR1. Three pools were generated as indicated for use in additional pull-down experiments. D , pull-down by TAT-TNFR1(229–244) from the pooled fractions generated in C . Note that the TNFR1 wild type sequence-containing peptide TAT-TNFR1(229–244) only pulled down CAV-1 from the light membrane fraction (pool 2) of the gradient. One of two independent experiments with similar results is shown. E , pull-down of lysates prepared from EA.hy926 cells pretreated with MβCD, a manipulation that disrupts low density membrane fractions by extraction of cholesterol. Note that the ability of TAT-TNFR1(229–244) to pull down interacting proteins, such as TNFR1 or CAV-1, was strongly inhibited by preincubation with MβCD. One of two independent experiments with similar results is shown. Ctr , cells without MβCD.

    Techniques Used: Sequencing, SDS Page, Staining, Fractionation, Generated

    8) Product Images from "Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors"

    Article Title: Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors

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

    doi: 10.1073/pnas.0606631103

    Purification of the A5 mAb by using protein A magnetic beads. ( A ) Plant-derived and standard mAbs migrating in a 10% polyacrylamide gel under nonreducing conditions. ( B ) Plant-derived and standard mAbs resolved in a 12% polyacrylamide gel under reducing conditions. ( B Top ) PAGE with Coomassie blue staining. ( B Middle ) Western blot probed with γ-chain-specific antibodies. ( B Bottom ) Western blot probed with λ-chain-specific antibodies. Lane 1, PVX-HC + TMV-LC, crude extract; lane 2, TMV-HC + PVX-LC, crude extract; lane 3, PVX-HC + TMV-LC, purified mAb; lane 4, TMV-HC + PVX-LC, purified mAb; lanes 5 and 6, A5 standard.
    Figure Legend Snippet: Purification of the A5 mAb by using protein A magnetic beads. ( A ) Plant-derived and standard mAbs migrating in a 10% polyacrylamide gel under nonreducing conditions. ( B ) Plant-derived and standard mAbs resolved in a 12% polyacrylamide gel under reducing conditions. ( B Top ) PAGE with Coomassie blue staining. ( B Middle ) Western blot probed with γ-chain-specific antibodies. ( B Bottom ) Western blot probed with λ-chain-specific antibodies. Lane 1, PVX-HC + TMV-LC, crude extract; lane 2, TMV-HC + PVX-LC, crude extract; lane 3, PVX-HC + TMV-LC, purified mAb; lane 4, TMV-HC + PVX-LC, purified mAb; lanes 5 and 6, A5 standard.

    Techniques Used: Purification, Magnetic Beads, Derivative Assay, Polyacrylamide Gel Electrophoresis, Staining, Western Blot

    9) Product Images from "Ypt1/Rab1 regulates Hrr25/CK1δ kinase activity in ER–Golgi traffic and macroautophagy"

    Article Title: Ypt1/Rab1 regulates Hrr25/CK1δ kinase activity in ER–Golgi traffic and macroautophagy

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201408075

    Ypt1 regulates Hrr25 kinase activity on vesicles . (A) Hrr25 kinase activity is ts in the  ypt1-3  mutant. WT (SFNY 2443) and mutant (SFNY 2445) cells were grown at 23°C or shifted to 37°C for 2 h. Hrr25-HA was precipitated from lysates onto Protein A–conjugated agarose beads and kinase activity was assayed using MBP as a substrate as described in the Materials and methods. (B) Quantitation of kinase activity from WT and the  ypt1-3  mutant from three separate experiments. The data were normalized to the amount of Hrr25-HA in the precipitate. Error bars represent SD;  n  = 3; *, P
    Figure Legend Snippet: Ypt1 regulates Hrr25 kinase activity on vesicles . (A) Hrr25 kinase activity is ts in the ypt1-3 mutant. WT (SFNY 2443) and mutant (SFNY 2445) cells were grown at 23°C or shifted to 37°C for 2 h. Hrr25-HA was precipitated from lysates onto Protein A–conjugated agarose beads and kinase activity was assayed using MBP as a substrate as described in the Materials and methods. (B) Quantitation of kinase activity from WT and the ypt1-3 mutant from three separate experiments. The data were normalized to the amount of Hrr25-HA in the precipitate. Error bars represent SD; n = 3; *, P

    Techniques Used: Activity Assay, Mutagenesis, Quantitation Assay

    10) Product Images from "Human Immunodeficiency Virus Type 2 Gag Interacts Specifically with PRP4, a Serine-Threonine Kinase, and Inhibits Phosphorylation of Splicing Factor SF2"

    Article Title: Human Immunodeficiency Virus Type 2 Gag Interacts Specifically with PRP4, a Serine-Threonine Kinase, and Inhibits Phosphorylation of Splicing Factor SF2

    Journal: Journal of Virology

    doi: 10.1128/JVI.78.20.11303-11312.2004

    PRP4 binds specifically with HIV-2 Gag. (A) In vitro binding of PRP4 to HIV-1 and HIV-2 Gag proteins. In vitro-transcribed and -translated HIV-1 Gag (lanes 2 to 4), HIV-2 Gag (lanes 6 to 8), or gelsolin (lanes 10 to 13) was analyzed by GST fusion pull-down assays for the ability to bind GST, GST-PRP4, GST-Gag1, or GST-Gag2 immobilized on glutathione-Sepharose beads. Inputs were 10% of the amount of HIV-1 Gag (lane 1), HIV-2 Gag (lane 5), or gelsolin (lane 9) used in each assay. (B) PRP4 cellular lysate binding to HIV-2 Gag protein. In vitro-transcribed and -translated HIV-2 Gag (lane 2) or gelsolin (lane 4) was analyzed by pull-down assay for the ability to bind PRP4 protein extract from transfected 293T cells immobilized on protein A-protein G beads. Inputs were 10% of the amount of HIV-2 Gag (lane 1) or gelsolin (lane 3) used in each assay. The positions of marker proteins are indicated in kilodaltons.
    Figure Legend Snippet: PRP4 binds specifically with HIV-2 Gag. (A) In vitro binding of PRP4 to HIV-1 and HIV-2 Gag proteins. In vitro-transcribed and -translated HIV-1 Gag (lanes 2 to 4), HIV-2 Gag (lanes 6 to 8), or gelsolin (lanes 10 to 13) was analyzed by GST fusion pull-down assays for the ability to bind GST, GST-PRP4, GST-Gag1, or GST-Gag2 immobilized on glutathione-Sepharose beads. Inputs were 10% of the amount of HIV-1 Gag (lane 1), HIV-2 Gag (lane 5), or gelsolin (lane 9) used in each assay. (B) PRP4 cellular lysate binding to HIV-2 Gag protein. In vitro-transcribed and -translated HIV-2 Gag (lane 2) or gelsolin (lane 4) was analyzed by pull-down assay for the ability to bind PRP4 protein extract from transfected 293T cells immobilized on protein A-protein G beads. Inputs were 10% of the amount of HIV-2 Gag (lane 1) or gelsolin (lane 3) used in each assay. The positions of marker proteins are indicated in kilodaltons.

    Techniques Used: In Vitro, Binding Assay, Pull Down Assay, Transfection, Marker

    11) Product Images from "CHIP E3 ligase mediates proteasomal degradation of the proliferation regulatory protein ALDH1L1 during the transition of NIH3T3 fibroblasts from G0/G1 to S-phase"

    Article Title: CHIP E3 ligase mediates proteasomal degradation of the proliferation regulatory protein ALDH1L1 during the transition of NIH3T3 fibroblasts from G0/G1 to S-phase

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0199699

    ALDH1L1 is ubiquitinated in NIH3T3 cells. A , ALDH1L1 pulled-down from NIH3T3 cell lysates using ALDH1L1-specific antibody and protein A beads; elution with glycine buffer ( lane 1 ), followed by elution with SDS-PAGE loading buffer ( lane 2 ). Proteins were resolved on a 7.5% SDS-PAGE gel followed by Western blot assay with ubiquitin-specific antibody ( left panel ) or ALDH1L1-specific antibody ( right panel ). Lane St is purified recombinant ALDH1L1. B , ALDH1L1 was immunoprecipitated from NIH3T3 cell lysates using an ALDH1L1-specific antibody and Protein A Magnetic beads; samples were resolved on a 7.5% SDS-PAGE followed by Western blot assay with anti-ubiquitin monoclonal antibody. Cells were harvested at different time points after splitting (as indicated); lysates were treated with deubiquitinase inhibitor (4.0 μM recombinant human ubiquitin aldehyde C-terminal derivative) prior to immunoprecipitation. After immunoprecipitation, eluates were treated with deubiquitinase (200 nM of recombinant human USP2 catalytic domain); control , untreated lysates. C , ALDH1L1 was immunoprecipitated from NIH3T3 cells as in B and treated with USP7. Cells were treated with 10 μM MG-132 for 4 h before the pull-down. After treatment with USP7, we have repeated the pull-down with ALDH1L1-specific antibody and detected ubiquitinated species as in B .
    Figure Legend Snippet: ALDH1L1 is ubiquitinated in NIH3T3 cells. A , ALDH1L1 pulled-down from NIH3T3 cell lysates using ALDH1L1-specific antibody and protein A beads; elution with glycine buffer ( lane 1 ), followed by elution with SDS-PAGE loading buffer ( lane 2 ). Proteins were resolved on a 7.5% SDS-PAGE gel followed by Western blot assay with ubiquitin-specific antibody ( left panel ) or ALDH1L1-specific antibody ( right panel ). Lane St is purified recombinant ALDH1L1. B , ALDH1L1 was immunoprecipitated from NIH3T3 cell lysates using an ALDH1L1-specific antibody and Protein A Magnetic beads; samples were resolved on a 7.5% SDS-PAGE followed by Western blot assay with anti-ubiquitin monoclonal antibody. Cells were harvested at different time points after splitting (as indicated); lysates were treated with deubiquitinase inhibitor (4.0 μM recombinant human ubiquitin aldehyde C-terminal derivative) prior to immunoprecipitation. After immunoprecipitation, eluates were treated with deubiquitinase (200 nM of recombinant human USP2 catalytic domain); control , untreated lysates. C , ALDH1L1 was immunoprecipitated from NIH3T3 cells as in B and treated with USP7. Cells were treated with 10 μM MG-132 for 4 h before the pull-down. After treatment with USP7, we have repeated the pull-down with ALDH1L1-specific antibody and detected ubiquitinated species as in B .

    Techniques Used: SDS Page, Western Blot, Purification, Recombinant, Immunoprecipitation, Magnetic Beads

    12) Product Images from "Immunoabsorbent nanoparticles based on a tobamovirus displaying protein A"

    Article Title: Immunoabsorbent nanoparticles based on a tobamovirus displaying protein A

    Journal:

    doi: 10.1073/pnas.0608869103

    High-level expression of protein A-viral particles. ( A ) Constructs used for transfection of  N. benthamiana  plants. Wild-type CP is expressed from an assembled vector (pICH17501). CP-protein A fusions are expressed from separate 5′ and 3′
    Figure Legend Snippet: High-level expression of protein A-viral particles. ( A ) Constructs used for transfection of N. benthamiana plants. Wild-type CP is expressed from an assembled vector (pICH17501). CP-protein A fusions are expressed from separate 5′ and 3′

    Techniques Used: Expressing, Construct, Transfection, Plasmid Preparation

    Efficient binding and release of immunoglobulins by using protein A nanoparticles (pICH20701-pICH21767). Samples from different steps were separated by SDS/PAGE, stained with Coomassie dye, or blotted onto PVDF membrane followed by detection of the heavy
    Figure Legend Snippet: Efficient binding and release of immunoglobulins by using protein A nanoparticles (pICH20701-pICH21767). Samples from different steps were separated by SDS/PAGE, stained with Coomassie dye, or blotted onto PVDF membrane followed by detection of the heavy

    Techniques Used: Binding Assay, SDS Page, Staining

    Binding and precipitation of immunoglobulins by using protein A particles (pICH20701-pICH21767). ( A ) Immunogold labeling of a mixed sample of protein A- and wild-type virions. Gold-conjugated antibodies are only bound to the protein A particle that is
    Figure Legend Snippet: Binding and precipitation of immunoglobulins by using protein A particles (pICH20701-pICH21767). ( A ) Immunogold labeling of a mixed sample of protein A- and wild-type virions. Gold-conjugated antibodies are only bound to the protein A particle that is

    Techniques Used: Binding Assay, Labeling

    Protein A nanoparticles (pICH20701-pICH21767) can be used for purification of plant-made mAbs. ( A ) Coomassie ( Left ) and silver-stained ( Right ) gels, samples from different steps of the purification procedure as described in . Numbers in parentheses
    Figure Legend Snippet: Protein A nanoparticles (pICH20701-pICH21767) can be used for purification of plant-made mAbs. ( A ) Coomassie ( Left ) and silver-stained ( Right ) gels, samples from different steps of the purification procedure as described in . Numbers in parentheses

    Techniques Used: Purification, Staining

    13) Product Images from "PRMT5-mediated histone arginine methylation antagonizes transcriptional repression by polycomb complex PRC2"

    Article Title: PRMT5-mediated histone arginine methylation antagonizes transcriptional repression by polycomb complex PRC2

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa065

    PRMT5 deletion or inhibition upregulates the global level of H3K27 tri-methylation in normal and malignant hematopoietic cells. ( A ) Histones were purified using Histone Extraction Kit from BM cells isolated from two control and two  Mx1-Cre +  Prmt5  conditional KO mice 7 days post poly (I:C) injection. Mono-, di and tri-methylation on H3K27, as well as tri-methylation on H3K4, H3K9, H3K36 and H4K20 were determined by western blot. ( B ) Lineage- and c-kit+ HSPC cells were isolated from BM of day 7 control and  Prmt5  KO mice. Cells were stimulated with or without cytokine mixture of murine SCF (100 ng/ml), IL-3 (20ng/ml), IL-6 (20ng/ml) and FLT3 ligand (100 ng/ml) for 10 min at 37°C. The level of PRMT5, H3K27me3, H3K27ac, H3S10ph and total H3 were determined by western blot with whole cell lysate. ( C ) Lineage- FL cells isolated from E14.5  Prmt5  control and  Vav1-Cre + KO embryos were subject to Western blot and an Odyssey Image System to determine the level of histone H3, H3K27me3 and EZH2 protein. A representative Western blot is shown at left, and a bar graph showing the increase in H3K27me3/H3 ratio at right ( n  = 3). ( D ) MV411 cells were transduced with lentiviruses expressing a scramble shRNA or shRNAs against PRMT5. Level of PRMT5 and H3K27me3 was shown with western blot, while H4 was used as a loading control. Leukemia cell lines HEL ( E ), MV411 and Molm13 ( F ) were treated with DMSO, PRMT5 Inhibitor 1 or 10 µM or EZH2 Inhibitor 1 µM for 4 days. Level of H3K27me3, EZH2 and PRMT5 was determined by western blot; while H3, H4 or β-actin was used as loading controls. The efficiency of PRMT5 Inhibitor was confirmed by the reduced level of cellular symmetric di-methylated arginine (SDR).
    Figure Legend Snippet: PRMT5 deletion or inhibition upregulates the global level of H3K27 tri-methylation in normal and malignant hematopoietic cells. ( A ) Histones were purified using Histone Extraction Kit from BM cells isolated from two control and two Mx1-Cre + Prmt5  conditional KO mice 7 days post poly (I:C) injection. Mono-, di and tri-methylation on H3K27, as well as tri-methylation on H3K4, H3K9, H3K36 and H4K20 were determined by western blot. ( B ) Lineage- and c-kit+ HSPC cells were isolated from BM of day 7 control and Prmt5  KO mice. Cells were stimulated with or without cytokine mixture of murine SCF (100 ng/ml), IL-3 (20ng/ml), IL-6 (20ng/ml) and FLT3 ligand (100 ng/ml) for 10 min at 37°C. The level of PRMT5, H3K27me3, H3K27ac, H3S10ph and total H3 were determined by western blot with whole cell lysate. ( C ) Lineage- FL cells isolated from E14.5 Prmt5  control and Vav1-Cre + KO embryos were subject to Western blot and an Odyssey Image System to determine the level of histone H3, H3K27me3 and EZH2 protein. A representative Western blot is shown at left, and a bar graph showing the increase in H3K27me3/H3 ratio at right ( n = 3). ( D ) MV411 cells were transduced with lentiviruses expressing a scramble shRNA or shRNAs against PRMT5. Level of PRMT5 and H3K27me3 was shown with western blot, while H4 was used as a loading control. Leukemia cell lines HEL ( E ), MV411 and Molm13 ( F ) were treated with DMSO, PRMT5 Inhibitor 1 or 10 µM or EZH2 Inhibitor 1 µM for 4 days. Level of H3K27me3, EZH2 and PRMT5 was determined by western blot; while H3, H4 or β-actin was used as loading controls. The efficiency of PRMT5 Inhibitor was confirmed by the reduced level of cellular symmetric di-methylated arginine (SDR).

    Techniques Used: Inhibition, Methylation, Purification, Isolation, Mouse Assay, Injection, Western Blot, Transduction, Expressing, shRNA

    14) Product Images from "Preassembled Single-Stranded RNA–Argonaute Complexes: A Novel Method to Silence Genes in Cryptosporidium"

    Article Title: Preassembled Single-Stranded RNA–Argonaute Complexes: A Novel Method to Silence Genes in Cryptosporidium

    Journal: The Journal of Infectious Diseases

    doi: 10.1093/infdis/jiv588

    Cryptosporidium transfection and viability assays. A , Fluorescent immunoglobulin G (IgG) labeled with phycoerythrin (red) was encapsulated within protein transfection reagent (PTR) and then used to transfect Cryptosporidium sporozoites within oocysts.
    Figure Legend Snippet: Cryptosporidium transfection and viability assays. A , Fluorescent immunoglobulin G (IgG) labeled with phycoerythrin (red) was encapsulated within protein transfection reagent (PTR) and then used to transfect Cryptosporidium sporozoites within oocysts.

    Techniques Used: Transfection, Labeling

    15) Product Images from "Improved production of Humira antibody in the genetically engineered Escherichia coli SHuffle, by co-expression of human PDI-GPx7 fusions"

    Article Title: Improved production of Humira antibody in the genetically engineered Escherichia coli SHuffle, by co-expression of human PDI-GPx7 fusions

    Journal: Applied Microbiology and Biotechnology

    doi: 10.1007/s00253-020-10920-5

    SHuffle expression of Humira IgG is improved by Gpx7-PDI fusions. a Effects of PDI-GPx7 fusions on the folding of Humira IgG is evaluated by protein A purification from soluble SHuffle lysates grown in shake-flask conditions. Samples were separated in non-reducing SDS-PAGE. The expected size of Humira IgG (150 kDa) is indicated with an arrow. Data are representative of three independent experiments. b Evaluation of PDI-GPx7 fusions on the Humira IgG folding conducted by quantifying the intensity of the band representing Humira to a contaminating band used to normalize loading amounts (*), ( n = 3). One-way ANOVA with alpha = 0.05, *** p value ≤ 0.001, ** p value ≤ 0.01, * p value ≤ 0.05. c Yields of protein A purified Humira antibody, produced in SHuffle or SHuffle2 strains grown in shake flasks from three independent cultures ( n = 3). Unpaired t test with alpha = 0.05, ** p value ≤ 0.01
    Figure Legend Snippet: SHuffle expression of Humira IgG is improved by Gpx7-PDI fusions. a Effects of PDI-GPx7 fusions on the folding of Humira IgG is evaluated by protein A purification from soluble SHuffle lysates grown in shake-flask conditions. Samples were separated in non-reducing SDS-PAGE. The expected size of Humira IgG (150 kDa) is indicated with an arrow. Data are representative of three independent experiments. b Evaluation of PDI-GPx7 fusions on the Humira IgG folding conducted by quantifying the intensity of the band representing Humira to a contaminating band used to normalize loading amounts (*), ( n = 3). One-way ANOVA with alpha = 0.05, *** p value ≤ 0.001, ** p value ≤ 0.01, * p value ≤ 0.05. c Yields of protein A purified Humira antibody, produced in SHuffle or SHuffle2 strains grown in shake flasks from three independent cultures ( n = 3). Unpaired t test with alpha = 0.05, ** p value ≤ 0.01

    Techniques Used: Expressing, Purification, SDS Page, Produced

    16) Product Images from "Robust transcriptome-wide discovery of RNA binding protein binding sites with enhanced CLIP (eCLIP)"

    Article Title: Robust transcriptome-wide discovery of RNA binding protein binding sites with enhanced CLIP (eCLIP)

    Journal: Nature methods

    doi: 10.1038/nmeth.3810

    Improved identification of RNA binding protein (RBP) targets by enhanced C ross L inking and I mmuno P recipitation followed by high-throughput sequencing (eCLIP-seq) (a) RBP-RNA interactions are stabilized with UV crosslinking, followed by limited RNase I digestion, immunoprecipitation of RBP-RNA complexes with a specific antibody of interest, and stringent washes. After dephosphorylation of RNA fragments, an “inline barcoded” RNA adapter is ligated to the 3′ end. After protein gel electrophoresis and nitrocellulose membrane transfer, a region 75 kDa (~220 nt of RNA) above the protein size is excised and proteinase K treated to isolate RNA. RNA is further prepared into paired-end high-throughput sequencing libraries, where read 1 begins with the inline barcode and read 2 begins with a random-mer sequence (added during the 3′ DNA adapter ligation) followed by sequence corresponding to the 5′ end of the original RNA fragment (which often marks reverse transcriptase termination at the crosslink site (red X)). (b) Bars indicate the number of reads remaining after processing steps. PCR duplicate reads that map to the same genomic position and have the same random-mer as previously considered reads are discarded, leaving only “Usable reads”. (c) Varying numbers of uniquely mapped reads were randomly sampled from RBFOX2 iCLIP and eCLIP experiments and PCR duplicate removal was performed. Points indicate the mean of 100 downsampling experiments (for all, s.e.m. is less than 0.1% of mean value). (d) RBFOX2 read density in reads per million usable (RPM). Shown are iCLIP, two biological replicates for eCLIP with paired size-matched input (SMInput) and IgG-only controls. CLIPper-identified clusters indicated as boxes below, with dark colored boxes indicating binding sites enriched above SMInput.
    Figure Legend Snippet: Improved identification of RNA binding protein (RBP) targets by enhanced C ross L inking and I mmuno P recipitation followed by high-throughput sequencing (eCLIP-seq) (a) RBP-RNA interactions are stabilized with UV crosslinking, followed by limited RNase I digestion, immunoprecipitation of RBP-RNA complexes with a specific antibody of interest, and stringent washes. After dephosphorylation of RNA fragments, an “inline barcoded” RNA adapter is ligated to the 3′ end. After protein gel electrophoresis and nitrocellulose membrane transfer, a region 75 kDa (~220 nt of RNA) above the protein size is excised and proteinase K treated to isolate RNA. RNA is further prepared into paired-end high-throughput sequencing libraries, where read 1 begins with the inline barcode and read 2 begins with a random-mer sequence (added during the 3′ DNA adapter ligation) followed by sequence corresponding to the 5′ end of the original RNA fragment (which often marks reverse transcriptase termination at the crosslink site (red X)). (b) Bars indicate the number of reads remaining after processing steps. PCR duplicate reads that map to the same genomic position and have the same random-mer as previously considered reads are discarded, leaving only “Usable reads”. (c) Varying numbers of uniquely mapped reads were randomly sampled from RBFOX2 iCLIP and eCLIP experiments and PCR duplicate removal was performed. Points indicate the mean of 100 downsampling experiments (for all, s.e.m. is less than 0.1% of mean value). (d) RBFOX2 read density in reads per million usable (RPM). Shown are iCLIP, two biological replicates for eCLIP with paired size-matched input (SMInput) and IgG-only controls. CLIPper-identified clusters indicated as boxes below, with dark colored boxes indicating binding sites enriched above SMInput.

    Techniques Used: RNA Binding Assay, Next-Generation Sequencing, Immunoprecipitation, De-Phosphorylation Assay, Nucleic Acid Electrophoresis, Sequencing, Ligation, Polymerase Chain Reaction, Binding Assay

    17) Product Images from "LYL1 Degradation by the Proteasome Is Directed by a N-Terminal PEST Rich Site in a Phosphorylation-Independent Manner"

    Article Title: LYL1 Degradation by the Proteasome Is Directed by a N-Terminal PEST Rich Site in a Phosphorylation-Independent Manner

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0012692

    LYL1 is phosphorylated by MAPK at Serine 36. A ) Dephosphorylation of LYL1 by Alkaline Phosphatase. Protein A/G beads with bound, immunoprecipitated LYL1-WT were treated with the Calf Intestinal Alkaline Phosphatase for 1 h. The samples were then analyzed by anti-V5 immunoblotting. The shown image is representative of four experiments.  B ) Mass fingerprinting of phosphorylated and non-phosphorylated LYL1. The upper, phosphorylated and the lower, non-phosphorylated bands of immunoprecipitated LYL1were excised, digested and mass fingerprinted by MALDI-TOF mass spectrometry. The histograms, representative of two experiments, show the measured m/z values of the peptide spanning residues 34 through 48 derived from the non-phosphorylated (LYL1) and the phosphorylated (LYL1-P) LYL1.  C ) Expression analysis of LYL1-WT and LYL1-S36A. LYL1 wild type and S36A variant were transiently expressed in 293T cells, immunoprecipitated and analyzed by western immunoblotting.  D ) Phosphorylation of LYL1 by the MAPK. Immunoprecipitated LYL1-WT and LYL1-S36 were first treated with Protein Phosphatase 1 to remove all phosphate modifications. Then, re-phosphorylation was attempted by treatment with the MAP kinase. The proteins were resolved and analyzed by anti-V5 immunoblotting. The images are representative of four experiments.
    Figure Legend Snippet: LYL1 is phosphorylated by MAPK at Serine 36. A ) Dephosphorylation of LYL1 by Alkaline Phosphatase. Protein A/G beads with bound, immunoprecipitated LYL1-WT were treated with the Calf Intestinal Alkaline Phosphatase for 1 h. The samples were then analyzed by anti-V5 immunoblotting. The shown image is representative of four experiments. B ) Mass fingerprinting of phosphorylated and non-phosphorylated LYL1. The upper, phosphorylated and the lower, non-phosphorylated bands of immunoprecipitated LYL1were excised, digested and mass fingerprinted by MALDI-TOF mass spectrometry. The histograms, representative of two experiments, show the measured m/z values of the peptide spanning residues 34 through 48 derived from the non-phosphorylated (LYL1) and the phosphorylated (LYL1-P) LYL1. C ) Expression analysis of LYL1-WT and LYL1-S36A. LYL1 wild type and S36A variant were transiently expressed in 293T cells, immunoprecipitated and analyzed by western immunoblotting. D ) Phosphorylation of LYL1 by the MAPK. Immunoprecipitated LYL1-WT and LYL1-S36 were first treated with Protein Phosphatase 1 to remove all phosphate modifications. Then, re-phosphorylation was attempted by treatment with the MAP kinase. The proteins were resolved and analyzed by anti-V5 immunoblotting. The images are representative of four experiments.

    Techniques Used: De-Phosphorylation Assay, Immunoprecipitation, Mass Spectrometry, Derivative Assay, Expressing, Variant Assay, Western Blot

    18) Product Images from "CHIP E3 ligase mediates proteasomal degradation of the proliferation regulatory protein ALDH1L1 during the transition of NIH3T3 fibroblasts from G0/G1 to S-phase"

    Article Title: CHIP E3 ligase mediates proteasomal degradation of the proliferation regulatory protein ALDH1L1 during the transition of NIH3T3 fibroblasts from G0/G1 to S-phase

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0199699

    ALDH1L1 is ubiquitinated in NIH3T3 cells. A , ALDH1L1 pulled-down from NIH3T3 cell lysates using ALDH1L1-specific antibody and protein A beads; elution with glycine buffer ( lane 1 ), followed by elution with SDS-PAGE loading buffer ( lane 2 ). Proteins were resolved on a 7.5% SDS-PAGE gel followed by Western blot assay with ubiquitin-specific antibody ( left panel ) or ALDH1L1-specific antibody ( right panel ). Lane St is purified recombinant ALDH1L1. B , ALDH1L1 was immunoprecipitated from NIH3T3 cell lysates using an ALDH1L1-specific antibody and Protein A Magnetic beads; samples were resolved on a 7.5% SDS-PAGE followed by Western blot assay with anti-ubiquitin monoclonal antibody. Cells were harvested at different time points after splitting (as indicated); lysates were treated with deubiquitinase inhibitor (4.0 μM recombinant human ubiquitin aldehyde C-terminal derivative) prior to immunoprecipitation. After immunoprecipitation, eluates were treated with deubiquitinase (200 nM of recombinant human USP2 catalytic domain); control , untreated lysates. C , ALDH1L1 was immunoprecipitated from NIH3T3 cells as in B and treated with USP7. Cells were treated with 10 μM MG-132 for 4 h before the pull-down. After treatment with USP7, we have repeated the pull-down with ALDH1L1-specific antibody and detected ubiquitinated species as in B .
    Figure Legend Snippet: ALDH1L1 is ubiquitinated in NIH3T3 cells. A , ALDH1L1 pulled-down from NIH3T3 cell lysates using ALDH1L1-specific antibody and protein A beads; elution with glycine buffer ( lane 1 ), followed by elution with SDS-PAGE loading buffer ( lane 2 ). Proteins were resolved on a 7.5% SDS-PAGE gel followed by Western blot assay with ubiquitin-specific antibody ( left panel ) or ALDH1L1-specific antibody ( right panel ). Lane St is purified recombinant ALDH1L1. B , ALDH1L1 was immunoprecipitated from NIH3T3 cell lysates using an ALDH1L1-specific antibody and Protein A Magnetic beads; samples were resolved on a 7.5% SDS-PAGE followed by Western blot assay with anti-ubiquitin monoclonal antibody. Cells were harvested at different time points after splitting (as indicated); lysates were treated with deubiquitinase inhibitor (4.0 μM recombinant human ubiquitin aldehyde C-terminal derivative) prior to immunoprecipitation. After immunoprecipitation, eluates were treated with deubiquitinase (200 nM of recombinant human USP2 catalytic domain); control , untreated lysates. C , ALDH1L1 was immunoprecipitated from NIH3T3 cells as in B and treated with USP7. Cells were treated with 10 μM MG-132 for 4 h before the pull-down. After treatment with USP7, we have repeated the pull-down with ALDH1L1-specific antibody and detected ubiquitinated species as in B .

    Techniques Used: SDS Page, Western Blot, Purification, Recombinant, Immunoprecipitation, Magnetic Beads

    19) Product Images from "GATA1 Binding Kinetics on Conformation-Specific Binding Sites Elicit Differential Transcriptional Regulation"

    Article Title: GATA1 Binding Kinetics on Conformation-Specific Binding Sites Elicit Differential Transcriptional Regulation

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00017-16

    Sequential oligonucleotide pulldown assays. (A to F) Detection of oligo-DNA probes containing Single-GATA (A, D), Pal-GATA (B, E), and Tandem-GATA (C, F), which bind to the respective GATA1 proteins, after the first (A to C) and the second (D to F) pulldowns.
    Figure Legend Snippet: Sequential oligonucleotide pulldown assays. (A to F) Detection of oligo-DNA probes containing Single-GATA (A, D), Pal-GATA (B, E), and Tandem-GATA (C, F), which bind to the respective GATA1 proteins, after the first (A to C) and the second (D to F) pulldowns.

    Techniques Used:

    Analysis of GATA1 binding to Single-GATA. (A) Alignment of the double-stranded DNA probes fixed on the sensor chip. GATA motifs are depicted in pink. Arrows indicate the 5′ to 3′ orientation of consensus GATA motifs. (B) Schematic diagram
    Figure Legend Snippet: Analysis of GATA1 binding to Single-GATA. (A) Alignment of the double-stranded DNA probes fixed on the sensor chip. GATA motifs are depicted in pink. Arrows indicate the 5′ to 3′ orientation of consensus GATA motifs. (B) Schematic diagram

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation

    20) Product Images from "Arf1p Provides an Unexpected Link between COPI Vesicles and mRNA in Saccharomyces cerevisiae D⃞"

    Article Title: Arf1p Provides an Unexpected Link between COPI Vesicles and mRNA in Saccharomyces cerevisiae D⃞

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E04-05-0411

    Arf1p and Pab1p are present in a ribonucleotide particle. (A and B) Pab1p and Arf1p coimmunoprecipitate. Pab1p and Arf1p were chromosomally appended with either a myc- or HA-tag. Yeast lysates were prepared from single- or double-tagged strains and subjected to immunoprecipitation with anti-myc or anti-HA antibodies. The precipitates were analyzed by immunoblot. Lanes 1 and 2 correspond to 1.7% of the lysate. (C) Pab1p–Arf1p interaction depends on mRNA. Yeast lysate from a wild-type strain was incubated with RNase A, DNase I, or mock treated. After the treatment an immunoprecipitation was performed with anti-Arf1p serum or a control serum and protein A-Sepharose. The precipitated proteins were detected by immunoblot. In lane 1, 1.7% of the lysate was loaded. (D) ASH1 mRNA is part of the Pab1p-Arf1p ribonucleotide particle even in the absence of the SHE machinery. A coimmunoprecipitation experiment was performed with affinity-purified anti-Arf1p antibodies. RNA was prepared from the precipitate and subjected to RT-PCR with primer specific for the indicated mRNAs. -RT indicates reactions in the absence of reverse transcriptase. In lanes 1 and 2, 1.7% of the lysate was loaded.
    Figure Legend Snippet: Arf1p and Pab1p are present in a ribonucleotide particle. (A and B) Pab1p and Arf1p coimmunoprecipitate. Pab1p and Arf1p were chromosomally appended with either a myc- or HA-tag. Yeast lysates were prepared from single- or double-tagged strains and subjected to immunoprecipitation with anti-myc or anti-HA antibodies. The precipitates were analyzed by immunoblot. Lanes 1 and 2 correspond to 1.7% of the lysate. (C) Pab1p–Arf1p interaction depends on mRNA. Yeast lysate from a wild-type strain was incubated with RNase A, DNase I, or mock treated. After the treatment an immunoprecipitation was performed with anti-Arf1p serum or a control serum and protein A-Sepharose. The precipitated proteins were detected by immunoblot. In lane 1, 1.7% of the lysate was loaded. (D) ASH1 mRNA is part of the Pab1p-Arf1p ribonucleotide particle even in the absence of the SHE machinery. A coimmunoprecipitation experiment was performed with affinity-purified anti-Arf1p antibodies. RNA was prepared from the precipitate and subjected to RT-PCR with primer specific for the indicated mRNAs. -RT indicates reactions in the absence of reverse transcriptase. In lanes 1 and 2, 1.7% of the lysate was loaded.

    Techniques Used: Immunoprecipitation, Incubation, Affinity Purification, Reverse Transcription Polymerase Chain Reaction

    21) Product Images from "The Architectural Chromatin Factor High Mobility Group A1 Enhances DNA Ligase IV Activity Influencing DNA Repair"

    Article Title: The Architectural Chromatin Factor High Mobility Group A1 Enhances DNA Ligase IV Activity Influencing DNA Repair

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0164258

    HMGA1a associate with the NHEJ DNA repair protein machinery and is a DNA-PK substrate. A. Co-immunoprecipitation (Co-IP) assay of endogenous HMGA1, Ku70, and Ku80 proteins on MDA-MB-231 cells in the presence of absence of Ethidium Bromide (EtBr). Cell lysates were immunoprecipitated with α-HMGA1. The cell lysates (input) and immunoprecipitates were analyzed by immunoblotting with antibodies as indicated. B. HMGA1a fused to MBP (MBP-A1a) or the MBP alone were produced by transient transfection in HEK 293T cells. Cellular lysates (input, lanes 1 and 3) were incubated with amylose resin and affinity captured MBP-HMGA1a and MBP proteins recovered. Bound proteins were eluted by SDS sample buffer, separated by SDS-PAGE (T = 10%), and analyzed by western blot using antibodies specific for Ku70, Ku80 (after Ku70 recognition), Ligase IV, and the catalytic subunit of DNA-PK, DNA-PKcs. C. Recombinant HMGA1a protein was subjected to a phosphorylation assay in presence of [γ- 32 P] ATP with DNA-PK for 0.5 and 16 h (lanes 3 and 4, respectively) and CK2 for 16 h (lane 1). D. Recombinant HMGA1a was subjected to a phosphorylation assay for 16 h with a complete DNA-PK reaction mix (lane 1), without activating DNA (lane 3), or without DNA-PK itself (lane 5). E. Time course phosphorylation assay (0.25, 0.5, 1, and 2 hours) performed with recombinant HMGA1a in the presence (lanes 1, 3, 5, and 7) or absence (lanes 2, 4, 6, and 8) of a specific DNA-PK inhibitor (NU7441–50 nM). Phosphorylated proteins were separated by SDS-PAGE (T = 15%) and 32 P incorporation visualized by autoradiography. Protein molecular markers (kDa) are indicated on the right.
    Figure Legend Snippet: HMGA1a associate with the NHEJ DNA repair protein machinery and is a DNA-PK substrate. A. Co-immunoprecipitation (Co-IP) assay of endogenous HMGA1, Ku70, and Ku80 proteins on MDA-MB-231 cells in the presence of absence of Ethidium Bromide (EtBr). Cell lysates were immunoprecipitated with α-HMGA1. The cell lysates (input) and immunoprecipitates were analyzed by immunoblotting with antibodies as indicated. B. HMGA1a fused to MBP (MBP-A1a) or the MBP alone were produced by transient transfection in HEK 293T cells. Cellular lysates (input, lanes 1 and 3) were incubated with amylose resin and affinity captured MBP-HMGA1a and MBP proteins recovered. Bound proteins were eluted by SDS sample buffer, separated by SDS-PAGE (T = 10%), and analyzed by western blot using antibodies specific for Ku70, Ku80 (after Ku70 recognition), Ligase IV, and the catalytic subunit of DNA-PK, DNA-PKcs. C. Recombinant HMGA1a protein was subjected to a phosphorylation assay in presence of [γ- 32 P] ATP with DNA-PK for 0.5 and 16 h (lanes 3 and 4, respectively) and CK2 for 16 h (lane 1). D. Recombinant HMGA1a was subjected to a phosphorylation assay for 16 h with a complete DNA-PK reaction mix (lane 1), without activating DNA (lane 3), or without DNA-PK itself (lane 5). E. Time course phosphorylation assay (0.25, 0.5, 1, and 2 hours) performed with recombinant HMGA1a in the presence (lanes 1, 3, 5, and 7) or absence (lanes 2, 4, 6, and 8) of a specific DNA-PK inhibitor (NU7441–50 nM). Phosphorylated proteins were separated by SDS-PAGE (T = 15%) and 32 P incorporation visualized by autoradiography. Protein molecular markers (kDa) are indicated on the right.

    Techniques Used: Non-Homologous End Joining, Co-Immunoprecipitation Assay, Multiple Displacement Amplification, Immunoprecipitation, Produced, Transfection, Incubation, SDS Page, Western Blot, Recombinant, Phosphorylation Assay, Autoradiography

    HMGA1a and HMGA2 are phosphorylated by DNA-PK. Different recombinant HMGA1a protein forms (full-length (FL), 1–89, and 34–106) and HMGA2 protein forms (full-length (FL) and 1–93) were phosphorylated by DNA-PK for 16 h and analyzed by LC-MS. Reconstructed mass spectra of the phosphorylated proteins are reported in panels A-E; P indicates the phosphate group. Each protein form was digested by trypsin and peptides analyzed by LC-MS/MS. For each HMGA form (panels A1-E1), a schematic view reports the mass/charge (m/z) relative intensity of phosphorylated peptides (red bars) in comparison with their unmodified counterparts (blue bars). The identities of these peptides (given by first and last aminoacid residue), together with their modified S/T residues, are indicated. A schematic representation of the various HMGA1a forms allows to map the phosphorylation sites with respect to HMGA functional domains (AT-hook: DNA-binding domain; Splicing region: the aminoacid region lacking in HMGA1b splicing isoform; P/P interaction: protein/protein interaction domain; Acidic tail: acidic C-terminal tail).
    Figure Legend Snippet: HMGA1a and HMGA2 are phosphorylated by DNA-PK. Different recombinant HMGA1a protein forms (full-length (FL), 1–89, and 34–106) and HMGA2 protein forms (full-length (FL) and 1–93) were phosphorylated by DNA-PK for 16 h and analyzed by LC-MS. Reconstructed mass spectra of the phosphorylated proteins are reported in panels A-E; P indicates the phosphate group. Each protein form was digested by trypsin and peptides analyzed by LC-MS/MS. For each HMGA form (panels A1-E1), a schematic view reports the mass/charge (m/z) relative intensity of phosphorylated peptides (red bars) in comparison with their unmodified counterparts (blue bars). The identities of these peptides (given by first and last aminoacid residue), together with their modified S/T residues, are indicated. A schematic representation of the various HMGA1a forms allows to map the phosphorylation sites with respect to HMGA functional domains (AT-hook: DNA-binding domain; Splicing region: the aminoacid region lacking in HMGA1b splicing isoform; P/P interaction: protein/protein interaction domain; Acidic tail: acidic C-terminal tail).

    Techniques Used: Recombinant, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Modification, Functional Assay, Binding Assay

    HMGA1 expression lowers the presence of DNA double-strands breaks. A. Quantitative evaluation of neutral comet assays performed on MDA-MB-231_shCTRL and MDA-MB-231_shA1_3 (A) and MCF7_CTRL and MCF7_HMGA1a cells (B) treated with doxorubicin (Dox) for 2 hours and left recover DNA damage for 3 and 5 hours. Not treated cells (NT). Box plot showed the tail moment. P value: **
    Figure Legend Snippet: HMGA1 expression lowers the presence of DNA double-strands breaks. A. Quantitative evaluation of neutral comet assays performed on MDA-MB-231_shCTRL and MDA-MB-231_shA1_3 (A) and MCF7_CTRL and MCF7_HMGA1a cells (B) treated with doxorubicin (Dox) for 2 hours and left recover DNA damage for 3 and 5 hours. Not treated cells (NT). Box plot showed the tail moment. P value: **

    Techniques Used: Expressing, Multiple Displacement Amplification

    HMGA1a enhances Ligase IV activity. A. DNA ligation was assayed using increasing quantities of DNA Ligase IV/XRCC4 (LX) complex (0, 0.3, 0.6, 1.2, 2.4, and 4.8 pmoles) either incubated with DNA alone (lanes 2–6) or with DNA pre-incubated with HMGA1a (1.2 pmoles, lanes 7–11). The DNA substrate (a double-stranded DNA fragment of 442 bp with 4 bp overhangs) and the ligated DNA multimers of different length were separated in an agarose gel. Lanes 1 and 12 show DNA or DNA/HMGA1a alone as controls, respectively. The figure shows a representative ligation assay. B. Quantification of ligation assay shown in A. The percentage of ligated DNA substrate is plotted as a function of the quantity of DNA Ligase IV/XRCC4 complexes (pmoles).
    Figure Legend Snippet: HMGA1a enhances Ligase IV activity. A. DNA ligation was assayed using increasing quantities of DNA Ligase IV/XRCC4 (LX) complex (0, 0.3, 0.6, 1.2, 2.4, and 4.8 pmoles) either incubated with DNA alone (lanes 2–6) or with DNA pre-incubated with HMGA1a (1.2 pmoles, lanes 7–11). The DNA substrate (a double-stranded DNA fragment of 442 bp with 4 bp overhangs) and the ligated DNA multimers of different length were separated in an agarose gel. Lanes 1 and 12 show DNA or DNA/HMGA1a alone as controls, respectively. The figure shows a representative ligation assay. B. Quantification of ligation assay shown in A. The percentage of ligated DNA substrate is plotted as a function of the quantity of DNA Ligase IV/XRCC4 complexes (pmoles).

    Techniques Used: Activity Assay, DNA Ligation, Incubation, Agarose Gel Electrophoresis, Ligation

    HMGA1a counteracts the repressive role of histone H1 with respect to Ligase IV/XRCC4 activity. A. DNA ligation was assayed using fixed amount of Ligase IV/XRCC4 (1.2 pmoles) and increasing quantities of histone H1 (0.1, 0.2, 0.4, 0.8, 1.2, and 2.4 pmoles, lanes 3–8). The unligated DNA substrate is shown in lane 1 and the activity of Ligase IV/XRCC4 alone is shown in lane 10. B. DNA ligation was assayed using fixed amount of Ligase IV/XRCC4 and histone H1 (1.2 and 2.4 pmoles, respectively) and increasing amounts of HMGA1a (0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 2.4, and 4.8 pmoles, lanes 5–12). The unligated DNA substrate is shown in lane 1, the Ligase IV/XRCC4 activity in the presence of histone H1 (2.4 pmoles) is shown in lane 3, and the Ligase IV/XRCC4 alone is shown in lane 10. The figure shows representative ligation assays.
    Figure Legend Snippet: HMGA1a counteracts the repressive role of histone H1 with respect to Ligase IV/XRCC4 activity. A. DNA ligation was assayed using fixed amount of Ligase IV/XRCC4 (1.2 pmoles) and increasing quantities of histone H1 (0.1, 0.2, 0.4, 0.8, 1.2, and 2.4 pmoles, lanes 3–8). The unligated DNA substrate is shown in lane 1 and the activity of Ligase IV/XRCC4 alone is shown in lane 10. B. DNA ligation was assayed using fixed amount of Ligase IV/XRCC4 and histone H1 (1.2 and 2.4 pmoles, respectively) and increasing amounts of HMGA1a (0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 2.4, and 4.8 pmoles, lanes 5–12). The unligated DNA substrate is shown in lane 1, the Ligase IV/XRCC4 activity in the presence of histone H1 (2.4 pmoles) is shown in lane 3, and the Ligase IV/XRCC4 alone is shown in lane 10. The figure shows representative ligation assays.

    Techniques Used: Activity Assay, DNA Ligation, Ligation

    HMGA1a overexpression alters the kinetic of DNA repair in MCF-7 breast cancer cells. A. Immunofluorescence analyses for the visualization of γ-H2AX foci formation in MCF7_CTRL and MCF7_HMGA1a cells exposed to 1 μM doxorubicin (Dox) at 2 hours after treatment removal. Not treated cells (NT) are shown as a control. Nuclei are stained with Hoechst (blue). Images were acquired using a Nikon Eclipse E800 epifluorescence microscope with a 40X objective coupled with a Nikon DXM1200 camera. The mean number of foci/nuclei after doxorubicin tretment is 20.0 and 20.6 for MCF7_CTRL and MCF7_HMGA1a, respectively. Nuclei were manually counted while foci were counted using ImageJ after applying the same threshold for all images, then using the Analyze Particles tool. B. Western blot analyses showing γ-H2AX and H2AX expression levels in MCF7_CTRL and MCF7_HMGA1a cells. Cells were treated with 1 μM doxorubicin for 2 hours and lysates were collected at different time points after treatment removal (not treated—NT and 2, 4, and 8 hours after treatment). Actin was used as an internal normalization. Protein molecular markers (kDa) are shown on the left. C. Graph showing the quantitative evaluation of γ-H2AX induction following the doxorubicin treatment described in B. The reported points are the mean percentage value of four independent experiments ± SD. For each time point, γ-H2AX percentage (%) of intensity (based on densitometric analysis of western blot data) is calculated with the following criteria: (γ-H2AX/H2AX / γ-H2AX NT /H2AX NT ) x 100, P value: *
    Figure Legend Snippet: HMGA1a overexpression alters the kinetic of DNA repair in MCF-7 breast cancer cells. A. Immunofluorescence analyses for the visualization of γ-H2AX foci formation in MCF7_CTRL and MCF7_HMGA1a cells exposed to 1 μM doxorubicin (Dox) at 2 hours after treatment removal. Not treated cells (NT) are shown as a control. Nuclei are stained with Hoechst (blue). Images were acquired using a Nikon Eclipse E800 epifluorescence microscope with a 40X objective coupled with a Nikon DXM1200 camera. The mean number of foci/nuclei after doxorubicin tretment is 20.0 and 20.6 for MCF7_CTRL and MCF7_HMGA1a, respectively. Nuclei were manually counted while foci were counted using ImageJ after applying the same threshold for all images, then using the Analyze Particles tool. B. Western blot analyses showing γ-H2AX and H2AX expression levels in MCF7_CTRL and MCF7_HMGA1a cells. Cells were treated with 1 μM doxorubicin for 2 hours and lysates were collected at different time points after treatment removal (not treated—NT and 2, 4, and 8 hours after treatment). Actin was used as an internal normalization. Protein molecular markers (kDa) are shown on the left. C. Graph showing the quantitative evaluation of γ-H2AX induction following the doxorubicin treatment described in B. The reported points are the mean percentage value of four independent experiments ± SD. For each time point, γ-H2AX percentage (%) of intensity (based on densitometric analysis of western blot data) is calculated with the following criteria: (γ-H2AX/H2AX / γ-H2AX NT /H2AX NT ) x 100, P value: *

    Techniques Used: Over Expression, Immunofluorescence, Staining, Microscopy, Western Blot, Expressing

    HMGA1 confers breast cancer cells a survival advantage with respect to DNA damaging agents. A. Colony formation assay performed on MDA-MB-231_shCTRL and MDA–MB–231_shA1_3 cells (A) and MCF7_CTRL and MCF7_HMGA1a cells (B). Cells were treated with doxorubicin (Dox) to induce DNA damage (not treated—NT), then left to grow, fixed, stained with 0.5% crystal violet, and counted. In the upper panel is shown the representative images of the colony assay, in the lower panel is shown the quantification of the colony formation assay as the mean number of colonies/cm 2 ± SD (n = 4). P value: **
    Figure Legend Snippet: HMGA1 confers breast cancer cells a survival advantage with respect to DNA damaging agents. A. Colony formation assay performed on MDA-MB-231_shCTRL and MDA–MB–231_shA1_3 cells (A) and MCF7_CTRL and MCF7_HMGA1a cells (B). Cells were treated with doxorubicin (Dox) to induce DNA damage (not treated—NT), then left to grow, fixed, stained with 0.5% crystal violet, and counted. In the upper panel is shown the representative images of the colony assay, in the lower panel is shown the quantification of the colony formation assay as the mean number of colonies/cm 2 ± SD (n = 4). P value: **

    Techniques Used: Colony Assay, Multiple Displacement Amplification, Staining

    22) Product Images from "Enhancement of DNA flexibility in vitro and in vivo by HMGB box A proteins carrying box B residues"

    Article Title: Enhancement of DNA flexibility in vitro and in vivo by HMGB box A proteins carrying box B residues

    Journal: Biochemistry

    doi: 10.1021/bi802269f

    Experimental strategy. A. In vitro measurement of HMGB protein enhancement of apparent DNA flexibility by ligase-catalyzed DNA cyclization kinetics. Top: HMGB protein was incubated for various times with ~200-bp radiolabeled DNA (M) in the presence of
    Figure Legend Snippet: Experimental strategy. A. In vitro measurement of HMGB protein enhancement of apparent DNA flexibility by ligase-catalyzed DNA cyclization kinetics. Top: HMGB protein was incubated for various times with ~200-bp radiolabeled DNA (M) in the presence of

    Techniques Used: In Vitro, Incubation

    In vitro assay of DNA flexibility enhancement by HMGB proteins and chimeras. A. Example data from T4 DNA ligase cyclization assay for 200-bp DNA probe in the absence (—) and presence of 40 nM HMGB constructs 16 and 5 (see ). B. Graphical
    Figure Legend Snippet: In vitro assay of DNA flexibility enhancement by HMGB proteins and chimeras. A. Example data from T4 DNA ligase cyclization assay for 200-bp DNA probe in the absence (—) and presence of 40 nM HMGB constructs 16 and 5 (see ). B. Graphical

    Techniques Used: In Vitro, Construct

    23) Product Images from "Dephosphorylation of the transcriptional cofactor NACA by the PP1A phosphatase enhances cJUN transcriptional activity and osteoblast differentiation"

    Article Title: Dephosphorylation of the transcriptional cofactor NACA by the PP1A phosphatase enhances cJUN transcriptional activity and osteoblast differentiation

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.006920

    PP1A associates with NACA. A , schematic overview of the proteomics approach used to identify NACA-interacting proteins. B , silver-stained SDS-PAGE gel showing expression and purification of NACA protein. C , mass spectrometry (MS/MS) analysis identified numerous NACA-associated proteins, including several components of the PP1A holoenzyme complex. D , validation of interaction of NACA-FLAG with endogenous PP1A complex proteins. HEK293T cells expressing NACA-FLAG or FLAG empty vector were lysed and immunoprecipitated ( IP ) with anti-FLAG antibody beads and blotted with anti-FLAG antibody or the indicated PP1A-complex antibodies. E , GST-pulldown analysis showing direct interaction between NACA and PPP1CA as well as PPP1R18. GST-NACA fusion protein or GST alone expressed in bacteria ( left panel ) was incubated with in vitro translated PPP1R9B, PPP1R12A, PPP1R18, and PPP1CA, and then subjected to SDS-PAGE and Western blot analysis ( right panel ).
    Figure Legend Snippet: PP1A associates with NACA. A , schematic overview of the proteomics approach used to identify NACA-interacting proteins. B , silver-stained SDS-PAGE gel showing expression and purification of NACA protein. C , mass spectrometry (MS/MS) analysis identified numerous NACA-associated proteins, including several components of the PP1A holoenzyme complex. D , validation of interaction of NACA-FLAG with endogenous PP1A complex proteins. HEK293T cells expressing NACA-FLAG or FLAG empty vector were lysed and immunoprecipitated ( IP ) with anti-FLAG antibody beads and blotted with anti-FLAG antibody or the indicated PP1A-complex antibodies. E , GST-pulldown analysis showing direct interaction between NACA and PPP1CA as well as PPP1R18. GST-NACA fusion protein or GST alone expressed in bacteria ( left panel ) was incubated with in vitro translated PPP1R9B, PPP1R12A, PPP1R18, and PPP1CA, and then subjected to SDS-PAGE and Western blot analysis ( right panel ).

    Techniques Used: Staining, SDS Page, Expressing, Purification, Mass Spectrometry, Tandem Mass Spectroscopy, Plasmid Preparation, Immunoprecipitation, Incubation, In Vitro, Western Blot

    24) Product Images from "The RNA binding of protein A from Wuhan nodavirus is mediated by mitochondrial membrane lipids"

    Article Title: The RNA binding of protein A from Wuhan nodavirus is mediated by mitochondrial membrane lipids

    Journal: Virology

    doi: 10.1016/j.virol.2014.05.022

    Characterization of the RNA probe used for determining the RNA binding of WhNV protein A in vitro. (A) Schematic of plasmids used for protein A GAA (prot A GAA ) and (+)RNA1E expression. RNA1E templates with authentic viral 5′ and 3′ termini of WhNV RNA1 and an inserting EGFP sequence were generated from pAC1E by precisely placing the Ac5 promoter start site and a hepatitisδribozyme (Rz), respectively, and by mutating the start codon at the indicated location to disrupt translation. The Ac5 promoter and SV40 polyadenylation signal (SV) flanking the protein A ORF in pA GAA thereof disrupt its activity as a viral RNA replication template and mutating the replication GDD sites into GAA but maintain its activity to recruit RNA ( Qiu et al., 2014 ). pA GAA -derived protein A GAA subsequently directs (+)RNA1E recruitment from (+)RNA1E template transcribed from pAC1E. (B) The secondary structure predicted for RNA1 nt 50–118 [RNA1 (50–118) ]. Del represents removing the RNA sequences formed the helices structure and Mut represents destroying the base pairing in the helices section. (C) RNA1 (50–118) mediates RNA1 recruitment in cells. Pr-E cells were transfected with the indicated plasmids, including pAC1E wt, Del or Mut (as shown in B) in the absence or in the presence of pA GAA (protein A GAA ). After transfection for 36 h, total RNA was extracted and analyzed by Northern blot with the probes against EGFP and 18S rRNA, respectively. (D) The levels of (+)RNA1E were determined from three experiments after normalization to 18S rRNA and are expressed as the level of protein A-stimulated (+)RNA1E accumulation relative to wt (+)RNA1E. (E) The secondary structure predicted for RNA2 nt 123–164 [RNA1 (123–164) ]. Del′ represents removing the RNA sequences formed the helices structure. (F) Schematic of plasmids used for protein A-mediated RNA2 recruitment. (G) RNA2 (123–164) mediates RNA2 recruitment in cells. Pr-E cells were transfected with the indicated plasmids, including pAC2 wt, pAC2 181–1562, pAC2 1–180, pAC2 Del′ (as shown in E and F) in the absence or in the presence of pA GAA . After transfection for 36 h, total RNA was extracted and analyzed by Northern blot with the probes against EGFP and 18S rRNA, respectively. (H) The levels of (+)RNA2 were determined from three experiments after normalization to 18S rRNA and are expressed as the level of protein A-stimulated (+)RNA2 accumulation relative to wt (+)RNA2.
    Figure Legend Snippet: Characterization of the RNA probe used for determining the RNA binding of WhNV protein A in vitro. (A) Schematic of plasmids used for protein A GAA (prot A GAA ) and (+)RNA1E expression. RNA1E templates with authentic viral 5′ and 3′ termini of WhNV RNA1 and an inserting EGFP sequence were generated from pAC1E by precisely placing the Ac5 promoter start site and a hepatitisδribozyme (Rz), respectively, and by mutating the start codon at the indicated location to disrupt translation. The Ac5 promoter and SV40 polyadenylation signal (SV) flanking the protein A ORF in pA GAA thereof disrupt its activity as a viral RNA replication template and mutating the replication GDD sites into GAA but maintain its activity to recruit RNA ( Qiu et al., 2014 ). pA GAA -derived protein A GAA subsequently directs (+)RNA1E recruitment from (+)RNA1E template transcribed from pAC1E. (B) The secondary structure predicted for RNA1 nt 50–118 [RNA1 (50–118) ]. Del represents removing the RNA sequences formed the helices structure and Mut represents destroying the base pairing in the helices section. (C) RNA1 (50–118) mediates RNA1 recruitment in cells. Pr-E cells were transfected with the indicated plasmids, including pAC1E wt, Del or Mut (as shown in B) in the absence or in the presence of pA GAA (protein A GAA ). After transfection for 36 h, total RNA was extracted and analyzed by Northern blot with the probes against EGFP and 18S rRNA, respectively. (D) The levels of (+)RNA1E were determined from three experiments after normalization to 18S rRNA and are expressed as the level of protein A-stimulated (+)RNA1E accumulation relative to wt (+)RNA1E. (E) The secondary structure predicted for RNA2 nt 123–164 [RNA1 (123–164) ]. Del′ represents removing the RNA sequences formed the helices structure. (F) Schematic of plasmids used for protein A-mediated RNA2 recruitment. (G) RNA2 (123–164) mediates RNA2 recruitment in cells. Pr-E cells were transfected with the indicated plasmids, including pAC2 wt, pAC2 181–1562, pAC2 1–180, pAC2 Del′ (as shown in E and F) in the absence or in the presence of pA GAA . After transfection for 36 h, total RNA was extracted and analyzed by Northern blot with the probes against EGFP and 18S rRNA, respectively. (H) The levels of (+)RNA2 were determined from three experiments after normalization to 18S rRNA and are expressed as the level of protein A-stimulated (+)RNA2 accumulation relative to wt (+)RNA2.

    Techniques Used: RNA Binding Assay, In Vitro, Expressing, Sequencing, Generated, Activity Assay, Derivative Assay, Transfection, Northern Blot

    The binding preference of recombinant protein A to RNA1. (A) SDS-PAGE analysis of purified recombinant protein A from E. coli . Protein A ORF was cloned into pMAL-c2X and expressed as C-terminal fusion proteins with MBP (MBP-protA) as described previously ( Qiu et al., 2014 ). Lane 1, Marker; lane 2, MBP protein alone; lane 3, MBP-protA. (B) Gel mobility shift assay showing interactions between MBP-protA and RNA1. The in vitro transcribed DIG-labeled RNA1 (50–118) was separately incubated with bovine serum albumin (BSA, lane 2), MBP alone (lane 3), boiled MBP-protA (lane 4) and MBP-protA (lane 5) (3 μM each), in a binding buffer at 27 °C for 30 min and then analyzed in 1% agarose gel. Gel was transferred to Hybond N nylon membranes via capillary transfer and then the membranes were incubated with anti-DIG antibody conjugated with alkaline phosphatase, exposed to film. The unbound, free RNA1 (50–118) probe and the shift (bound) RNA-protein complex are marked on the right. (C) Unlabeled competitor RNAs at increasing concentrations (in 1-, 10-, 60-fold excess) were added to the mixture containing the DIG-labeled RNA1 (50–118) and 3 μM MBP-protA, and the bound complexes were analyzed in a gel mobility shift assay. The tRNA was from yeast. (D) Gel mobility shift assay showing interactions between MBP-protA and RNA2. The in vitro transcribed DIG-labeled RNA2 (123–164) was incubated with MBP-protA (lane 2) and in a binding buffer at 27 °C for 30 min and then analyzed in 1% agarose gel. Unlabeled competitor RNAs at increasing concentrations (in 5-, 50-, 100-fold excess) were added to the mixture containing the DIG-labeled RNA2 (123–164) and 3 μM MBP-protA, and the bound complexes were analyzed in a gel mobility shift assay. (E) Cooperative binding of MBP-protA to RNA1 (50–118) . Gel mobility shift assays were performed using increasing molar concentrations of MBP-protA incubated with 20nM RNA1 (50–118) probe. The molar concentrations of MBP-protA (0.1–6 μM) are indicated above each lane. (F) The plot of the percent of RNA bound versus molar concentration of MBP-protA. (G) The Hill coefficients of the RNA binding of protein A based on Fig. 2 E at low and high protein concentrations are indicated.
    Figure Legend Snippet: The binding preference of recombinant protein A to RNA1. (A) SDS-PAGE analysis of purified recombinant protein A from E. coli . Protein A ORF was cloned into pMAL-c2X and expressed as C-terminal fusion proteins with MBP (MBP-protA) as described previously ( Qiu et al., 2014 ). Lane 1, Marker; lane 2, MBP protein alone; lane 3, MBP-protA. (B) Gel mobility shift assay showing interactions between MBP-protA and RNA1. The in vitro transcribed DIG-labeled RNA1 (50–118) was separately incubated with bovine serum albumin (BSA, lane 2), MBP alone (lane 3), boiled MBP-protA (lane 4) and MBP-protA (lane 5) (3 μM each), in a binding buffer at 27 °C for 30 min and then analyzed in 1% agarose gel. Gel was transferred to Hybond N nylon membranes via capillary transfer and then the membranes were incubated with anti-DIG antibody conjugated with alkaline phosphatase, exposed to film. The unbound, free RNA1 (50–118) probe and the shift (bound) RNA-protein complex are marked on the right. (C) Unlabeled competitor RNAs at increasing concentrations (in 1-, 10-, 60-fold excess) were added to the mixture containing the DIG-labeled RNA1 (50–118) and 3 μM MBP-protA, and the bound complexes were analyzed in a gel mobility shift assay. The tRNA was from yeast. (D) Gel mobility shift assay showing interactions between MBP-protA and RNA2. The in vitro transcribed DIG-labeled RNA2 (123–164) was incubated with MBP-protA (lane 2) and in a binding buffer at 27 °C for 30 min and then analyzed in 1% agarose gel. Unlabeled competitor RNAs at increasing concentrations (in 5-, 50-, 100-fold excess) were added to the mixture containing the DIG-labeled RNA2 (123–164) and 3 μM MBP-protA, and the bound complexes were analyzed in a gel mobility shift assay. (E) Cooperative binding of MBP-protA to RNA1 (50–118) . Gel mobility shift assays were performed using increasing molar concentrations of MBP-protA incubated with 20nM RNA1 (50–118) probe. The molar concentrations of MBP-protA (0.1–6 μM) are indicated above each lane. (F) The plot of the percent of RNA bound versus molar concentration of MBP-protA. (G) The Hill coefficients of the RNA binding of protein A based on Fig. 2 E at low and high protein concentrations are indicated.

    Techniques Used: Binding Assay, Recombinant, SDS Page, Purification, Clone Assay, Marker, Mobility Shift, In Vitro, Labeling, Incubation, Agarose Gel Electrophoresis, Concentration Assay, RNA Binding Assay

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    New England Biolabs rna
    Improved identification of <t>RNA</t> binding protein (RBP) targets by enhanced C ross L inking and I mmuno P recipitation followed by high-throughput sequencing (eCLIP-seq) (a) RBP-RNA interactions are stabilized with UV crosslinking, followed by limited RNase I digestion, immunoprecipitation of RBP-RNA complexes with a specific antibody of interest, and stringent washes. After dephosphorylation of RNA fragments, an “inline barcoded” RNA adapter is ligated to the 3′ end. After protein gel electrophoresis and nitrocellulose membrane transfer, a region 75 <t>kDa</t> (~220 nt of RNA) above the protein size is excised and proteinase K treated to isolate RNA. RNA is further prepared into paired-end high-throughput sequencing libraries, where read 1 begins with the inline barcode and read 2 begins with a random-mer sequence (added during the 3′ DNA adapter ligation) followed by sequence corresponding to the 5′ end of the original RNA fragment (which often marks reverse transcriptase termination at the crosslink site (red X)). (b) Bars indicate the number of reads remaining after processing steps. PCR duplicate reads that map to the same genomic position and have the same random-mer as previously considered reads are discarded, leaving only “Usable reads”. (c) Varying numbers of uniquely mapped reads were randomly sampled from RBFOX2 iCLIP and eCLIP experiments and PCR duplicate removal was performed. Points indicate the mean of 100 downsampling experiments (for all, s.e.m. is less than 0.1% of mean value). (d) RBFOX2 read density in reads per million usable (RPM). Shown are iCLIP, two biological replicates for eCLIP with paired size-matched input (SMInput) and IgG-only controls. CLIPper-identified clusters indicated as boxes below, with dark colored boxes indicating binding sites enriched above SMInput.
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    Improved identification of RNA binding protein (RBP) targets by enhanced C ross L inking and I mmuno P recipitation followed by high-throughput sequencing (eCLIP-seq) (a) RBP-RNA interactions are stabilized with UV crosslinking, followed by limited RNase I digestion, immunoprecipitation of RBP-RNA complexes with a specific antibody of interest, and stringent washes. After dephosphorylation of RNA fragments, an “inline barcoded” RNA adapter is ligated to the 3′ end. After protein gel electrophoresis and nitrocellulose membrane transfer, a region 75 kDa (~220 nt of RNA) above the protein size is excised and proteinase K treated to isolate RNA. RNA is further prepared into paired-end high-throughput sequencing libraries, where read 1 begins with the inline barcode and read 2 begins with a random-mer sequence (added during the 3′ DNA adapter ligation) followed by sequence corresponding to the 5′ end of the original RNA fragment (which often marks reverse transcriptase termination at the crosslink site (red X)). (b) Bars indicate the number of reads remaining after processing steps. PCR duplicate reads that map to the same genomic position and have the same random-mer as previously considered reads are discarded, leaving only “Usable reads”. (c) Varying numbers of uniquely mapped reads were randomly sampled from RBFOX2 iCLIP and eCLIP experiments and PCR duplicate removal was performed. Points indicate the mean of 100 downsampling experiments (for all, s.e.m. is less than 0.1% of mean value). (d) RBFOX2 read density in reads per million usable (RPM). Shown are iCLIP, two biological replicates for eCLIP with paired size-matched input (SMInput) and IgG-only controls. CLIPper-identified clusters indicated as boxes below, with dark colored boxes indicating binding sites enriched above SMInput.

    Journal: Nature methods

    Article Title: Robust transcriptome-wide discovery of RNA binding protein binding sites with enhanced CLIP (eCLIP)

    doi: 10.1038/nmeth.3810

    Figure Lengend Snippet: Improved identification of RNA binding protein (RBP) targets by enhanced C ross L inking and I mmuno P recipitation followed by high-throughput sequencing (eCLIP-seq) (a) RBP-RNA interactions are stabilized with UV crosslinking, followed by limited RNase I digestion, immunoprecipitation of RBP-RNA complexes with a specific antibody of interest, and stringent washes. After dephosphorylation of RNA fragments, an “inline barcoded” RNA adapter is ligated to the 3′ end. After protein gel electrophoresis and nitrocellulose membrane transfer, a region 75 kDa (~220 nt of RNA) above the protein size is excised and proteinase K treated to isolate RNA. RNA is further prepared into paired-end high-throughput sequencing libraries, where read 1 begins with the inline barcode and read 2 begins with a random-mer sequence (added during the 3′ DNA adapter ligation) followed by sequence corresponding to the 5′ end of the original RNA fragment (which often marks reverse transcriptase termination at the crosslink site (red X)). (b) Bars indicate the number of reads remaining after processing steps. PCR duplicate reads that map to the same genomic position and have the same random-mer as previously considered reads are discarded, leaving only “Usable reads”. (c) Varying numbers of uniquely mapped reads were randomly sampled from RBFOX2 iCLIP and eCLIP experiments and PCR duplicate removal was performed. Points indicate the mean of 100 downsampling experiments (for all, s.e.m. is less than 0.1% of mean value). (d) RBFOX2 read density in reads per million usable (RPM). Shown are iCLIP, two biological replicates for eCLIP with paired size-matched input (SMInput) and IgG-only controls. CLIPper-identified clusters indicated as boxes below, with dark colored boxes indicating binding sites enriched above SMInput.

    Article Snippet: Samples were then run on standard protein gels and transferred to nitrocellulose membranes, and a region 75 kDa (~150 nt of RNA) above the protein size was isolated and proteinase K (NEB) treated to isolate RNA.

    Techniques: RNA Binding Assay, Next-Generation Sequencing, Immunoprecipitation, De-Phosphorylation Assay, Nucleic Acid Electrophoresis, Sequencing, Ligation, Polymerase Chain Reaction, Binding Assay

    DNA-PKcs interacts with the tandem BRCT domain of BRCA1.  (A)  FLAG-tagged fragments of BRCA1 were transiently expressed in HeLa cells and subsequently IPed using an antiserum specific for the FLAG-tag. The immunoprecipitates were analyzed by western blot analysis using anti-DNA-PKcs or anti-FLAG antibodies.  (B)  DNA-PKcs was IP from HeLa cells, washed three times and the IP DNA-PKcs which was still bound to the protein A-sepharose was incubated with purified GST or GST-tagged tandem BRCT domain of BRCA1 or MDC1. The pull-downs were then washed and western blot analysis was performed using antibodies against GST or DNA-PKcs.  (C)  Representation of BRCA1 fragments used in (D and E) and their ability to interact with DNA-PKcs.

    Journal: Nucleic Acids Research

    Article Title: BRCA1 modulates the autophosphorylation status of DNA-PKcs in S phase of the cell cycle

    doi: 10.1093/nar/gku824

    Figure Lengend Snippet: DNA-PKcs interacts with the tandem BRCT domain of BRCA1. (A) FLAG-tagged fragments of BRCA1 were transiently expressed in HeLa cells and subsequently IPed using an antiserum specific for the FLAG-tag. The immunoprecipitates were analyzed by western blot analysis using anti-DNA-PKcs or anti-FLAG antibodies. (B) DNA-PKcs was IP from HeLa cells, washed three times and the IP DNA-PKcs which was still bound to the protein A-sepharose was incubated with purified GST or GST-tagged tandem BRCT domain of BRCA1 or MDC1. The pull-downs were then washed and western blot analysis was performed using antibodies against GST or DNA-PKcs. (C) Representation of BRCA1 fragments used in (D and E) and their ability to interact with DNA-PKcs.

    Article Snippet: The protein A beads were washed three times with wash buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 10% glycerol and protease inhibitors), and untreated or treated with 1200 U of λ-phosphatase (New England Biolabs) for 30 min at 30ºC and then incubated with purified GST or GST-tagged BRCA1 fragments in wash buffer supplemented with 10 μg bovine serum albumin (BSA) at ambient temperatures for 2 h with mixing.

    Techniques: FLAG-tag, Western Blot, Incubation, Purification

    The interaction between DNA-PKcs and the tandem BRCT domain of BRCA1 is phospho-independent.  (A)  Exponentially HeLa cells were mock or irradiated with 20 Gy and allowed to recover for 1 h. DNA-PKcs was IPed, washed three times and the IP DNA-PKcs which was still bound to the protein A-sepharose was incubated with GST or GST-tagged BRCA1 tandem BRCT domain (BRCT) protein fragment in the presence or absence of lambda phosphatase (PPase). The pull-downs were then washed and western blot analysis was performed using antibodies against GST or DNA-PKcs. Antibodies against phosphorylated serine 2056 were used to show IR-induced phosphorylation of DNA-PKcs.  (B)  Purified DNA-PKcs was incubated with GST or GST-tagged protein fragments of BRCA1 encoding the RING finger domain (RING) or the tandem BRCT domain (BRCT) in the presence or absence of lambda phosphatase (PPase). The pull-downs were then washed and western blot analysis was performed using antibodies against GST or DNA-PKcs.

    Journal: Nucleic Acids Research

    Article Title: BRCA1 modulates the autophosphorylation status of DNA-PKcs in S phase of the cell cycle

    doi: 10.1093/nar/gku824

    Figure Lengend Snippet: The interaction between DNA-PKcs and the tandem BRCT domain of BRCA1 is phospho-independent. (A) Exponentially HeLa cells were mock or irradiated with 20 Gy and allowed to recover for 1 h. DNA-PKcs was IPed, washed three times and the IP DNA-PKcs which was still bound to the protein A-sepharose was incubated with GST or GST-tagged BRCA1 tandem BRCT domain (BRCT) protein fragment in the presence or absence of lambda phosphatase (PPase). The pull-downs were then washed and western blot analysis was performed using antibodies against GST or DNA-PKcs. Antibodies against phosphorylated serine 2056 were used to show IR-induced phosphorylation of DNA-PKcs. (B) Purified DNA-PKcs was incubated with GST or GST-tagged protein fragments of BRCA1 encoding the RING finger domain (RING) or the tandem BRCT domain (BRCT) in the presence or absence of lambda phosphatase (PPase). The pull-downs were then washed and western blot analysis was performed using antibodies against GST or DNA-PKcs.

    Article Snippet: The protein A beads were washed three times with wash buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 10% glycerol and protease inhibitors), and untreated or treated with 1200 U of λ-phosphatase (New England Biolabs) for 30 min at 30ºC and then incubated with purified GST or GST-tagged BRCA1 fragments in wash buffer supplemented with 10 μg bovine serum albumin (BSA) at ambient temperatures for 2 h with mixing.

    Techniques: Irradiation, Incubation, Western Blot, Purification

    BRCA1 preferentially interacts with the N-terminal region of DNA-PKcs near the 2056 cluster.  (A)  FLAG-tagged N-terminal fragment (N-PKcs) and C-terminal fragment (C-PKcs) of DNA-PKcs were expressed and IPed from Sf9 cells using FLAG antibody, washed three times and the IP DNA-PKcs fragments still bound to the protein A-sepharose were incubated with GST or BRCT. Western blot analysis was then performed using antibodies against GST or FLAG.  (B)  GST-tagged fragments of DNA-PKcs encoding the amino acids indicated in the figure were purified from bacteria and left bound on the glutathione-agarose. These fragments were then incubated with purified His-tagged BRCA1 tandem BRCT domain protein fragment (BRCT). Following the incubation, the GST pull-downs were washed and western blot analysis was performed using anti-His and GST antibodies.

    Journal: Nucleic Acids Research

    Article Title: BRCA1 modulates the autophosphorylation status of DNA-PKcs in S phase of the cell cycle

    doi: 10.1093/nar/gku824

    Figure Lengend Snippet: BRCA1 preferentially interacts with the N-terminal region of DNA-PKcs near the 2056 cluster. (A) FLAG-tagged N-terminal fragment (N-PKcs) and C-terminal fragment (C-PKcs) of DNA-PKcs were expressed and IPed from Sf9 cells using FLAG antibody, washed three times and the IP DNA-PKcs fragments still bound to the protein A-sepharose were incubated with GST or BRCT. Western blot analysis was then performed using antibodies against GST or FLAG. (B) GST-tagged fragments of DNA-PKcs encoding the amino acids indicated in the figure were purified from bacteria and left bound on the glutathione-agarose. These fragments were then incubated with purified His-tagged BRCA1 tandem BRCT domain protein fragment (BRCT). Following the incubation, the GST pull-downs were washed and western blot analysis was performed using anti-His and GST antibodies.

    Article Snippet: The protein A beads were washed three times with wash buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 10% glycerol and protease inhibitors), and untreated or treated with 1200 U of λ-phosphatase (New England Biolabs) for 30 min at 30ºC and then incubated with purified GST or GST-tagged BRCA1 fragments in wash buffer supplemented with 10 μg bovine serum albumin (BSA) at ambient temperatures for 2 h with mixing.

    Techniques: Incubation, Western Blot, Purification

    Phosphorylation of the CTD is required for efficient transcription through terminator sequences. (A) Structure of templates. The pW1 template carries a terminator formed by an RNA stem-loop, followed by nine uridines (τ). pH3.3 contains two tandem arrest sequences (τ′ 1a and τ′ 1b ) that induce DNA bending. In pΔTerm, the terminator sequence was replaced by the original HIV sequence. (B) Transcription reactions were performed with three different immobilized DNA templates (pW1, pH3.3, or pΔTerm). Reactions contained 100 ng of LacR and were performed in the absence (−) or presence (+) of 20 ng of Tat. After labeling for 20 min with [α- 32 P]UTP (−PP1), the immobilized templates were purified and treated with RNase H in the presence of the RHX1 and RHLAC oligonucleotides to remove the labeled RNA transcripts from transcription complexes that had read through the LacR site. The arrested complexes were then dephosphorylated by PP1 treatment (+PP1). After the complexes were washed with TMZ buffer, the dephosphorylated complexes were chased by the addition of 250 μM ATP, GTP, and CTP; 5 μM UTP; and 25 mM IPTG in the absence (−) or presence (+) of 100 μM DRB. Positions of transcripts at the runoffs (ρ), terminator (τ), and lac repressor (LacR) in pW1 template are indicated. τ′ 1a and τ′ 1b indicate the stop sites from the pH3.3 template. (C) Immunoblot. Samples of the reactions shown in panel B were immunoblotted with the N-20 antibody against RNA Pol II and the anti-Spt5 antibody. Tat-dependent hyperphosphorylation of RNA polymerase CTD and Spt5 was observed during the chase of dephosphorylated complexes assembled on each template.

    Journal: Molecular and Cellular Biology

    Article Title: Phosphorylation of the RNA Polymerase II Carboxyl-Terminal Domain by CDK9 Is Directly Responsible for Human Immunodeficiency Virus Type 1 Tat-Activated Transcriptional Elongation

    doi: 10.1128/MCB.22.13.4622-4637.2002

    Figure Lengend Snippet: Phosphorylation of the CTD is required for efficient transcription through terminator sequences. (A) Structure of templates. The pW1 template carries a terminator formed by an RNA stem-loop, followed by nine uridines (τ). pH3.3 contains two tandem arrest sequences (τ′ 1a and τ′ 1b ) that induce DNA bending. In pΔTerm, the terminator sequence was replaced by the original HIV sequence. (B) Transcription reactions were performed with three different immobilized DNA templates (pW1, pH3.3, or pΔTerm). Reactions contained 100 ng of LacR and were performed in the absence (−) or presence (+) of 20 ng of Tat. After labeling for 20 min with [α- 32 P]UTP (−PP1), the immobilized templates were purified and treated with RNase H in the presence of the RHX1 and RHLAC oligonucleotides to remove the labeled RNA transcripts from transcription complexes that had read through the LacR site. The arrested complexes were then dephosphorylated by PP1 treatment (+PP1). After the complexes were washed with TMZ buffer, the dephosphorylated complexes were chased by the addition of 250 μM ATP, GTP, and CTP; 5 μM UTP; and 25 mM IPTG in the absence (−) or presence (+) of 100 μM DRB. Positions of transcripts at the runoffs (ρ), terminator (τ), and lac repressor (LacR) in pW1 template are indicated. τ′ 1a and τ′ 1b indicate the stop sites from the pH3.3 template. (C) Immunoblot. Samples of the reactions shown in panel B were immunoblotted with the N-20 antibody against RNA Pol II and the anti-Spt5 antibody. Tat-dependent hyperphosphorylation of RNA polymerase CTD and Spt5 was observed during the chase of dephosphorylated complexes assembled on each template.

    Article Snippet: Elongation complexes arrested by lac repressor were treated with 2.5 U of protein phosphatase 1 (PP1; New England Biolabs) for 1 h at 30°C with occasional mixing.

    Techniques: Sequencing, Labeling, Purification

    Tat and TAR stimulate hyperphosphorylation of CTD in transcription elongation complexes. (A) Rephosphorylation by CDK9. Elongation complexes assembled on the pW1 templates were dephosphorylated by PP1 treatment (Pol II a ). Chase of the dephosphorylated complexes from the LacR site in the presence of all four nucleotides and IPTG permits rephosphorylation of the RNA polymerase CTD (Pol II o *) in the presence (+) of 20 ng of Tat but not in the absence (−) of Tat. (B) Inhibition of CDK9 by DRB. Between 0 and 10 μM DRB was included in the chase reactions containing dephosphorylated elongation complexes. Transcription was performed on the pW1 DNA templates in the absence (−) and presence (+) of 20 ng of Tat. (C) Activation of CDK9 by Tat and TAR. Elongation complexes were assembled on templates carrying wild-type TAR (WT) or mutant TAR elements in the Tat-binding site (mGC) or in the CycT1-binding site (mLG) in the absence (−) or presence (+) of 20 ng of Tat. After dephosphorylation of RNA polymerase CTD, the complexes were chased as described above.

    Journal: Molecular and Cellular Biology

    Article Title: Phosphorylation of the RNA Polymerase II Carboxyl-Terminal Domain by CDK9 Is Directly Responsible for Human Immunodeficiency Virus Type 1 Tat-Activated Transcriptional Elongation

    doi: 10.1128/MCB.22.13.4622-4637.2002

    Figure Lengend Snippet: Tat and TAR stimulate hyperphosphorylation of CTD in transcription elongation complexes. (A) Rephosphorylation by CDK9. Elongation complexes assembled on the pW1 templates were dephosphorylated by PP1 treatment (Pol II a ). Chase of the dephosphorylated complexes from the LacR site in the presence of all four nucleotides and IPTG permits rephosphorylation of the RNA polymerase CTD (Pol II o *) in the presence (+) of 20 ng of Tat but not in the absence (−) of Tat. (B) Inhibition of CDK9 by DRB. Between 0 and 10 μM DRB was included in the chase reactions containing dephosphorylated elongation complexes. Transcription was performed on the pW1 DNA templates in the absence (−) and presence (+) of 20 ng of Tat. (C) Activation of CDK9 by Tat and TAR. Elongation complexes were assembled on templates carrying wild-type TAR (WT) or mutant TAR elements in the Tat-binding site (mGC) or in the CycT1-binding site (mLG) in the absence (−) or presence (+) of 20 ng of Tat. After dephosphorylation of RNA polymerase CTD, the complexes were chased as described above.

    Article Snippet: Elongation complexes arrested by lac repressor were treated with 2.5 U of protein phosphatase 1 (PP1; New England Biolabs) for 1 h at 30°C with occasional mixing.

    Techniques: Inhibition, Activation Assay, Mutagenesis, Binding Assay, De-Phosphorylation Assay

    Strategy used for analyzing transcription elongation complexes. (A) Structure of HIV-LTR template. DNA templates containing the lac operator (lacO) binding site for the lac repressor protein (LacR) and a terminator (τ) sequence were biotinylated and bound to streptavidin beads. (B) Elongation complexes were trapped by the lac repressor (LacR) after incubation of the immobilized templates with HeLa nuclear extract (NE) in the presence of nucleotide triphosphates and LacR and in the absence or presence of Tat. The CTD of the RNA polymerase was phosphorylated during the elongation reaction due to the activity of CDK7 and CDK9. (C) Elongation complexes arrested by LacR were treated with PP1 to remove phosphate groups from the CTD. (D) The phosphatase-treated complexes can resume transcription elongation after the addition of nucleotides and IPTG. During the chase reaction the CTD became phosphorylated by CDK9. The addition of DRB blocked the rephosphorylation of the CTD and induced pausing of the transcription complex at the terminator sequences.

    Journal: Molecular and Cellular Biology

    Article Title: Phosphorylation of the RNA Polymerase II Carboxyl-Terminal Domain by CDK9 Is Directly Responsible for Human Immunodeficiency Virus Type 1 Tat-Activated Transcriptional Elongation

    doi: 10.1128/MCB.22.13.4622-4637.2002

    Figure Lengend Snippet: Strategy used for analyzing transcription elongation complexes. (A) Structure of HIV-LTR template. DNA templates containing the lac operator (lacO) binding site for the lac repressor protein (LacR) and a terminator (τ) sequence were biotinylated and bound to streptavidin beads. (B) Elongation complexes were trapped by the lac repressor (LacR) after incubation of the immobilized templates with HeLa nuclear extract (NE) in the presence of nucleotide triphosphates and LacR and in the absence or presence of Tat. The CTD of the RNA polymerase was phosphorylated during the elongation reaction due to the activity of CDK7 and CDK9. (C) Elongation complexes arrested by LacR were treated with PP1 to remove phosphate groups from the CTD. (D) The phosphatase-treated complexes can resume transcription elongation after the addition of nucleotides and IPTG. During the chase reaction the CTD became phosphorylated by CDK9. The addition of DRB blocked the rephosphorylation of the CTD and induced pausing of the transcription complex at the terminator sequences.

    Article Snippet: Elongation complexes arrested by lac repressor were treated with 2.5 U of protein phosphatase 1 (PP1; New England Biolabs) for 1 h at 30°C with occasional mixing.

    Techniques: Binding Assay, Sequencing, Incubation, Activity Assay

    CDK9 phosphorylates Ser5 and Ser2 of the CTD in elongation complexes. (A) Transcription reactions. Preinitiation complexes (PIC) were assembled on immobilized wild-type template (WT) by using hexokinase/glucose-treated HeLa nuclear extract in the presence of 50 μM dATP and in the absence (−) or presence (+) of 20 ng of Tat. Transcription complexes paused at the uridine residue at position 14 were obtained after elongation of the preinitiation complexes in the absence of ATP. Standard transcription reactions were performed in parallel with templates carrying either the wild-type TAR element (WT) or a mutation in the Tat-binding site (mGC). Protein composition and phosphorylation of RNA Pol II CTD were analyzed from different transcription complexes by immunoblotting with the N-20, H5, and H14 antibodies directed against RNA Pol II (RNAP) and antibodies against CDK9, CycT1, CDK7, and CycH. (B) Rephosphorylation reactions. Transcription elongation complexes arrested by LacR were dephosphorylated by PP1, washed with EBCD buffer containing 0.1% Sarkosyl, and chased in the presence of 25 mM IPTG and all four nucleotide triphosphates. The proteins were detected by immunoblotting with the N-20, 8WG16, H5, and H14 antibodies directed against RNA Pol II and antibodies against CDK9 and CycT1.

    Journal: Molecular and Cellular Biology

    Article Title: Phosphorylation of the RNA Polymerase II Carboxyl-Terminal Domain by CDK9 Is Directly Responsible for Human Immunodeficiency Virus Type 1 Tat-Activated Transcriptional Elongation

    doi: 10.1128/MCB.22.13.4622-4637.2002

    Figure Lengend Snippet: CDK9 phosphorylates Ser5 and Ser2 of the CTD in elongation complexes. (A) Transcription reactions. Preinitiation complexes (PIC) were assembled on immobilized wild-type template (WT) by using hexokinase/glucose-treated HeLa nuclear extract in the presence of 50 μM dATP and in the absence (−) or presence (+) of 20 ng of Tat. Transcription complexes paused at the uridine residue at position 14 were obtained after elongation of the preinitiation complexes in the absence of ATP. Standard transcription reactions were performed in parallel with templates carrying either the wild-type TAR element (WT) or a mutation in the Tat-binding site (mGC). Protein composition and phosphorylation of RNA Pol II CTD were analyzed from different transcription complexes by immunoblotting with the N-20, H5, and H14 antibodies directed against RNA Pol II (RNAP) and antibodies against CDK9, CycT1, CDK7, and CycH. (B) Rephosphorylation reactions. Transcription elongation complexes arrested by LacR were dephosphorylated by PP1, washed with EBCD buffer containing 0.1% Sarkosyl, and chased in the presence of 25 mM IPTG and all four nucleotide triphosphates. The proteins were detected by immunoblotting with the N-20, 8WG16, H5, and H14 antibodies directed against RNA Pol II and antibodies against CDK9 and CycT1.

    Article Snippet: Elongation complexes arrested by lac repressor were treated with 2.5 U of protein phosphatase 1 (PP1; New England Biolabs) for 1 h at 30°C with occasional mixing.

    Techniques: Mutagenesis, Binding Assay

    Interactions of native FXN isoforms with native NFS1 and ISCU. Lymphoblastoid cell lysate was analyzed by Superdex 75 size exclusion chromatography. Fractions comprising the entire molecular mass fractionation range of the column were analyzed by Western blotting with ( A ) anti-NFS1 monoclonal antibody MyBioSource, ( B ) anti-FXN, or ( C ) polyclonal antibodies. The high and low molecular weight fractions were pooled ( HMW and LMW box , respectively), and immunoprecipitation was performed with PAC 2517 anti-FXN antibody immobilized on Protein A Magnetic beads, as described under “Experimental Procedures.” Aliquots of each pool (HMW or LMW) before immunoprecipitation ( Input , ∼5% of total volume), the flow-through fraction ( Not bound ; ∼5% of total volume), and the affinity-purified fraction ( Bound ; 100% of total volume) were analyzed by Western blotting ( WB ) with anti-NFS1 monoclonal antibody ( D ) or anti-FXN monoclonal antibody ( E ) or anti-Isu1 polyclonal antibody ( F ). FXN and ISCU were detected in individual HMW fractions ( B and C ) but not in the HMW fraction pool ( E and F , input and not Bound ) because ∼4 times less total protein was loaded on the gel in the latter analysis. For the control (MOCK), the Protein A Magnetic beads without antibody were incubated overnight with non-fractionated lymphoblastoid cell lysate, and Input , Not bound , and Bound proteins were analyzed as described above.

    Journal: The Journal of Biological Chemistry

    Article Title: Normal and Friedreich Ataxia Cells Express Different Isoforms of Frataxin with Complementary Roles in Iron-Sulfur Cluster Assembly *

    doi: 10.1074/jbc.M110.145144

    Figure Lengend Snippet: Interactions of native FXN isoforms with native NFS1 and ISCU. Lymphoblastoid cell lysate was analyzed by Superdex 75 size exclusion chromatography. Fractions comprising the entire molecular mass fractionation range of the column were analyzed by Western blotting with ( A ) anti-NFS1 monoclonal antibody MyBioSource, ( B ) anti-FXN, or ( C ) polyclonal antibodies. The high and low molecular weight fractions were pooled ( HMW and LMW box , respectively), and immunoprecipitation was performed with PAC 2517 anti-FXN antibody immobilized on Protein A Magnetic beads, as described under “Experimental Procedures.” Aliquots of each pool (HMW or LMW) before immunoprecipitation ( Input , ∼5% of total volume), the flow-through fraction ( Not bound ; ∼5% of total volume), and the affinity-purified fraction ( Bound ; 100% of total volume) were analyzed by Western blotting ( WB ) with anti-NFS1 monoclonal antibody ( D ) or anti-FXN monoclonal antibody ( E ) or anti-Isu1 polyclonal antibody ( F ). FXN and ISCU were detected in individual HMW fractions ( B and C ) but not in the HMW fraction pool ( E and F , input and not Bound ) because ∼4 times less total protein was loaded on the gel in the latter analysis. For the control (MOCK), the Protein A Magnetic beads without antibody were incubated overnight with non-fractionated lymphoblastoid cell lysate, and Input , Not bound , and Bound proteins were analyzed as described above.

    Article Snippet: Polyclonal antibodies were immobilized on Protein A magnetic beads (New England BioLabs) by cross-linking with dimethyl pimelidate dihydrochloride, per the manufacturer's protocol.

    Techniques: Size-exclusion Chromatography, Fractionation, Western Blot, Molecular Weight, Immunoprecipitation, Magnetic Beads, Flow Cytometry, Affinity Purification, Incubation