streptavidin agarose beads Search Results


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  • 94
    Thermo Fisher streptavidin sepharose 4b conjugate beads
    Streptavidin Sepharose 4b Conjugate Beads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin conjugated agarose beads
    Retrograde trafficking of β-DG from the PM to the ER. ( A ) ER was purified using density gradient techniques (OptiPrep) and then ER fractions were immunoblotted for the ER marker calnexin or β-DG on the same membrane. ( B ) Verification of the purity of ER fractions: Aliquots from each step of the ER purification were analyzed by Western blotting using primary antibodies against EEA1 (early endosomal marker), GAPDH (cytosolic marker) and Sp3 (nuclear marker). As a PM marker, ER was isolated from biotinylated cells at 4 °C, the lysates were pulldown using <t>streptavidin-agarose</t> beads and then blotted with HRP-streptavidin. NF: Nuclear fraction; NN: Non-nuclear fraction; CS: Cytosolic fraction; ER: Endoplasmic reticulum fraction. ( C ) Cells were subjected to cell surface biotinylation and subsequently to ER purification using the OptiPrep gradient. The ER fractions were combined and biotinylated proteins were precipitated using streptavidin-agarose beads and then analyzed by SDS-PAGE/Western blotting with antibodies against β-DG and calnexin.
    Streptavidin Conjugated Agarose Beads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 260 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cell Signaling Technology Inc streptavidin agarose
    Creld1 knockdown leads to reduced AChR expression at the plasma membrane in vitro . ( A–E ) Measurement of mouse CRELD1 and AChR α subunit protein levels. C2C12 cells expressing shRNA against Creld1 ( shCreld1 ) or scrambled ( shScramble ) sequences were differentiated for 5 days and then subjected to surface labeling with αBT-biotin for AChRα. N.T. = non transfected cells. <t>Streptavidin</t> precipitates (surface) and total lysates were separated by SDS/PAGE and probed for indicated proteins ( A and C ). CRELD1 protein levels were reduced by 75% in cells expressing shCreld1 as compared to shScramble ( B ). Quantitation of total AChRα levels ( D ) and of the surface to total AChRα ratio ( E ) in shScramble (100%) and shCreld1 cells from n = 5 independent experiments. Error bars, SEM; **p=0,0079, Mann-Whitney test. ( F–G ) Measurement of mouse Creld1 and AChRα subunit mRNA levels. C2C12 cells expressing shScramble or shCreld1 were differentiated for 5 days and then subjected to RNA extraction. Quantitative real-time PCR measurements of mRNA levels for Creld1 ( F ) and AChRα subunit ( G ). Creld1 mRNA is decreased in shCreld1 cells compared to shScramble cells, whereas AChRα subunit mRNA is not significantly decreased in shCreld-1 cells. Mean ± SEM is shown in six independent experiments. *p=0,0411; p=0,4740, ns (not significant), Mann–Whitney test.
    Streptavidin Agarose, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher streptavidin sepharose
    JMJD6 interacts with and hydroxylates histone H3 and H4 in vitro . A and B , in vitro pulldown assay. Biotin-labeled histone H3 1–21 peptides ( A ) or recombinant histone H4 ( B ) were incubated with or without GST-JMJD6, pulled down by <t>streptavidin-Sepharose,</t>
    Streptavidin Sepharose, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 764 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher affinity streptavidin agarose beads
    LANA and MCMs are part of the replication complex. (A) Schematic of two-step iPOND performed on KSHV-positive cells. Approximately 100 million KSHV-positive cells were labeled with 5-ethynyl-2′deoxyuridine (EdU) for 30 min, harvested, and washed with 1× PBS (1). Protein and DNA were cross-linked using 1% formaldehyde for 20 min and quenched using 125 mM glycine (2). The cells were permeabilized with 0.25% Triton X-100 in PBS for 30 min at room temperature (3), and nuclei were isolated following centrifugation (4). Click chemistry was performed on the nuclei to conjugate biotin-azide to EdU, and DMSO was used as a negative control (5). The chromatin was sheared using sonication to generate fragments of 100 to 300 bp (6). Protein-DNA complex bound to LANA was captured using monoclonal LANA antibody (7). Protein A/G beads were used to capture antigen-antibody complexes, which were eluted from the beads using peptide specific for LANA (8). <t>Streptavidin</t> beads were used to capture EdU-labeled (replicated) DNA-protein complexes (9). Proteins bound to streptavidin beads were eluted by boiling the beads at 95°C and detected using specific antibodies (10). (B) Immunoblot of LANA, MCM6, and PCNA obtained through two-step iPOND using respective antibodies from KSHV-positive BCBL-1 and BrK.219 cells. A total of 100 million cells were labeled with EdU and permeabilized, and a click reaction was performed using biotin-azide and DMSO. The cells were lysed and sonicated, and proteins were pulled down using LANA antibody. The proteins associated with DNA were pulled down using streptavidin beads and eluted by boiling in the loading buffer.
    Affinity Streptavidin Agarose Beads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Merck & Co streptavidin agarose beads
    LANA and MCMs are part of the replication complex. (A) Schematic of two-step iPOND performed on KSHV-positive cells. Approximately 100 million KSHV-positive cells were labeled with 5-ethynyl-2′deoxyuridine (EdU) for 30 min, harvested, and washed with 1× PBS (1). Protein and DNA were cross-linked using 1% formaldehyde for 20 min and quenched using 125 mM glycine (2). The cells were permeabilized with 0.25% Triton X-100 in PBS for 30 min at room temperature (3), and nuclei were isolated following centrifugation (4). Click chemistry was performed on the nuclei to conjugate biotin-azide to EdU, and DMSO was used as a negative control (5). The chromatin was sheared using sonication to generate fragments of 100 to 300 bp (6). Protein-DNA complex bound to LANA was captured using monoclonal LANA antibody (7). Protein A/G beads were used to capture antigen-antibody complexes, which were eluted from the beads using peptide specific for LANA (8). <t>Streptavidin</t> beads were used to capture EdU-labeled (replicated) DNA-protein complexes (9). Proteins bound to streptavidin beads were eluted by boiling the beads at 95°C and detected using specific antibodies (10). (B) Immunoblot of LANA, MCM6, and PCNA obtained through two-step iPOND using respective antibodies from KSHV-positive BCBL-1 and BrK.219 cells. A total of 100 million cells were labeled with EdU and permeabilized, and a click reaction was performed using biotin-azide and DMSO. The cells were lysed and sonicated, and proteins were pulled down using LANA antibody. The proteins associated with DNA were pulled down using streptavidin beads and eluted by boiling in the loading buffer.
    Streptavidin Agarose Beads, supplied by Merck & Co, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GE Healthcare streptavidin agarose beads
    Histone tail binding specificity of MBP-SpPHD and mutants thereof. The indicated recombinant MBP-fusion proteins were incubated with biotinylated synthetic histone tails, retrieved from the binding reaction using <t>streptavidin</t> beads, and bound proteins were resolved and visualized by SDS/PAGE and Western blotting with anti-MBP antibodies. One representative blot is shown for each recombinant protein tested. The MBP-tag by itself (panel e) served as a control. Lanes 1 contain 5% of the total binding reaction prior to the addition of streptavidin beads. The histone tails used are indicated above the lanes (3-6), and samples in lanes 2 (resin) did not contain any histone tails serving as negative controls.
    Streptavidin Agarose Beads, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 201 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Roche streptavidin agarose beads
    Interaction with SORLA controls trafficking and synaptic exposure of TrkB. ( A ) Colocalization of endogenous SORLA (red) and TrkB (green) in primary cortical neurons as shown by confocal immunofluorescence microscopy. Scale bar: 10 µm. ( B ) Co-immunoprecipitation of endogenous SORLA and TrkB from brain lysates is seen using anti-SORLA (IP anti-SORLA; lane 2) and anti-Trk antisera (IP anti-TrkB; lane 3). Panel Input (lane 1) indicates presence of endogenous SORLA and TrkB in brain lysate prior to co-immunoprecipitation. Lane 4 indicates lack co-immunoprecipitation in the absence of primary antibody (No IgG). ( C ) SH-SY5Y neuroblastoma cells stably overexpressing SORLA (SY5Y-S) or parental control cells (SY5Y) were transfected with expression constructs for TrkB. Subsequently, proteins at the cell surface were biotinylated and immunoprecipitated using <t>streptavidin</t> beads. Western blot analysis documents reduced levels of biotinylated TrkB at the cell surface in SY5Y-S compared to SY5Y cells (panel Surface). Panel Input represents levels of TrkB and SORLA in cell lysates prior to precipitation. Detection of tubulin (tub.), β-integrin (β -integ.), and PDGF-β receptor (PDGF-R) served as controls for loading and immunoprecipitation, respectively. ( D ) Densitometric quantification of replicate Western blots as exemplified in (C) (n = 6, Student’s t -test). ( E ) Subcellullar fractionations of wild type and SORLA-deficient mouse brain extracts were probed for the indicated proteins using Western blot analysis. Elevated levels of TrkB receptors (pan-Trk antibody) are seen in the synaptosomal plasma membrane (pm) fraction in SORLA-deficient (lanes 7 and 8) as compared to control brains (lanes 5 and 6). In contrast, levels of Trk receptors in the postsynaptic density (PSD) are reduced in receptor-deficient (lane 10) compared with wild type brains (lane 9). Levels of Trk in total brain lysates prior to subcellular fractionation are similar between genotypes (lanes 1–4). Detection of synaptophysin (Synapt), AMPA receptors, and PSD95 served as respective loading controls. ( F ) Densitometric quantification of replicate Western blots as exemplified in (E) (n = 4–6, Student’s t -test).
    Streptavidin Agarose Beads, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 79 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Solulink streptavidin agarose beads
    Cep170 is an FHDC1-interacting protein. BirA*-tagged FHDC1 was expressed in NIH 3T3 cells by transient transfection. Ciliogenesis was induced as before in low-serum media. BirA*-FHDC1 (red) was detected by virtue of the encoded myc-tag. (A) BirA*-FHDC1 is recruited to the elongated cilia and, following exogenous biotin treatment, induces biotinylation of cytoplasmic microtubules (green) and the cilia (acetylated tubulin, white). Specific punctate structures within the cytoplasmic microtubule network were also labeled (arrows). Biotinylated proteins were detected with <t>Alexa488-streptavidin.</t> (B) BirA*-FHDC1 (red) induces biotinylation of puncta (green) that colocalize with γ-tubulin puncta detected with anti–γ-tubulin (white). (C) BirA*-FHDC1 induces biotinylation of endogenous Cep170. As in B, BirA*-FHDC1 was expressed by transient transfection and its ability to induce biotinylation of the endogenous Cep170 was assessed by IB. myc-FHDC1 and FMNL2-BirA* were included as negative controls. Total biotinylated proteins were isolated from each sample using streptavidin–agarose beads. The eluted proteins were immunoblotted using the indicated antibodies. Endogenous Cep170 is only present in the pool of biotinylated proteins induced by BirA*-FHDC1 (arrow).
    Streptavidin Agarose Beads, supplied by Solulink, used in various techniques. Bioz Stars score: 91/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Upstate Biotechnology Inc streptavidin agarose beads
    Biochemical analysis of gp49 expression. Cell surface proteins were biotinylated and precipitated with <t>streptavidin-agarose,</t> separated by SDS-PAGE, blotted onto nitrocellulose, and probed with antiserum specific for gp49A or gp49B, as indicated. BMMC and NK cells from wild-type (+/+) and gp49B 0 (−/−) mice were analyzed. Included is a comparison of the level of gp49A expression in BMMC and NK cells from a C57BL/6 mouse.
    Streptavidin Agarose Beads, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 92/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Fisher Scientific streptavidin agarose beads
    Biochemical analysis of gp49 expression. Cell surface proteins were biotinylated and precipitated with <t>streptavidin-agarose,</t> separated by SDS-PAGE, blotted onto nitrocellulose, and probed with antiserum specific for gp49A or gp49B, as indicated. BMMC and NK cells from wild-type (+/+) and gp49B 0 (−/−) mice were analyzed. Included is a comparison of the level of gp49A expression in BMMC and NK cells from a C57BL/6 mouse.
    Streptavidin Agarose Beads, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 92/100, based on 58 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    EY Laboratories streptavidin agarose beads
    (A) Time course of gp15 mRNA expression in C. parvum -infected MDCK cell monolayers. Total RNA was isolated from infected MDCK cells at various times postinfection, DNase treated, and used as a template for RT-PCR (lanes 2 to 12) or control PCR (lanes 13 to 23), both primed with the gp15ATG and gp15STOP primers. The arrowhead indicates the position of the 1-kb gp15 amplicon. RNA template was isolated from infected MDCK cells at 0.5 h (lanes 3 and 14), 2 h (lanes 4 and 15), 4 h (lanes 5 and 16), 6 h (lanes 6 and 17), 9 h (lanes 7 and 18), 11 h (lanes 8 and 19), 24 h (lanes 9 and 20), and 48 h (lanes 10 and 21) postinfection; from uninfected MDCK cells (lanes 2 and 13); and directly from purified sporozoites (2 μg; lanes 11 and 22). Additional controls for the RT-PCR and PCRs lacked template nucleic acid (lanes 12 and 23 and 24, respectively). (B) Intracellular expression of gp15 protein occurs late in merogony. C. parvum ), or with anti-gp15 MAb 11A5 (F and H), CrA1 (J and L) or CrA2 (N and P) followed by biotinylated secondary antibodies and Cy3-conjugated <t>streptavidin.</t> Parasite nuclei in the same microscopic fields were stained with DAPI (lanes A, E, I, M, C, G, K, and O).
    Streptavidin Agarose Beads, supplied by EY Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim streptavidin agarose beads
    All of the Sm proteins associate in a specific and stable manner with a biotinylated Sm site oligonucleotide. (A) Reconstitution with TPs was carried out with biotinylated Sm site oligonucleotide (AAUUUUUGA [lane 2]) or, as negative controls, in the absence of biotinylated RNA (lane 3) or with biotinylated SmC3-C7 (AACCCCCGA) oligonucleotide (lane 4). Following <t>streptavidin-agarose</t> precipitation, samples were washed extensively with buffer containing 150 mM KCl. Bound proteins were analyzed by SDS-polyacrylamide gel electrophoresis and stained with Coomassie blue. (B) Reconstitution with TPs was performed in either the presence or absence of biotinylated Sm site oligonucleotide (b-Sm site), as indicated above each lane. The salt stability of the coprecipitation of U1-specific and Sm proteins was analyzed by extensive washing with buffers containing various concentrations of salt (0.15 to 2 M KCl, as indicated above the lanes). Following SDS-polyacrylamide gel electrophoresis, proteins were visualized by staining first with Coomassie blue and then with silver.
    Streptavidin Agarose Beads, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    BioVision streptavidin agarose beads
    All of the Sm proteins associate in a specific and stable manner with a biotinylated Sm site oligonucleotide. (A) Reconstitution with TPs was carried out with biotinylated Sm site oligonucleotide (AAUUUUUGA [lane 2]) or, as negative controls, in the absence of biotinylated RNA (lane 3) or with biotinylated SmC3-C7 (AACCCCCGA) oligonucleotide (lane 4). Following <t>streptavidin-agarose</t> precipitation, samples were washed extensively with buffer containing 150 mM KCl. Bound proteins were analyzed by SDS-polyacrylamide gel electrophoresis and stained with Coomassie blue. (B) Reconstitution with TPs was performed in either the presence or absence of biotinylated Sm site oligonucleotide (b-Sm site), as indicated above each lane. The salt stability of the coprecipitation of U1-specific and Sm proteins was analyzed by extensive washing with buffers containing various concentrations of salt (0.15 to 2 M KCl, as indicated above the lanes). Following SDS-polyacrylamide gel electrophoresis, proteins were visualized by staining first with Coomassie blue and then with silver.
    Streptavidin Agarose Beads, supplied by BioVision, used in various techniques. Bioz Stars score: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermal Scientific streptavidin agarose beads
    All of the Sm proteins associate in a specific and stable manner with a biotinylated Sm site oligonucleotide. (A) Reconstitution with TPs was carried out with biotinylated Sm site oligonucleotide (AAUUUUUGA [lane 2]) or, as negative controls, in the absence of biotinylated RNA (lane 3) or with biotinylated SmC3-C7 (AACCCCCGA) oligonucleotide (lane 4). Following <t>streptavidin-agarose</t> precipitation, samples were washed extensively with buffer containing 150 mM KCl. Bound proteins were analyzed by SDS-polyacrylamide gel electrophoresis and stained with Coomassie blue. (B) Reconstitution with TPs was performed in either the presence or absence of biotinylated Sm site oligonucleotide (b-Sm site), as indicated above each lane. The salt stability of the coprecipitation of U1-specific and Sm proteins was analyzed by extensive washing with buffers containing various concentrations of salt (0.15 to 2 M KCl, as indicated above the lanes). Following SDS-polyacrylamide gel electrophoresis, proteins were visualized by staining first with Coomassie blue and then with silver.
    Streptavidin Agarose Beads, supplied by Thermal Scientific, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    ProZyme streptavidin agarose beads
    Mutant p53 can bind to and transactivate the GRO1 promoter. A , schematic presentation of the GRO1 ( top ), p21 ( middle ), and GAPDH ( bottom ) promoters and the location of PCR primers used for ChIP assay. B , the binding of mutant p53 to the GRO1 promoter in SW480 cells was measured by ChIP. Mutant p53-DNA complexes were captured with anti-p53 antibody along with rabbit IgG as a control. The binding of mutant p53 protein to the p21 and GAPDH promoters was also measured as negative controls. C , pull-down assay of mutant p53 ( top ) and NF-κB (p65) ( bottom ) binding to the GRO1 promoter in vitro . The biotinated GRO1 promoter probe bound to <t>streptavidin-agarose</t> beads was used to precipitate p65 and mutant p53 from nuclear protein extracts from SW480 cells uninduced (-) or induced (+) to knock down mutant p53 for 3 days. The biotinated GAPDH promoter probe was used as a negative binding control. The nuclear proteins pulled down by biotinated promoter probes were analyzed by Western blot assay with antibodies against p53 and p65, respectively. D , schematic presentation of the luciferase reporter constructs under the control of the GRO1 ( top ) and p21 ( bottom ) promoters, respectively. E , the GRO1 promoter is responsive to mutant p53-R273H ( left ) but not wild-type p53 ( right ). The response of the p21 promoter to wild-type p53 was measured as a positive control. The luciferase activity for the promoterless pGL2 plasmid in the presence of wild-type and mutant p53 was measured as a negative control. Luciferase assay was performed as described under “Experimental Procedures.”
    Streptavidin Agarose Beads, supplied by ProZyme, used in various techniques. Bioz Stars score: 92/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Retrograde trafficking of β-DG from the PM to the ER. ( A ) ER was purified using density gradient techniques (OptiPrep) and then ER fractions were immunoblotted for the ER marker calnexin or β-DG on the same membrane. ( B ) Verification of the purity of ER fractions: Aliquots from each step of the ER purification were analyzed by Western blotting using primary antibodies against EEA1 (early endosomal marker), GAPDH (cytosolic marker) and Sp3 (nuclear marker). As a PM marker, ER was isolated from biotinylated cells at 4 °C, the lysates were pulldown using streptavidin-agarose beads and then blotted with HRP-streptavidin. NF: Nuclear fraction; NN: Non-nuclear fraction; CS: Cytosolic fraction; ER: Endoplasmic reticulum fraction. ( C ) Cells were subjected to cell surface biotinylation and subsequently to ER purification using the OptiPrep gradient. The ER fractions were combined and biotinylated proteins were precipitated using streptavidin-agarose beads and then analyzed by SDS-PAGE/Western blotting with antibodies against β-DG and calnexin.

    Journal: Scientific Reports

    Article Title: Retrograde trafficking of β-dystroglycan from the plasma membrane to the nucleus

    doi: 10.1038/s41598-017-09972-x

    Figure Lengend Snippet: Retrograde trafficking of β-DG from the PM to the ER. ( A ) ER was purified using density gradient techniques (OptiPrep) and then ER fractions were immunoblotted for the ER marker calnexin or β-DG on the same membrane. ( B ) Verification of the purity of ER fractions: Aliquots from each step of the ER purification were analyzed by Western blotting using primary antibodies against EEA1 (early endosomal marker), GAPDH (cytosolic marker) and Sp3 (nuclear marker). As a PM marker, ER was isolated from biotinylated cells at 4 °C, the lysates were pulldown using streptavidin-agarose beads and then blotted with HRP-streptavidin. NF: Nuclear fraction; NN: Non-nuclear fraction; CS: Cytosolic fraction; ER: Endoplasmic reticulum fraction. ( C ) Cells were subjected to cell surface biotinylation and subsequently to ER purification using the OptiPrep gradient. The ER fractions were combined and biotinylated proteins were precipitated using streptavidin-agarose beads and then analyzed by SDS-PAGE/Western blotting with antibodies against β-DG and calnexin.

    Article Snippet: To isolate biotinylated proteins, treated-cells were subjected to cell fractionation (see above) and the total, nuclear or cytoplasmic extracts (250 µg) were incubated overnight at 4 °C with 20 uL of Streptavidin-conjugated agarose beads (Thermo-Fisher Scientific, Rockford, IL).

    Techniques: Purification, Marker, Western Blot, Isolation, SDS Page

    Nuclear β-DG derives from the PM. ( A ) C2C12 cells were incubated with biotin for the indicated time intervals to label cell surface proteins (see Methods). Cells were then subjected to subcellular fractionation to isolate nuclear and non-nuclear fractions and biotinylated proteins were pulled-down using streptavidin-agarose beads and analyzed by SDS-PAGE/Western blotting using primary antibodies for non-phosphorylated-β-DG (upper panel) and phosphorylated β-DG (lower panel). Lower panel blot was reorganized so that the time points order matched the ones shown in upper panel, original blot is shown in Supp. Figure 1 . Input: immunoblotting analysis of cellular fractions prior to streptavidin-mediated pull-down. B: Bound/precipitated fraction. Membranes were stripped and reprobed for lamin A/C and GAPDH/calnexin as purity controls for nuclear and non-nuclear fractions respectively. ( B ) C2C12 cells cultured on glass coverslips were double-immunostained for total β-DG and the early endosomal marker EEA1. Nuclei were counterstained with DAPI prior to CLSM analysis. A typical single Z-section from three independent experiments is shown. Scale bar 20 µm.

    Journal: Scientific Reports

    Article Title: Retrograde trafficking of β-dystroglycan from the plasma membrane to the nucleus

    doi: 10.1038/s41598-017-09972-x

    Figure Lengend Snippet: Nuclear β-DG derives from the PM. ( A ) C2C12 cells were incubated with biotin for the indicated time intervals to label cell surface proteins (see Methods). Cells were then subjected to subcellular fractionation to isolate nuclear and non-nuclear fractions and biotinylated proteins were pulled-down using streptavidin-agarose beads and analyzed by SDS-PAGE/Western blotting using primary antibodies for non-phosphorylated-β-DG (upper panel) and phosphorylated β-DG (lower panel). Lower panel blot was reorganized so that the time points order matched the ones shown in upper panel, original blot is shown in Supp. Figure 1 . Input: immunoblotting analysis of cellular fractions prior to streptavidin-mediated pull-down. B: Bound/precipitated fraction. Membranes were stripped and reprobed for lamin A/C and GAPDH/calnexin as purity controls for nuclear and non-nuclear fractions respectively. ( B ) C2C12 cells cultured on glass coverslips were double-immunostained for total β-DG and the early endosomal marker EEA1. Nuclei were counterstained with DAPI prior to CLSM analysis. A typical single Z-section from three independent experiments is shown. Scale bar 20 µm.

    Article Snippet: To isolate biotinylated proteins, treated-cells were subjected to cell fractionation (see above) and the total, nuclear or cytoplasmic extracts (250 µg) were incubated overnight at 4 °C with 20 uL of Streptavidin-conjugated agarose beads (Thermo-Fisher Scientific, Rockford, IL).

    Techniques: Incubation, Fractionation, SDS Page, Western Blot, Cell Culture, Marker, Confocal Laser Scanning Microscopy

    Inhibition of dynamin-dependent endocytosis reduces nuclear localization of β-DG. ( A , left panel) Serum-starved C2C12 cells, seeded on coverslips, were treated with 40 µM dynasore (endocytosis inhibitor) or 0.05% DMSO (vehicle) for 30 min at 37 °C and then incubated for 5 min at 20 °C with Alexa594-transferrin (red). After 15 min at 37 °C, cells were fixed, stained with DAPI (nuclei) and transferrin uptake was monitored by CLSM analysis, with typical images shown (scale bar is 20 µm). ( A , right panel) Cells treated with dynasore or DMSO, as above, were immunolabeled for β-DG and counterstained with DAPI prior to be imaged by CLSM, with typical Z-sections shown (scale bar is 20 µm). (A, lower panel). Nuclear accumulation of β-DG (F n/c) was estimated as described in Methods. Data correspond to mean +/− SD from a series of three separate experiments (n = 30 cells). ( B ) Cytoplasmic and nuclear extracts obtained from dynasore- or DMSO-treated cells were analyzed by Western blotting using anti-β-DG antibodies. Stripped membranes were reprobed for lamin A/C and GAPDH as purity and loading controls for nuclear and cytoplasmic extracts respectively. The nuclear/cytoplasmic ratio (n/c) of β-DG was estimated by densitometry analysis. Results represent the mean +/− SD for 3 separate experiments, with significant differences denoted by p values (Student t-test). ( C ) DMSO- and dynasore-treated cells were subjected to biotinylation assays as in Fig. 2 . At 1 h post-biotinyation time, cells were fractionated into nuclear and non-nuclear fractions and pulled-down using streptavidin-agarose beads. Recovered and unbound proteins were analyzed by SDS-PAGE/Western blotting using primary antibodies for total β-DG. Membranes were stripped and reprobed for lamin A/C and calnexin; markers for nuclear and non-nuclear fractions respectively. Input: immunoblotting analysis of cellular fractions prior to pull-down. B, bound/precipitated fraction. Un, unbound fraction.

    Journal: Scientific Reports

    Article Title: Retrograde trafficking of β-dystroglycan from the plasma membrane to the nucleus

    doi: 10.1038/s41598-017-09972-x

    Figure Lengend Snippet: Inhibition of dynamin-dependent endocytosis reduces nuclear localization of β-DG. ( A , left panel) Serum-starved C2C12 cells, seeded on coverslips, were treated with 40 µM dynasore (endocytosis inhibitor) or 0.05% DMSO (vehicle) for 30 min at 37 °C and then incubated for 5 min at 20 °C with Alexa594-transferrin (red). After 15 min at 37 °C, cells were fixed, stained with DAPI (nuclei) and transferrin uptake was monitored by CLSM analysis, with typical images shown (scale bar is 20 µm). ( A , right panel) Cells treated with dynasore or DMSO, as above, were immunolabeled for β-DG and counterstained with DAPI prior to be imaged by CLSM, with typical Z-sections shown (scale bar is 20 µm). (A, lower panel). Nuclear accumulation of β-DG (F n/c) was estimated as described in Methods. Data correspond to mean +/− SD from a series of three separate experiments (n = 30 cells). ( B ) Cytoplasmic and nuclear extracts obtained from dynasore- or DMSO-treated cells were analyzed by Western blotting using anti-β-DG antibodies. Stripped membranes were reprobed for lamin A/C and GAPDH as purity and loading controls for nuclear and cytoplasmic extracts respectively. The nuclear/cytoplasmic ratio (n/c) of β-DG was estimated by densitometry analysis. Results represent the mean +/− SD for 3 separate experiments, with significant differences denoted by p values (Student t-test). ( C ) DMSO- and dynasore-treated cells were subjected to biotinylation assays as in Fig. 2 . At 1 h post-biotinyation time, cells were fractionated into nuclear and non-nuclear fractions and pulled-down using streptavidin-agarose beads. Recovered and unbound proteins were analyzed by SDS-PAGE/Western blotting using primary antibodies for total β-DG. Membranes were stripped and reprobed for lamin A/C and calnexin; markers for nuclear and non-nuclear fractions respectively. Input: immunoblotting analysis of cellular fractions prior to pull-down. B, bound/precipitated fraction. Un, unbound fraction.

    Article Snippet: To isolate biotinylated proteins, treated-cells were subjected to cell fractionation (see above) and the total, nuclear or cytoplasmic extracts (250 µg) were incubated overnight at 4 °C with 20 uL of Streptavidin-conjugated agarose beads (Thermo-Fisher Scientific, Rockford, IL).

    Techniques: Inhibition, Incubation, Staining, Confocal Laser Scanning Microscopy, Immunolabeling, Western Blot, SDS Page

    Characterization of the endogenously expressed Bmpr2 mutant product in pulmonary endothelial cells from Bmpr2 ΔEx2/+ mice. Studies were performed using conditionally immortalized PECs (ciPECs) isolated from wild type control and Bmpr2 ΔEx2/+ mice and replicated at least three times. A, Western blot using the Clone 18 anti-BMPR2 antibody in wild type (WT) and Bmpr2 ΔEx2/+ (ΔEx2/+) ciPEC lysates. Both cell lines expressed a 150 kDa wild type Bmpr2 product (WT). Bmpr2 ΔEx2/+ ciPECs also expressed a 130 kDa product (ΔEx2). B, Western blot using ASQ anti-BMPR2 antibody. Wild type control and Bmpr2 ΔEx2/+ ciPECs expressed 150 kDa WT Bmpr2, but the 130 kDa product was not detected. C, Cell surface expression of Bmpr2 in ciPECs. ciPECs were labeled with membrane impermeable biotin and cell surface expression of Bmpr2 detected in streptavidin pull-down of cell lysates. Anti-BMPR2 Clone 18 antibody detected the 150 kDa wild type Bmpr2 in control and Bmpr2 ΔEx2/+ ciPECs after streptavidin pull-down but not the 130kDa Bmpr2ΔEx2 mutant product. Lower panel, Western blot for Bmpr2 in the supernatant remaining after depletion of cell surface proteins by streptavidin pull-down. The ratios of 150 kDa WT Bmpr2 and the 130 kDa Bmpr2ΔEx2 mutant band intensities in Bmpr2 ΔEx2/+ ciPECs supernatants after depletion of cell surface proteins are indicated below the lower panel.

    Journal: PLoS ONE

    Article Title: Abnormal Trafficking of Endogenously Expressed BMPR2 Mutant Allelic Products in Patients with Heritable Pulmonary Arterial Hypertension

    doi: 10.1371/journal.pone.0080319

    Figure Lengend Snippet: Characterization of the endogenously expressed Bmpr2 mutant product in pulmonary endothelial cells from Bmpr2 ΔEx2/+ mice. Studies were performed using conditionally immortalized PECs (ciPECs) isolated from wild type control and Bmpr2 ΔEx2/+ mice and replicated at least three times. A, Western blot using the Clone 18 anti-BMPR2 antibody in wild type (WT) and Bmpr2 ΔEx2/+ (ΔEx2/+) ciPEC lysates. Both cell lines expressed a 150 kDa wild type Bmpr2 product (WT). Bmpr2 ΔEx2/+ ciPECs also expressed a 130 kDa product (ΔEx2). B, Western blot using ASQ anti-BMPR2 antibody. Wild type control and Bmpr2 ΔEx2/+ ciPECs expressed 150 kDa WT Bmpr2, but the 130 kDa product was not detected. C, Cell surface expression of Bmpr2 in ciPECs. ciPECs were labeled with membrane impermeable biotin and cell surface expression of Bmpr2 detected in streptavidin pull-down of cell lysates. Anti-BMPR2 Clone 18 antibody detected the 150 kDa wild type Bmpr2 in control and Bmpr2 ΔEx2/+ ciPECs after streptavidin pull-down but not the 130kDa Bmpr2ΔEx2 mutant product. Lower panel, Western blot for Bmpr2 in the supernatant remaining after depletion of cell surface proteins by streptavidin pull-down. The ratios of 150 kDa WT Bmpr2 and the 130 kDa Bmpr2ΔEx2 mutant band intensities in Bmpr2 ΔEx2/+ ciPECs supernatants after depletion of cell surface proteins are indicated below the lower panel.

    Article Snippet: Chemicals and Reagents Sulfo-NHS-LC-Biotin and Streptavidin agarose conjugated beads (Pierce/Thermo Scientific); Dio-Ac-LDL (Biomedical Technologies Inc.); Endo-H and PNGase-F glycosidases (New England Biolabs); Human recombinant BMP-2 (R & D Systems); chemical chaperones sodium phenylbutyrate (4-PBA) and Sodium taurourdeoxycholic acid (TUDCA) (Sigma-Aldrich).

    Techniques: Mutagenesis, Mouse Assay, Isolation, Western Blot, Expressing, Labeling

    Chemical chaperones partially restore Bmpr2ΔEx2 mutant product expression at the cell surface. A, Cell surface expression of Bmpr2ΔEx2 in Bmpr2 ΔEx2/+ ciPECs treated with 4-PBA. Bmpr2 ΔEx2/+ ciPECs were treated for 48 hours with 100µM, 250µM, 500µM or 1mM of 4-PBA. Monolayers were then labeled with membrane impermeable biotin and biotinylated cell surface proteins pulled-down with streptavidin agarose beads. Western Blot was performed with Clone 18 anti-BMPR2 antibody. The 150 kDa wild type Bmpr2 product was detected in the streptavidin pull-down in control and Bmpr2 ΔEx2/+ ciPECs, but the 130 kDa Bmpr2ΔEx2 mutant product was not detected. After treating with the chemical chaperone 4-PBA, the 130kDa Bmpr2ΔEx2 mutant product was detected and there was increased expression of the wild type Bmpr2 product in the streptavidin pull-down. Numbers shown below the upper panel indicate the ratio of the 130 kDa Bmpr2ΔEx2 band before and after treatment with 4-PBA. Lower panel, expression of wild type Bmpr2 and Bmpr2ΔEx2 in ciPEC cell lysates with 4-PBA treatment. B, Quantification of wild type Bmpr2 and Bmpr2ΔEx2 band densities after 4-PBA treatment relative to untreated controls from three independent experiments, standard error is indicated. C, Cell surface expression of Bmpr2ΔEx2 in Bmpr2 ΔEx2/+ ciPECs treated with TUDCA. Wild type and Bmpr2 ΔEx2/+ ciPECs were treated for 5 hours with 50µM 100µM, 250µM or 500µM of TUDCA. Streptavidin pull-down shows that the 130kDa Bmpr2ΔEx2 mutant product was partially restored at the cell surface and there was a slight increase in wild type Bmpr2 with TUDCA treatment (1.4 fold increase with 500µM TUDCA versus untreated cells). Numbers shown below the upper panel indicate the ratio of the 130 kDa Bmpr2ΔEx2 band before and after treatment with TUDCA Lower panel, expression of wild type Bmpr2 and Bmpr2ΔEx2 in ciPEC cell lysates with TUDCA treatment. D, Quantification of wild type Bmpr2 and Bmpr2ΔEx2 band densities after TUDCA treatment relative to untreated controls from three independent experiments.

    Journal: PLoS ONE

    Article Title: Abnormal Trafficking of Endogenously Expressed BMPR2 Mutant Allelic Products in Patients with Heritable Pulmonary Arterial Hypertension

    doi: 10.1371/journal.pone.0080319

    Figure Lengend Snippet: Chemical chaperones partially restore Bmpr2ΔEx2 mutant product expression at the cell surface. A, Cell surface expression of Bmpr2ΔEx2 in Bmpr2 ΔEx2/+ ciPECs treated with 4-PBA. Bmpr2 ΔEx2/+ ciPECs were treated for 48 hours with 100µM, 250µM, 500µM or 1mM of 4-PBA. Monolayers were then labeled with membrane impermeable biotin and biotinylated cell surface proteins pulled-down with streptavidin agarose beads. Western Blot was performed with Clone 18 anti-BMPR2 antibody. The 150 kDa wild type Bmpr2 product was detected in the streptavidin pull-down in control and Bmpr2 ΔEx2/+ ciPECs, but the 130 kDa Bmpr2ΔEx2 mutant product was not detected. After treating with the chemical chaperone 4-PBA, the 130kDa Bmpr2ΔEx2 mutant product was detected and there was increased expression of the wild type Bmpr2 product in the streptavidin pull-down. Numbers shown below the upper panel indicate the ratio of the 130 kDa Bmpr2ΔEx2 band before and after treatment with 4-PBA. Lower panel, expression of wild type Bmpr2 and Bmpr2ΔEx2 in ciPEC cell lysates with 4-PBA treatment. B, Quantification of wild type Bmpr2 and Bmpr2ΔEx2 band densities after 4-PBA treatment relative to untreated controls from three independent experiments, standard error is indicated. C, Cell surface expression of Bmpr2ΔEx2 in Bmpr2 ΔEx2/+ ciPECs treated with TUDCA. Wild type and Bmpr2 ΔEx2/+ ciPECs were treated for 5 hours with 50µM 100µM, 250µM or 500µM of TUDCA. Streptavidin pull-down shows that the 130kDa Bmpr2ΔEx2 mutant product was partially restored at the cell surface and there was a slight increase in wild type Bmpr2 with TUDCA treatment (1.4 fold increase with 500µM TUDCA versus untreated cells). Numbers shown below the upper panel indicate the ratio of the 130 kDa Bmpr2ΔEx2 band before and after treatment with TUDCA Lower panel, expression of wild type Bmpr2 and Bmpr2ΔEx2 in ciPEC cell lysates with TUDCA treatment. D, Quantification of wild type Bmpr2 and Bmpr2ΔEx2 band densities after TUDCA treatment relative to untreated controls from three independent experiments.

    Article Snippet: Chemicals and Reagents Sulfo-NHS-LC-Biotin and Streptavidin agarose conjugated beads (Pierce/Thermo Scientific); Dio-Ac-LDL (Biomedical Technologies Inc.); Endo-H and PNGase-F glycosidases (New England Biolabs); Human recombinant BMP-2 (R & D Systems); chemical chaperones sodium phenylbutyrate (4-PBA) and Sodium taurourdeoxycholic acid (TUDCA) (Sigma-Aldrich).

    Techniques: Mutagenesis, Expressing, Labeling, Western Blot

    HPAH patient-derived lymphocytes express mutant BMPR2 products. A, Schematic of the BMPR2 protein. Areas recognized by the anti-BMPR2 antibodies clone 18 and ASQ are indicated by black arrows. Exons 1-13 are represented by alternating gray and white boxes, and numbers indicate corresponding amino acids. LBD, represents the ligand binding domain, TM the transmembrane domain, KD the kinase domain and CT the cytoplasmic tail. B, Detection of BMPR2 products in HPAH patient-derived lymphocytes, image representative of three experiments. Western blot using anti-BMPR2 antibody, Clone 18. A 130-145 kDa wild type BMPR2 product (WT) was detected in normal control and Family 108 (F108) HPAH patient-derived lymphoblasts. F108 cells expressed an additional 120 kDa BMPR2 mutant product (ΔEx2). C, Representative western blot from three experiments using the ASQ anti-BMPR2 antibody. The 130-145 kDa product was detected in both control and F108 lymphocytes, but the 120 kDa band was not detected. D, Cell surface expression of BMPR2 in HPAH patient-derived lymphocytes labeled with a membrane impermeable biotin. Experiment replicated three times. Left panel, input cell lysates before the streptavidin pull-down. Right panel, cell surface proteins detected in streptavidin pull-down by Western blot using Clone 18 anti-BMPR2 antibody. A 130-145 kDa wild type BMPR2 product was detected in control and F108 cultured lymphocytes in streptavidin pull-down but not 120kDa BMPR2ΔEx2 mutant product.

    Journal: PLoS ONE

    Article Title: Abnormal Trafficking of Endogenously Expressed BMPR2 Mutant Allelic Products in Patients with Heritable Pulmonary Arterial Hypertension

    doi: 10.1371/journal.pone.0080319

    Figure Lengend Snippet: HPAH patient-derived lymphocytes express mutant BMPR2 products. A, Schematic of the BMPR2 protein. Areas recognized by the anti-BMPR2 antibodies clone 18 and ASQ are indicated by black arrows. Exons 1-13 are represented by alternating gray and white boxes, and numbers indicate corresponding amino acids. LBD, represents the ligand binding domain, TM the transmembrane domain, KD the kinase domain and CT the cytoplasmic tail. B, Detection of BMPR2 products in HPAH patient-derived lymphocytes, image representative of three experiments. Western blot using anti-BMPR2 antibody, Clone 18. A 130-145 kDa wild type BMPR2 product (WT) was detected in normal control and Family 108 (F108) HPAH patient-derived lymphoblasts. F108 cells expressed an additional 120 kDa BMPR2 mutant product (ΔEx2). C, Representative western blot from three experiments using the ASQ anti-BMPR2 antibody. The 130-145 kDa product was detected in both control and F108 lymphocytes, but the 120 kDa band was not detected. D, Cell surface expression of BMPR2 in HPAH patient-derived lymphocytes labeled with a membrane impermeable biotin. Experiment replicated three times. Left panel, input cell lysates before the streptavidin pull-down. Right panel, cell surface proteins detected in streptavidin pull-down by Western blot using Clone 18 anti-BMPR2 antibody. A 130-145 kDa wild type BMPR2 product was detected in control and F108 cultured lymphocytes in streptavidin pull-down but not 120kDa BMPR2ΔEx2 mutant product.

    Article Snippet: Chemicals and Reagents Sulfo-NHS-LC-Biotin and Streptavidin agarose conjugated beads (Pierce/Thermo Scientific); Dio-Ac-LDL (Biomedical Technologies Inc.); Endo-H and PNGase-F glycosidases (New England Biolabs); Human recombinant BMP-2 (R & D Systems); chemical chaperones sodium phenylbutyrate (4-PBA) and Sodium taurourdeoxycholic acid (TUDCA) (Sigma-Aldrich).

    Techniques: Derivative Assay, Mutagenesis, Ligand Binding Assay, Western Blot, Expressing, Labeling, Cell Culture

    Creld1 knockdown leads to reduced AChR expression at the plasma membrane in vitro . ( A–E ) Measurement of mouse CRELD1 and AChR α subunit protein levels. C2C12 cells expressing shRNA against Creld1 ( shCreld1 ) or scrambled ( shScramble ) sequences were differentiated for 5 days and then subjected to surface labeling with αBT-biotin for AChRα. N.T. = non transfected cells. Streptavidin precipitates (surface) and total lysates were separated by SDS/PAGE and probed for indicated proteins ( A and C ). CRELD1 protein levels were reduced by 75% in cells expressing shCreld1 as compared to shScramble ( B ). Quantitation of total AChRα levels ( D ) and of the surface to total AChRα ratio ( E ) in shScramble (100%) and shCreld1 cells from n = 5 independent experiments. Error bars, SEM; **p=0,0079, Mann-Whitney test. ( F–G ) Measurement of mouse Creld1 and AChRα subunit mRNA levels. C2C12 cells expressing shScramble or shCreld1 were differentiated for 5 days and then subjected to RNA extraction. Quantitative real-time PCR measurements of mRNA levels for Creld1 ( F ) and AChRα subunit ( G ). Creld1 mRNA is decreased in shCreld1 cells compared to shScramble cells, whereas AChRα subunit mRNA is not significantly decreased in shCreld-1 cells. Mean ± SEM is shown in six independent experiments. *p=0,0411; p=0,4740, ns (not significant), Mann–Whitney test.

    Journal: eLife

    Article Title: CRELD1 is an evolutionarily-conserved maturational enhancer of ionotropic acetylcholine receptors

    doi: 10.7554/eLife.39649

    Figure Lengend Snippet: Creld1 knockdown leads to reduced AChR expression at the plasma membrane in vitro . ( A–E ) Measurement of mouse CRELD1 and AChR α subunit protein levels. C2C12 cells expressing shRNA against Creld1 ( shCreld1 ) or scrambled ( shScramble ) sequences were differentiated for 5 days and then subjected to surface labeling with αBT-biotin for AChRα. N.T. = non transfected cells. Streptavidin precipitates (surface) and total lysates were separated by SDS/PAGE and probed for indicated proteins ( A and C ). CRELD1 protein levels were reduced by 75% in cells expressing shCreld1 as compared to shScramble ( B ). Quantitation of total AChRα levels ( D ) and of the surface to total AChRα ratio ( E ) in shScramble (100%) and shCreld1 cells from n = 5 independent experiments. Error bars, SEM; **p=0,0079, Mann-Whitney test. ( F–G ) Measurement of mouse Creld1 and AChRα subunit mRNA levels. C2C12 cells expressing shScramble or shCreld1 were differentiated for 5 days and then subjected to RNA extraction. Quantitative real-time PCR measurements of mRNA levels for Creld1 ( F ) and AChRα subunit ( G ). Creld1 mRNA is decreased in shCreld1 cells compared to shScramble cells, whereas AChRα subunit mRNA is not significantly decreased in shCreld-1 cells. Mean ± SEM is shown in six independent experiments. *p=0,0411; p=0,4740, ns (not significant), Mann–Whitney test.

    Article Snippet: Streptavidin-agarose (Cell Signaling Technology) was used to recover biotinylated proteins.

    Techniques: Expressing, In Vitro, shRNA, Labeling, Transfection, SDS Page, Quantitation Assay, MANN-WHITNEY, RNA Extraction, Real-time Polymerase Chain Reaction

    JMJD6 interacts with and hydroxylates histone H3 and H4 in vitro . A and B , in vitro pulldown assay. Biotin-labeled histone H3 1–21 peptides ( A ) or recombinant histone H4 ( B ) were incubated with or without GST-JMJD6, pulled down by streptavidin-Sepharose,

    Journal: The Journal of Biological Chemistry

    Article Title: Lysyl 5-Hydroxylation, a Novel Histone Modification, by Jumonji Domain Containing 6 (JMJD6) *

    doi: 10.1074/jbc.M112.433284

    Figure Lengend Snippet: JMJD6 interacts with and hydroxylates histone H3 and H4 in vitro . A and B , in vitro pulldown assay. Biotin-labeled histone H3 1–21 peptides ( A ) or recombinant histone H4 ( B ) were incubated with or without GST-JMJD6, pulled down by streptavidin-Sepharose,

    Article Snippet: The biotin-labeled histone H31–21 peptides were pulled down with interacting proteins by streptavidin Sepharose (S951, Invitrogen).

    Techniques: In Vitro, Labeling, Recombinant, Incubation

    Comparison of BSA- 7b and BSA- 7c linker cleavage (A). Lane 1, BSA- 7b (20 µg) captured on streptavidin-agarose beads ; lane 2, BSA- 7c (20 µg) captured on streptavidin-agarose beads; lane 3, supernatant from BSA- 7b bead sample in lane 1 incubated in 1% TFA at 37 °C for 1 hour; lane 4, supernatant from BSA- 7c bead sample in lane 2 incubated in 1% TFA at 37 °C for 1 hour; lane 5, streptavidin-agarose beads with captured BSA- 7b from lane 1 after TFA incubation; lane 6, streptavidin-agarose beads with captured BSA- 7c from lane 2 after TFA incubation. (B) Capture and cleavage of BSA- 7c in the presence of bacterial whole cell lysates; lane 1, cell lysate + BSA- 7c (20 µg); lane 2, BSA- 7c captured on streptavidin-agarose beads from cell lysate sample in lane 1; lane 3, supernatant from BSA- 7c bead sample in lane 2 incubated in 1% TFA at 37 °C for 1 hour; lane 4, streptavidin-agarose beads with captured BSA- 7c from lane 2 after TFA incubation.

    Journal: Organic & biomolecular chemistry

    Article Title: Cyclic acetals as cleavable linkers for affinity capture

    doi: 10.1039/c5ob01056j

    Figure Lengend Snippet: Comparison of BSA- 7b and BSA- 7c linker cleavage (A). Lane 1, BSA- 7b (20 µg) captured on streptavidin-agarose beads ; lane 2, BSA- 7c (20 µg) captured on streptavidin-agarose beads; lane 3, supernatant from BSA- 7b bead sample in lane 1 incubated in 1% TFA at 37 °C for 1 hour; lane 4, supernatant from BSA- 7c bead sample in lane 2 incubated in 1% TFA at 37 °C for 1 hour; lane 5, streptavidin-agarose beads with captured BSA- 7b from lane 1 after TFA incubation; lane 6, streptavidin-agarose beads with captured BSA- 7c from lane 2 after TFA incubation. (B) Capture and cleavage of BSA- 7c in the presence of bacterial whole cell lysates; lane 1, cell lysate + BSA- 7c (20 µg); lane 2, BSA- 7c captured on streptavidin-agarose beads from cell lysate sample in lane 1; lane 3, supernatant from BSA- 7c bead sample in lane 2 incubated in 1% TFA at 37 °C for 1 hour; lane 4, streptavidin-agarose beads with captured BSA- 7c from lane 2 after TFA incubation.

    Article Snippet: NHS-LC-biotin and streptavidin agarose beads were purchased from Thermo Scientific.

    Techniques: Incubation

    Evaluation of biotin probes 7a and 7b cleavage and comparison to non-cleavable biotin probe 8 . (A) BSA (20 µg) conjugated to each probe, 7a , 7b , or 8 , was incubated in 1% TFA at 37 °C in the absence of streptavidin resin for the indicated time. (B) Test of BSA- 7b linker cleavage. Lane 1, BSA- 7b (20 µg) captured on streptavidin-ultralink beads; Lane 2, BSA- 8 (20 µg) captured on streptavidin-ultralink beads; lane 3, supernatant from BSA- 7b (20 µg) bead sample in lane 1 incubated in 1% TFA at 37 °C for 1 hour; lane 4, supernatant from BSA- 8 (20 µg) bead sample in lane 2 incubated in 1% TFA at 37 °C for 1 hour; lane 5, streptavidin-ultralink beads with captured BSA- 7b from lane 3 after TFA incubation; lane 6, streptavidin-ultralink beads with captured BSA- 8 from lane 4 after TFA incubation.

    Journal: Organic & biomolecular chemistry

    Article Title: Cyclic acetals as cleavable linkers for affinity capture

    doi: 10.1039/c5ob01056j

    Figure Lengend Snippet: Evaluation of biotin probes 7a and 7b cleavage and comparison to non-cleavable biotin probe 8 . (A) BSA (20 µg) conjugated to each probe, 7a , 7b , or 8 , was incubated in 1% TFA at 37 °C in the absence of streptavidin resin for the indicated time. (B) Test of BSA- 7b linker cleavage. Lane 1, BSA- 7b (20 µg) captured on streptavidin-ultralink beads; Lane 2, BSA- 8 (20 µg) captured on streptavidin-ultralink beads; lane 3, supernatant from BSA- 7b (20 µg) bead sample in lane 1 incubated in 1% TFA at 37 °C for 1 hour; lane 4, supernatant from BSA- 8 (20 µg) bead sample in lane 2 incubated in 1% TFA at 37 °C for 1 hour; lane 5, streptavidin-ultralink beads with captured BSA- 7b from lane 3 after TFA incubation; lane 6, streptavidin-ultralink beads with captured BSA- 8 from lane 4 after TFA incubation.

    Article Snippet: NHS-LC-biotin and streptavidin agarose beads were purchased from Thermo Scientific.

    Techniques: Incubation

    Further modification of the BSA aldehyde tag. Lane 1, BSA- 7c (20 µg); lane 2, BSA- 7c sample from lane 1 captured on streptavidin-agarose beads; lane 3, supernatant from BSA- 7c bead sample in lane 2 incubated in 1% TFA at 37 °C for 1 hour; lane 4, BSA-aldehyde from sample in lane 3 after reaction with alkoxyamine-PEG-biotin at pH 5, 37 °C for 4 hours. Exact amounts used in each step are given in the Materials and Methods.

    Journal: Organic & biomolecular chemistry

    Article Title: Cyclic acetals as cleavable linkers for affinity capture

    doi: 10.1039/c5ob01056j

    Figure Lengend Snippet: Further modification of the BSA aldehyde tag. Lane 1, BSA- 7c (20 µg); lane 2, BSA- 7c sample from lane 1 captured on streptavidin-agarose beads; lane 3, supernatant from BSA- 7c bead sample in lane 2 incubated in 1% TFA at 37 °C for 1 hour; lane 4, BSA-aldehyde from sample in lane 3 after reaction with alkoxyamine-PEG-biotin at pH 5, 37 °C for 4 hours. Exact amounts used in each step are given in the Materials and Methods.

    Article Snippet: NHS-LC-biotin and streptavidin agarose beads were purchased from Thermo Scientific.

    Techniques: Modification, Incubation

    RNase A- 7c capture and elution. (A) Capture of RNase A- 7c in the presence of bacterial whole cell lysate and release; lane 1, RNase A-maleimide alkyne; lane 2, RNase A- 7c ; lane 3, cell lysate + RNase A- 7c (20 µg); lane 4, supernatant after RNase A- 7c streptavidin capture from sample in lane 3; lane 5, RNase A- 7c captured on streptavidin-agarose beads from cell lysate sample in lane 3; lane 6, supernatant from RNase A- 7c bead sample in lane 5 incubated in 1% TFA at 37 °C for 1 hour; lane 7, streptavidin-agarose beads with captured RNase A- 7c from lane 5 after TFA incubation. (B) Test of streptavidin monomer release under cleavage conditions used for other cleavable linkers. Supernatant after treating streptavidin beads: lane 1, Na 2 S 2 O 4 for 1 hour at 25 °C; lane 2, 2% 2-mercaptoethanol for 1 hour at 25 °C; lane 3, 5% formic acid for 2 hours at 25 °C; lane 4, 1M guanidine hydrochloride in 1% TFA at 37°C for 1 hour. (C) Effect of guanidine concentration on streptavidin monomer release from the beads. Lane 1, cell lysate + RNase A- 7c ; lane 2, supernatant after RNase A- 7c streptavidin capture. Note: the remaining RNase A is from incomplete reaction with maleimide alkyne; lane 3, RNase A- 7c captured on streptavidin-agarose beads from cell lysate; lane 4, supernatant from RNase A- 7c incubated in 1M guanidine/1% TFA at 37 °C for 1 hour; lane 5, supernatant from RNase A- 7c incubated in 3 M guanidine/1% TFA at 37 °C for 1 hour; lane 6, streptavidin-agarose beads with captured RNase A- 7c after 1M guanidine/TFA incubation; lane 7, streptavidin-agarose beads with captured RNase A- 7c after 3M guanidine/TFA incubation. Exact amounts used in each step are given in the Materials and Methods.

    Journal: Organic & biomolecular chemistry

    Article Title: Cyclic acetals as cleavable linkers for affinity capture

    doi: 10.1039/c5ob01056j

    Figure Lengend Snippet: RNase A- 7c capture and elution. (A) Capture of RNase A- 7c in the presence of bacterial whole cell lysate and release; lane 1, RNase A-maleimide alkyne; lane 2, RNase A- 7c ; lane 3, cell lysate + RNase A- 7c (20 µg); lane 4, supernatant after RNase A- 7c streptavidin capture from sample in lane 3; lane 5, RNase A- 7c captured on streptavidin-agarose beads from cell lysate sample in lane 3; lane 6, supernatant from RNase A- 7c bead sample in lane 5 incubated in 1% TFA at 37 °C for 1 hour; lane 7, streptavidin-agarose beads with captured RNase A- 7c from lane 5 after TFA incubation. (B) Test of streptavidin monomer release under cleavage conditions used for other cleavable linkers. Supernatant after treating streptavidin beads: lane 1, Na 2 S 2 O 4 for 1 hour at 25 °C; lane 2, 2% 2-mercaptoethanol for 1 hour at 25 °C; lane 3, 5% formic acid for 2 hours at 25 °C; lane 4, 1M guanidine hydrochloride in 1% TFA at 37°C for 1 hour. (C) Effect of guanidine concentration on streptavidin monomer release from the beads. Lane 1, cell lysate + RNase A- 7c ; lane 2, supernatant after RNase A- 7c streptavidin capture. Note: the remaining RNase A is from incomplete reaction with maleimide alkyne; lane 3, RNase A- 7c captured on streptavidin-agarose beads from cell lysate; lane 4, supernatant from RNase A- 7c incubated in 1M guanidine/1% TFA at 37 °C for 1 hour; lane 5, supernatant from RNase A- 7c incubated in 3 M guanidine/1% TFA at 37 °C for 1 hour; lane 6, streptavidin-agarose beads with captured RNase A- 7c after 1M guanidine/TFA incubation; lane 7, streptavidin-agarose beads with captured RNase A- 7c after 3M guanidine/TFA incubation. Exact amounts used in each step are given in the Materials and Methods.

    Article Snippet: NHS-LC-biotin and streptavidin agarose beads were purchased from Thermo Scientific.

    Techniques: Incubation, Concentration Assay

    CaMKIV is GlcNAcylated. A–D, lysates from HEK293 cells transfected with empty HA plasmid or HA-CaMKIV were immunoprecipitated for HA. A, subjected to galactosyltransferase labeling in the presence of UDP-[ 3 H]galactose for autoradiography. B, labeled in the presence of UDP-GalNAz and reacted with TAMRA alkyne for detection by in-gel fluorescence. C, treated with β-elimination. IP , immunoprecipitation; IB , immunoblot. D, treated with γ-phosphatase (γ- PPase ) or GlcNAcase prior to immunoblotting for O -GlcNAc and HA. A, prior to autoradiography, the gel was stained for total protein using Coomassie Brilliant Blue G-250 ( CBB G250 ). B, TAMRA fluorescence was detected in-gel prior to staining for total protein using SYPRO Ruby. E, rat cerebellum extract was immunoprecipitated for CaMKIV, subjected to galactosyltransferase labeling in the presence of UDP-GalNAz, reacted with biotin alkyne, and immunoblotted for CaMKIV or biotin (using streptavidin-horseradish peroxidase). F, lysates from Jurkat cells were immunoprecipitated for CaMKIV or using nonspecific ( ns ) mouse antibodies and immunoblotted for O -GlcNAc or CaMKIV.

    Journal: The Journal of Biological Chemistry

    Article Title: Regulation of Calcium/Calmodulin-dependent Kinase IV by O-GlcNAc Modification *

    doi: 10.1074/jbc.M109.007310

    Figure Lengend Snippet: CaMKIV is GlcNAcylated. A–D, lysates from HEK293 cells transfected with empty HA plasmid or HA-CaMKIV were immunoprecipitated for HA. A, subjected to galactosyltransferase labeling in the presence of UDP-[ 3 H]galactose for autoradiography. B, labeled in the presence of UDP-GalNAz and reacted with TAMRA alkyne for detection by in-gel fluorescence. C, treated with β-elimination. IP , immunoprecipitation; IB , immunoblot. D, treated with γ-phosphatase (γ- PPase ) or GlcNAcase prior to immunoblotting for O -GlcNAc and HA. A, prior to autoradiography, the gel was stained for total protein using Coomassie Brilliant Blue G-250 ( CBB G250 ). B, TAMRA fluorescence was detected in-gel prior to staining for total protein using SYPRO Ruby. E, rat cerebellum extract was immunoprecipitated for CaMKIV, subjected to galactosyltransferase labeling in the presence of UDP-GalNAz, reacted with biotin alkyne, and immunoblotted for CaMKIV or biotin (using streptavidin-horseradish peroxidase). F, lysates from Jurkat cells were immunoprecipitated for CaMKIV or using nonspecific ( ns ) mouse antibodies and immunoblotted for O -GlcNAc or CaMKIV.

    Article Snippet: Biotin-containing peptides were eluted from the streptavidin-agarose by β-elimination followed by Michael addition (BEMAD) , to replace the GlcNAc-GalNAz-biotin moiety with DTT ( A ), and analyzed by an LTQ mass spectrometer (Thermo Finnigan) coupled with a nano-two-dimensional liquid chromatography pump (Eksigent Technologies).

    Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Labeling, Autoradiography, Fluorescence, Staining

    In vitro dephosphorylation assays and generation of RPTP chimeras. ( A ) The indicated PTPRK and PTPRM domains were assayed for phosphatase activity using the pNPP colorimetric assay. Control wells contained pNPP only. Protein amounts used are shown. ( B ) Pervanadate-treated MCF10A lysates were incubated with predetermined amounts of the indicated domains to give equal phosphatase-activity, prior to phosphotyrosine immunoprecipitation and immunoblot analysis. ( C ) Recombinant proteins consisting of combinations of PTPRK and PTPRM D1 and D2 domains were expressed in and using Ni-NTA affinity resin. Purified proteins were then subjected to size exclusion chromatography. ( D ) Recombinant His- and Avi-tagged PTPRK and PTPRM chimeric domains were purified from E. coli cultured in biotin-supplemented media, incubated ±streptavidin and subjected to SDS-PAGE and Coomassie staining, to determine the extent of biotinylation. Arrows indicate the purified domains and the respective streptavidin-induced mobility shift. ( E ) The indicated recombinant PTPRK and PTPRM chimeric domains were incubated were assayed for phosphatase activity using the pNPP colorimetric assay. Control wells contained pNPP. Protein amounts used are shown.

    Journal: eLife

    Article Title: The homophilic receptor PTPRK selectively dephosphorylates multiple junctional regulators to promote cell–cell adhesion

    doi: 10.7554/eLife.44597

    Figure Lengend Snippet: In vitro dephosphorylation assays and generation of RPTP chimeras. ( A ) The indicated PTPRK and PTPRM domains were assayed for phosphatase activity using the pNPP colorimetric assay. Control wells contained pNPP only. Protein amounts used are shown. ( B ) Pervanadate-treated MCF10A lysates were incubated with predetermined amounts of the indicated domains to give equal phosphatase-activity, prior to phosphotyrosine immunoprecipitation and immunoblot analysis. ( C ) Recombinant proteins consisting of combinations of PTPRK and PTPRM D1 and D2 domains were expressed in and using Ni-NTA affinity resin. Purified proteins were then subjected to size exclusion chromatography. ( D ) Recombinant His- and Avi-tagged PTPRK and PTPRM chimeric domains were purified from E. coli cultured in biotin-supplemented media, incubated ±streptavidin and subjected to SDS-PAGE and Coomassie staining, to determine the extent of biotinylation. Arrows indicate the purified domains and the respective streptavidin-induced mobility shift. ( E ) The indicated recombinant PTPRK and PTPRM chimeric domains were incubated were assayed for phosphatase activity using the pNPP colorimetric assay. Control wells contained pNPP. Protein amounts used are shown.

    Article Snippet: Added to each fraction was 10 μl of rabbit anti-pY-1000 antibody (Cell Signal Technologies, New England Biolabs) pre-conjugated to 5 μl of protein G agarose bead suspension (Merck Millipore) and 2.4 μg each of biotinylated Src and Grb2 SH2 mutant domains, pre-conjugated to 5 μl of streptavidin agarose bead suspension (Thermo Fisher Scientific) and ice-cold IAP buffer up to 1 ml.

    Techniques: In Vitro, De-Phosphorylation Assay, Activity Assay, Colorimetric Assay, Incubation, Immunoprecipitation, Recombinant, Purification, Size-exclusion Chromatography, Cell Culture, SDS Page, Staining, Mobility Shift

    Purification of biotinylated recombinant PTPRK domains. ( A ) His- and Avi-tagged PTPRK domains were expressed in E. coli cultured in biotin-supplemented media and purified using Nickel-NTA beads, followed by size exclusion chromatography (SEC). DA = D1057A mutant. CS = C1089S mutant. ( B ) SEC-purified proteins bound to streptavidin resin were eluted and resolved by SDS-PAGE followed by Coomassie staining. In; input, B; beads. ( C ) The phosphatase activity of indicated amounts of purified proteins was assessed using the Biomol green assay with two tyrosine phosphorylated peptides as substrates and was quantified at 620 nm. ( D ) Recombinant proteins bound to streptavidin resin were used in pull down assays from pervanadate treated Hs27 fibroblast lysates. After extensive washing, bound proteins were eluted in sample buffer and analyzed by immunoblot.

    Journal: eLife

    Article Title: The homophilic receptor PTPRK selectively dephosphorylates multiple junctional regulators to promote cell–cell adhesion

    doi: 10.7554/eLife.44597

    Figure Lengend Snippet: Purification of biotinylated recombinant PTPRK domains. ( A ) His- and Avi-tagged PTPRK domains were expressed in E. coli cultured in biotin-supplemented media and purified using Nickel-NTA beads, followed by size exclusion chromatography (SEC). DA = D1057A mutant. CS = C1089S mutant. ( B ) SEC-purified proteins bound to streptavidin resin were eluted and resolved by SDS-PAGE followed by Coomassie staining. In; input, B; beads. ( C ) The phosphatase activity of indicated amounts of purified proteins was assessed using the Biomol green assay with two tyrosine phosphorylated peptides as substrates and was quantified at 620 nm. ( D ) Recombinant proteins bound to streptavidin resin were used in pull down assays from pervanadate treated Hs27 fibroblast lysates. After extensive washing, bound proteins were eluted in sample buffer and analyzed by immunoblot.

    Article Snippet: Added to each fraction was 10 μl of rabbit anti-pY-1000 antibody (Cell Signal Technologies, New England Biolabs) pre-conjugated to 5 μl of protein G agarose bead suspension (Merck Millipore) and 2.4 μg each of biotinylated Src and Grb2 SH2 mutant domains, pre-conjugated to 5 μl of streptavidin agarose bead suspension (Thermo Fisher Scientific) and ice-cold IAP buffer up to 1 ml.

    Techniques: Purification, Recombinant, Cell Culture, Size-exclusion Chromatography, Mutagenesis, SDS Page, Staining, Activity Assay

    The PTPRK-dependent tyrosine phosphoproteome. ( A ) After SEC, proteins were incubated with or without streptavidin and subjected to SDS PAGE followed by Coomassie staining to determine the extent of biotinylation. Arrows indicate the purified domains and the respective streptavidin-induced mobility shift. ( B ) Volcano plot of tyrosine phosphosites detected in PTPRK KO and wildtype MCF10As. Phosphosites > 50% enriched in (p

    Journal: eLife

    Article Title: The homophilic receptor PTPRK selectively dephosphorylates multiple junctional regulators to promote cell–cell adhesion

    doi: 10.7554/eLife.44597

    Figure Lengend Snippet: The PTPRK-dependent tyrosine phosphoproteome. ( A ) After SEC, proteins were incubated with or without streptavidin and subjected to SDS PAGE followed by Coomassie staining to determine the extent of biotinylation. Arrows indicate the purified domains and the respective streptavidin-induced mobility shift. ( B ) Volcano plot of tyrosine phosphosites detected in PTPRK KO and wildtype MCF10As. Phosphosites > 50% enriched in (p

    Article Snippet: Added to each fraction was 10 μl of rabbit anti-pY-1000 antibody (Cell Signal Technologies, New England Biolabs) pre-conjugated to 5 μl of protein G agarose bead suspension (Merck Millipore) and 2.4 μg each of biotinylated Src and Grb2 SH2 mutant domains, pre-conjugated to 5 μl of streptavidin agarose bead suspension (Thermo Fisher Scientific) and ice-cold IAP buffer up to 1 ml.

    Techniques: Size-exclusion Chromatography, Incubation, SDS Page, Staining, Purification, Mobility Shift

    LANA and MCMs are part of the replication complex. (A) Schematic of two-step iPOND performed on KSHV-positive cells. Approximately 100 million KSHV-positive cells were labeled with 5-ethynyl-2′deoxyuridine (EdU) for 30 min, harvested, and washed with 1× PBS (1). Protein and DNA were cross-linked using 1% formaldehyde for 20 min and quenched using 125 mM glycine (2). The cells were permeabilized with 0.25% Triton X-100 in PBS for 30 min at room temperature (3), and nuclei were isolated following centrifugation (4). Click chemistry was performed on the nuclei to conjugate biotin-azide to EdU, and DMSO was used as a negative control (5). The chromatin was sheared using sonication to generate fragments of 100 to 300 bp (6). Protein-DNA complex bound to LANA was captured using monoclonal LANA antibody (7). Protein A/G beads were used to capture antigen-antibody complexes, which were eluted from the beads using peptide specific for LANA (8). Streptavidin beads were used to capture EdU-labeled (replicated) DNA-protein complexes (9). Proteins bound to streptavidin beads were eluted by boiling the beads at 95°C and detected using specific antibodies (10). (B) Immunoblot of LANA, MCM6, and PCNA obtained through two-step iPOND using respective antibodies from KSHV-positive BCBL-1 and BrK.219 cells. A total of 100 million cells were labeled with EdU and permeabilized, and a click reaction was performed using biotin-azide and DMSO. The cells were lysed and sonicated, and proteins were pulled down using LANA antibody. The proteins associated with DNA were pulled down using streptavidin beads and eluted by boiling in the loading buffer.

    Journal: Journal of Virology

    Article Title: Minichromosome Maintenance Proteins Cooperate with LANA during the G1/S Phase of the Cell Cycle To Support Viral DNA Replication

    doi: 10.1128/JVI.02256-18

    Figure Lengend Snippet: LANA and MCMs are part of the replication complex. (A) Schematic of two-step iPOND performed on KSHV-positive cells. Approximately 100 million KSHV-positive cells were labeled with 5-ethynyl-2′deoxyuridine (EdU) for 30 min, harvested, and washed with 1× PBS (1). Protein and DNA were cross-linked using 1% formaldehyde for 20 min and quenched using 125 mM glycine (2). The cells were permeabilized with 0.25% Triton X-100 in PBS for 30 min at room temperature (3), and nuclei were isolated following centrifugation (4). Click chemistry was performed on the nuclei to conjugate biotin-azide to EdU, and DMSO was used as a negative control (5). The chromatin was sheared using sonication to generate fragments of 100 to 300 bp (6). Protein-DNA complex bound to LANA was captured using monoclonal LANA antibody (7). Protein A/G beads were used to capture antigen-antibody complexes, which were eluted from the beads using peptide specific for LANA (8). Streptavidin beads were used to capture EdU-labeled (replicated) DNA-protein complexes (9). Proteins bound to streptavidin beads were eluted by boiling the beads at 95°C and detected using specific antibodies (10). (B) Immunoblot of LANA, MCM6, and PCNA obtained through two-step iPOND using respective antibodies from KSHV-positive BCBL-1 and BrK.219 cells. A total of 100 million cells were labeled with EdU and permeabilized, and a click reaction was performed using biotin-azide and DMSO. The cells were lysed and sonicated, and proteins were pulled down using LANA antibody. The proteins associated with DNA were pulled down using streptavidin beads and eluted by boiling in the loading buffer.

    Article Snippet: Elution was performed twice, and the eluted proteins were then incubated with 100 μl of Pierce streptavidin agarose Beads (ThermoFisher) overnight for 12 to 16 h in dark.

    Techniques: Labeling, Isolation, Centrifugation, Negative Control, Sonication

    Binding of carboxy-terminal L2 peptides to retromer. A . The top lines show the sequences of biotinylated peptides, where B indicates position of biotin. Putative retromer recognition motifs in the carboxy-terminal peptide are shown in boxes. Mutant versions of the carboxy-terminal peptide are also shown, with the mutations in red. B . Left panel. L2-N, L2-M, or L2-C peptide was incubated with uninfected HeLa cell RIPA lysate. The samples were analyzed by streptavidin pull-down, SDS-PAGE, and immunoblotting with an anti-Vps35 antibody. Molecular weight markers in kDa are shown at the left. Right panel. The wild-type or a mutant carboxy-terminal peptide were incubated with uninfected HeLa cell HEPES lysate and processed as in panel A. Similar results were obtained in three or more independent experiments. C . Experiments performed as in panel B with HaCaT cell RIPA lysates.

    Journal: PLoS Pathogens

    Article Title: Direct Binding of Retromer to Human Papillomavirus Type 16 Minor Capsid Protein L2 Mediates Endosome Exit during Viral Infection

    doi: 10.1371/journal.ppat.1004699

    Figure Lengend Snippet: Binding of carboxy-terminal L2 peptides to retromer. A . The top lines show the sequences of biotinylated peptides, where B indicates position of biotin. Putative retromer recognition motifs in the carboxy-terminal peptide are shown in boxes. Mutant versions of the carboxy-terminal peptide are also shown, with the mutations in red. B . Left panel. L2-N, L2-M, or L2-C peptide was incubated with uninfected HeLa cell RIPA lysate. The samples were analyzed by streptavidin pull-down, SDS-PAGE, and immunoblotting with an anti-Vps35 antibody. Molecular weight markers in kDa are shown at the left. Right panel. The wild-type or a mutant carboxy-terminal peptide were incubated with uninfected HeLa cell HEPES lysate and processed as in panel A. Similar results were obtained in three or more independent experiments. C . Experiments performed as in panel B with HaCaT cell RIPA lysates.

    Article Snippet: 40 μl of streptavidin agarose beads slurry (Pierce, cat# 20349) was added, and the mixture was gently rocked for 45 min at 4°C.

    Techniques: Binding Assay, Mutagenesis, Incubation, SDS Page, Molecular Weight

    Histone tail binding specificity of MBP-SpPHD and mutants thereof. The indicated recombinant MBP-fusion proteins were incubated with biotinylated synthetic histone tails, retrieved from the binding reaction using streptavidin beads, and bound proteins were resolved and visualized by SDS/PAGE and Western blotting with anti-MBP antibodies. One representative blot is shown for each recombinant protein tested. The MBP-tag by itself (panel e) served as a control. Lanes 1 contain 5% of the total binding reaction prior to the addition of streptavidin beads. The histone tails used are indicated above the lanes (3-6), and samples in lanes 2 (resin) did not contain any histone tails serving as negative controls.

    Journal: Developmental and comparative immunology

    Article Title: The PHD domain of the sea urchin RAG2 homolog, SpRAG2L, recognizes dimethylated lysine 4 in histone H3 tails

    doi: 10.1016/j.dci.2008.03.012

    Figure Lengend Snippet: Histone tail binding specificity of MBP-SpPHD and mutants thereof. The indicated recombinant MBP-fusion proteins were incubated with biotinylated synthetic histone tails, retrieved from the binding reaction using streptavidin beads, and bound proteins were resolved and visualized by SDS/PAGE and Western blotting with anti-MBP antibodies. One representative blot is shown for each recombinant protein tested. The MBP-tag by itself (panel e) served as a control. Lanes 1 contain 5% of the total binding reaction prior to the addition of streptavidin beads. The histone tails used are indicated above the lanes (3-6), and samples in lanes 2 (resin) did not contain any histone tails serving as negative controls.

    Article Snippet: Afterwards, 100μl of a 50% suspension of streptavidin agarose beads (Amersham) were added and mixed by rotation for one hour at 4°C.

    Techniques: Binding Assay, Recombinant, Incubation, SDS Page, Western Blot

    Interaction with SORLA controls trafficking and synaptic exposure of TrkB. ( A ) Colocalization of endogenous SORLA (red) and TrkB (green) in primary cortical neurons as shown by confocal immunofluorescence microscopy. Scale bar: 10 µm. ( B ) Co-immunoprecipitation of endogenous SORLA and TrkB from brain lysates is seen using anti-SORLA (IP anti-SORLA; lane 2) and anti-Trk antisera (IP anti-TrkB; lane 3). Panel Input (lane 1) indicates presence of endogenous SORLA and TrkB in brain lysate prior to co-immunoprecipitation. Lane 4 indicates lack co-immunoprecipitation in the absence of primary antibody (No IgG). ( C ) SH-SY5Y neuroblastoma cells stably overexpressing SORLA (SY5Y-S) or parental control cells (SY5Y) were transfected with expression constructs for TrkB. Subsequently, proteins at the cell surface were biotinylated and immunoprecipitated using streptavidin beads. Western blot analysis documents reduced levels of biotinylated TrkB at the cell surface in SY5Y-S compared to SY5Y cells (panel Surface). Panel Input represents levels of TrkB and SORLA in cell lysates prior to precipitation. Detection of tubulin (tub.), β-integrin (β -integ.), and PDGF-β receptor (PDGF-R) served as controls for loading and immunoprecipitation, respectively. ( D ) Densitometric quantification of replicate Western blots as exemplified in (C) (n = 6, Student’s t -test). ( E ) Subcellullar fractionations of wild type and SORLA-deficient mouse brain extracts were probed for the indicated proteins using Western blot analysis. Elevated levels of TrkB receptors (pan-Trk antibody) are seen in the synaptosomal plasma membrane (pm) fraction in SORLA-deficient (lanes 7 and 8) as compared to control brains (lanes 5 and 6). In contrast, levels of Trk receptors in the postsynaptic density (PSD) are reduced in receptor-deficient (lane 10) compared with wild type brains (lane 9). Levels of Trk in total brain lysates prior to subcellular fractionation are similar between genotypes (lanes 1–4). Detection of synaptophysin (Synapt), AMPA receptors, and PSD95 served as respective loading controls. ( F ) Densitometric quantification of replicate Western blots as exemplified in (E) (n = 4–6, Student’s t -test).

    Journal: PLoS ONE

    Article Title: SORLA-Mediated Trafficking of TrkB Enhances the Response of Neurons to BDNF

    doi: 10.1371/journal.pone.0072164

    Figure Lengend Snippet: Interaction with SORLA controls trafficking and synaptic exposure of TrkB. ( A ) Colocalization of endogenous SORLA (red) and TrkB (green) in primary cortical neurons as shown by confocal immunofluorescence microscopy. Scale bar: 10 µm. ( B ) Co-immunoprecipitation of endogenous SORLA and TrkB from brain lysates is seen using anti-SORLA (IP anti-SORLA; lane 2) and anti-Trk antisera (IP anti-TrkB; lane 3). Panel Input (lane 1) indicates presence of endogenous SORLA and TrkB in brain lysate prior to co-immunoprecipitation. Lane 4 indicates lack co-immunoprecipitation in the absence of primary antibody (No IgG). ( C ) SH-SY5Y neuroblastoma cells stably overexpressing SORLA (SY5Y-S) or parental control cells (SY5Y) were transfected with expression constructs for TrkB. Subsequently, proteins at the cell surface were biotinylated and immunoprecipitated using streptavidin beads. Western blot analysis documents reduced levels of biotinylated TrkB at the cell surface in SY5Y-S compared to SY5Y cells (panel Surface). Panel Input represents levels of TrkB and SORLA in cell lysates prior to precipitation. Detection of tubulin (tub.), β-integrin (β -integ.), and PDGF-β receptor (PDGF-R) served as controls for loading and immunoprecipitation, respectively. ( D ) Densitometric quantification of replicate Western blots as exemplified in (C) (n = 6, Student’s t -test). ( E ) Subcellullar fractionations of wild type and SORLA-deficient mouse brain extracts were probed for the indicated proteins using Western blot analysis. Elevated levels of TrkB receptors (pan-Trk antibody) are seen in the synaptosomal plasma membrane (pm) fraction in SORLA-deficient (lanes 7 and 8) as compared to control brains (lanes 5 and 6). In contrast, levels of Trk receptors in the postsynaptic density (PSD) are reduced in receptor-deficient (lane 10) compared with wild type brains (lane 9). Levels of Trk in total brain lysates prior to subcellular fractionation are similar between genotypes (lanes 1–4). Detection of synaptophysin (Synapt), AMPA receptors, and PSD95 served as respective loading controls. ( F ) Densitometric quantification of replicate Western blots as exemplified in (E) (n = 4–6, Student’s t -test).

    Article Snippet: Co-immunoprecipitation of endogenous proteins from brain lysates was conducted according to standard protocols utilizing streptavidin agarose beads (Roche).

    Techniques: Immunofluorescence, Microscopy, Immunoprecipitation, Stable Transfection, Transfection, Expressing, Construct, Western Blot, Fractionation

    Cep170 is an FHDC1-interacting protein. BirA*-tagged FHDC1 was expressed in NIH 3T3 cells by transient transfection. Ciliogenesis was induced as before in low-serum media. BirA*-FHDC1 (red) was detected by virtue of the encoded myc-tag. (A) BirA*-FHDC1 is recruited to the elongated cilia and, following exogenous biotin treatment, induces biotinylation of cytoplasmic microtubules (green) and the cilia (acetylated tubulin, white). Specific punctate structures within the cytoplasmic microtubule network were also labeled (arrows). Biotinylated proteins were detected with Alexa488-streptavidin. (B) BirA*-FHDC1 (red) induces biotinylation of puncta (green) that colocalize with γ-tubulin puncta detected with anti–γ-tubulin (white). (C) BirA*-FHDC1 induces biotinylation of endogenous Cep170. As in B, BirA*-FHDC1 was expressed by transient transfection and its ability to induce biotinylation of the endogenous Cep170 was assessed by IB. myc-FHDC1 and FMNL2-BirA* were included as negative controls. Total biotinylated proteins were isolated from each sample using streptavidin–agarose beads. The eluted proteins were immunoblotted using the indicated antibodies. Endogenous Cep170 is only present in the pool of biotinylated proteins induced by BirA*-FHDC1 (arrow).

    Journal: Molecular Biology of the Cell

    Article Title: Actin-dependent regulation of cilia length by the inverted formin FHDC1

    doi: 10.1091/mbc.E18-02-0088

    Figure Lengend Snippet: Cep170 is an FHDC1-interacting protein. BirA*-tagged FHDC1 was expressed in NIH 3T3 cells by transient transfection. Ciliogenesis was induced as before in low-serum media. BirA*-FHDC1 (red) was detected by virtue of the encoded myc-tag. (A) BirA*-FHDC1 is recruited to the elongated cilia and, following exogenous biotin treatment, induces biotinylation of cytoplasmic microtubules (green) and the cilia (acetylated tubulin, white). Specific punctate structures within the cytoplasmic microtubule network were also labeled (arrows). Biotinylated proteins were detected with Alexa488-streptavidin. (B) BirA*-FHDC1 (red) induces biotinylation of puncta (green) that colocalize with γ-tubulin puncta detected with anti–γ-tubulin (white). (C) BirA*-FHDC1 induces biotinylation of endogenous Cep170. As in B, BirA*-FHDC1 was expressed by transient transfection and its ability to induce biotinylation of the endogenous Cep170 was assessed by IB. myc-FHDC1 and FMNL2-BirA* were included as negative controls. Total biotinylated proteins were isolated from each sample using streptavidin–agarose beads. The eluted proteins were immunoblotted using the indicated antibodies. Endogenous Cep170 is only present in the pool of biotinylated proteins induced by BirA*-FHDC1 (arrow).

    Article Snippet: Protein concentration was determined by BCA assay and equal amounts of each lysate were incubated separately with streptavidin agarose beads (Solulink) for 3 h at 4°C.

    Techniques: Transfection, Labeling, Isolation

    Biochemical analysis of gp49 expression. Cell surface proteins were biotinylated and precipitated with streptavidin-agarose, separated by SDS-PAGE, blotted onto nitrocellulose, and probed with antiserum specific for gp49A or gp49B, as indicated. BMMC and NK cells from wild-type (+/+) and gp49B 0 (−/−) mice were analyzed. Included is a comparison of the level of gp49A expression in BMMC and NK cells from a C57BL/6 mouse.

    Journal: Molecular and Cellular Biology

    Article Title: Natural Killer Cells and Mast Cells from gp49B Null Mutant Mice Are Functional

    doi:

    Figure Lengend Snippet: Biochemical analysis of gp49 expression. Cell surface proteins were biotinylated and precipitated with streptavidin-agarose, separated by SDS-PAGE, blotted onto nitrocellulose, and probed with antiserum specific for gp49A or gp49B, as indicated. BMMC and NK cells from wild-type (+/+) and gp49B 0 (−/−) mice were analyzed. Included is a comparison of the level of gp49A expression in BMMC and NK cells from a C57BL/6 mouse.

    Article Snippet: Cleared lysates were incubated with streptavidin-agarose beads (Upstate Biotechnology) for 2 h at 4°C.

    Techniques: Expressing, SDS Page, Mouse Assay

    Analysis of NK and mast cells in wild-type and gp49B 0 mice. (A) Unstimulated splenocytes prepared from wild-type (+/+) and gp49B 0 (−/−) mice double-stained with FITC-conjugated anti-CD3ɛ and phycoerythrin (PE)-conjugated DX5. (B) IL-2-expanded DX5 + NK1.1 + cell populations from wild-type and gp49B 0 mice double-stained with PE-conjugated anti-CD3ɛ and anti-gp49 antiserum followed by goat anti-rabbit IgG-FITC. (C) Expression of c-kit and FcɛRI in cultured BMMC from wild-type and gp49B 0 mice. FcɛRI expression was determined by binding of biotinylated IgE followed by streptavidin-PE (thick line). The control for this experiment was streptavidin-PE alone (thin line). FL1-H, green fluorescence; FL2-H, red fluorescence.

    Journal: Molecular and Cellular Biology

    Article Title: Natural Killer Cells and Mast Cells from gp49B Null Mutant Mice Are Functional

    doi:

    Figure Lengend Snippet: Analysis of NK and mast cells in wild-type and gp49B 0 mice. (A) Unstimulated splenocytes prepared from wild-type (+/+) and gp49B 0 (−/−) mice double-stained with FITC-conjugated anti-CD3ɛ and phycoerythrin (PE)-conjugated DX5. (B) IL-2-expanded DX5 + NK1.1 + cell populations from wild-type and gp49B 0 mice double-stained with PE-conjugated anti-CD3ɛ and anti-gp49 antiserum followed by goat anti-rabbit IgG-FITC. (C) Expression of c-kit and FcɛRI in cultured BMMC from wild-type and gp49B 0 mice. FcɛRI expression was determined by binding of biotinylated IgE followed by streptavidin-PE (thick line). The control for this experiment was streptavidin-PE alone (thin line). FL1-H, green fluorescence; FL2-H, red fluorescence.

    Article Snippet: Cleared lysates were incubated with streptavidin-agarose beads (Upstate Biotechnology) for 2 h at 4°C.

    Techniques: Mouse Assay, Staining, Expressing, Cell Culture, Binding Assay, Fluorescence

    (A) Time course of gp15 mRNA expression in C. parvum -infected MDCK cell monolayers. Total RNA was isolated from infected MDCK cells at various times postinfection, DNase treated, and used as a template for RT-PCR (lanes 2 to 12) or control PCR (lanes 13 to 23), both primed with the gp15ATG and gp15STOP primers. The arrowhead indicates the position of the 1-kb gp15 amplicon. RNA template was isolated from infected MDCK cells at 0.5 h (lanes 3 and 14), 2 h (lanes 4 and 15), 4 h (lanes 5 and 16), 6 h (lanes 6 and 17), 9 h (lanes 7 and 18), 11 h (lanes 8 and 19), 24 h (lanes 9 and 20), and 48 h (lanes 10 and 21) postinfection; from uninfected MDCK cells (lanes 2 and 13); and directly from purified sporozoites (2 μg; lanes 11 and 22). Additional controls for the RT-PCR and PCRs lacked template nucleic acid (lanes 12 and 23 and 24, respectively). (B) Intracellular expression of gp15 protein occurs late in merogony. C. parvum ), or with anti-gp15 MAb 11A5 (F and H), CrA1 (J and L) or CrA2 (N and P) followed by biotinylated secondary antibodies and Cy3-conjugated streptavidin. Parasite nuclei in the same microscopic fields were stained with DAPI (lanes A, E, I, M, C, G, K, and O).

    Journal: Infection and Immunity

    Article Title: Cloning and Sequence Analysis of a Highly Polymorphic Cryptosporidium parvum Gene Encoding a 60-Kilodalton Glycoprotein and Characterization of Its 15- and 45-Kilodalton Zoite Surface Antigen Products

    doi:

    Figure Lengend Snippet: (A) Time course of gp15 mRNA expression in C. parvum -infected MDCK cell monolayers. Total RNA was isolated from infected MDCK cells at various times postinfection, DNase treated, and used as a template for RT-PCR (lanes 2 to 12) or control PCR (lanes 13 to 23), both primed with the gp15ATG and gp15STOP primers. The arrowhead indicates the position of the 1-kb gp15 amplicon. RNA template was isolated from infected MDCK cells at 0.5 h (lanes 3 and 14), 2 h (lanes 4 and 15), 4 h (lanes 5 and 16), 6 h (lanes 6 and 17), 9 h (lanes 7 and 18), 11 h (lanes 8 and 19), 24 h (lanes 9 and 20), and 48 h (lanes 10 and 21) postinfection; from uninfected MDCK cells (lanes 2 and 13); and directly from purified sporozoites (2 μg; lanes 11 and 22). Additional controls for the RT-PCR and PCRs lacked template nucleic acid (lanes 12 and 23 and 24, respectively). (B) Intracellular expression of gp15 protein occurs late in merogony. C. parvum ), or with anti-gp15 MAb 11A5 (F and H), CrA1 (J and L) or CrA2 (N and P) followed by biotinylated secondary antibodies and Cy3-conjugated streptavidin. Parasite nuclei in the same microscopic fields were stained with DAPI (lanes A, E, I, M, C, G, K, and O).

    Article Snippet: CrA1/2 and 11A5 immunoprecipitations were performed by incubating a preformed ternary complex composed of primary MAb, biotin-conjugated secondary antibody (goat anti-mouse IgA or goat anti-mouse IgG), and streptavidin-agarose beads (EY Laboratories) with the extracts on a rotator for 1 h at room temperature.

    Techniques: Expressing, Infection, Isolation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Purification, Staining

    All of the Sm proteins associate in a specific and stable manner with a biotinylated Sm site oligonucleotide. (A) Reconstitution with TPs was carried out with biotinylated Sm site oligonucleotide (AAUUUUUGA [lane 2]) or, as negative controls, in the absence of biotinylated RNA (lane 3) or with biotinylated SmC3-C7 (AACCCCCGA) oligonucleotide (lane 4). Following streptavidin-agarose precipitation, samples were washed extensively with buffer containing 150 mM KCl. Bound proteins were analyzed by SDS-polyacrylamide gel electrophoresis and stained with Coomassie blue. (B) Reconstitution with TPs was performed in either the presence or absence of biotinylated Sm site oligonucleotide (b-Sm site), as indicated above each lane. The salt stability of the coprecipitation of U1-specific and Sm proteins was analyzed by extensive washing with buffers containing various concentrations of salt (0.15 to 2 M KCl, as indicated above the lanes). Following SDS-polyacrylamide gel electrophoresis, proteins were visualized by staining first with Coomassie blue and then with silver.

    Journal: Molecular and Cellular Biology

    Article Title: Spliceosomal U snRNP Core Assembly: Sm Proteins Assemble onto an Sm Site RNA Nonanucleotide in a Specific and Thermodynamically Stable Manner

    doi:

    Figure Lengend Snippet: All of the Sm proteins associate in a specific and stable manner with a biotinylated Sm site oligonucleotide. (A) Reconstitution with TPs was carried out with biotinylated Sm site oligonucleotide (AAUUUUUGA [lane 2]) or, as negative controls, in the absence of biotinylated RNA (lane 3) or with biotinylated SmC3-C7 (AACCCCCGA) oligonucleotide (lane 4). Following streptavidin-agarose precipitation, samples were washed extensively with buffer containing 150 mM KCl. Bound proteins were analyzed by SDS-polyacrylamide gel electrophoresis and stained with Coomassie blue. (B) Reconstitution with TPs was performed in either the presence or absence of biotinylated Sm site oligonucleotide (b-Sm site), as indicated above each lane. The salt stability of the coprecipitation of U1-specific and Sm proteins was analyzed by extensive washing with buffers containing various concentrations of salt (0.15 to 2 M KCl, as indicated above the lanes). Following SDS-polyacrylamide gel electrophoresis, proteins were visualized by staining first with Coomassie blue and then with silver.

    Article Snippet: Reconstitution assay mixtures containing biotinylated RNA oligonucleotide or mock assay mixtures without RNA oligonucleotide were incubated with 10 μg of prewashed streptavidin-agarose beads (Boehringer Mannheim) in 400 μl of phosphate-buffered saline (pH 8.0).

    Techniques: Polyacrylamide Gel Electrophoresis, Staining

    Mutant p53 can bind to and transactivate the GRO1 promoter. A , schematic presentation of the GRO1 ( top ), p21 ( middle ), and GAPDH ( bottom ) promoters and the location of PCR primers used for ChIP assay. B , the binding of mutant p53 to the GRO1 promoter in SW480 cells was measured by ChIP. Mutant p53-DNA complexes were captured with anti-p53 antibody along with rabbit IgG as a control. The binding of mutant p53 protein to the p21 and GAPDH promoters was also measured as negative controls. C , pull-down assay of mutant p53 ( top ) and NF-κB (p65) ( bottom ) binding to the GRO1 promoter in vitro . The biotinated GRO1 promoter probe bound to streptavidin-agarose beads was used to precipitate p65 and mutant p53 from nuclear protein extracts from SW480 cells uninduced (-) or induced (+) to knock down mutant p53 for 3 days. The biotinated GAPDH promoter probe was used as a negative binding control. The nuclear proteins pulled down by biotinated promoter probes were analyzed by Western blot assay with antibodies against p53 and p65, respectively. D , schematic presentation of the luciferase reporter constructs under the control of the GRO1 ( top ) and p21 ( bottom ) promoters, respectively. E , the GRO1 promoter is responsive to mutant p53-R273H ( left ) but not wild-type p53 ( right ). The response of the p21 promoter to wild-type p53 was measured as a positive control. The luciferase activity for the promoterless pGL2 plasmid in the presence of wild-type and mutant p53 was measured as a negative control. Luciferase assay was performed as described under “Experimental Procedures.”

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of GRO1 as a Critical Determinant for Mutant p53 Gain of Function *

    doi: 10.1074/jbc.M900994200

    Figure Lengend Snippet: Mutant p53 can bind to and transactivate the GRO1 promoter. A , schematic presentation of the GRO1 ( top ), p21 ( middle ), and GAPDH ( bottom ) promoters and the location of PCR primers used for ChIP assay. B , the binding of mutant p53 to the GRO1 promoter in SW480 cells was measured by ChIP. Mutant p53-DNA complexes were captured with anti-p53 antibody along with rabbit IgG as a control. The binding of mutant p53 protein to the p21 and GAPDH promoters was also measured as negative controls. C , pull-down assay of mutant p53 ( top ) and NF-κB (p65) ( bottom ) binding to the GRO1 promoter in vitro . The biotinated GRO1 promoter probe bound to streptavidin-agarose beads was used to precipitate p65 and mutant p53 from nuclear protein extracts from SW480 cells uninduced (-) or induced (+) to knock down mutant p53 for 3 days. The biotinated GAPDH promoter probe was used as a negative binding control. The nuclear proteins pulled down by biotinated promoter probes were analyzed by Western blot assay with antibodies against p53 and p65, respectively. D , schematic presentation of the luciferase reporter constructs under the control of the GRO1 ( top ) and p21 ( bottom ) promoters, respectively. E , the GRO1 promoter is responsive to mutant p53-R273H ( left ) but not wild-type p53 ( right ). The response of the p21 promoter to wild-type p53 was measured as a positive control. The luciferase activity for the promoterless pGL2 plasmid in the presence of wild-type and mutant p53 was measured as a negative control. Luciferase assay was performed as described under “Experimental Procedures.”

    Article Snippet: The binding assay was performed by mixing 200 μg of nuclear protein extracts, 2 μg of a biotin-labeled probe, 20 μl of 50% streptavidin-agarose beads (Prozyme), and 500 μl of phosphate-buffered saline with various protein inhibitors (PBSI).

    Techniques: Mutagenesis, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Binding Assay, Pull Down Assay, In Vitro, Western Blot, Luciferase, Construct, Positive Control, Activity Assay, Plasmid Preparation, Negative Control