streptavidin agarose beads  (Millipore)

 
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    Name:
    Streptavidin Mag Sepharose
    Description:
    Streptavidin Mag Sepharose R is a magnetic bead for simple and efficient enrichment of target proteins by immunoprecipitation and purification of biotinylated biomolecules Streptavidin Mag Sepharose R utilizes the strong interaction between biotin and streptavidin ligand which is immobilized on magnetic beads Magnetic beads simplify sample handling in small scale purifications
    Catalog Number:
    GE28-9857-38
    Price:
    None
    Applications:
    Streptavidin Mag Sepharose(R) is a magnetic bead for simple and efficient enrichment of target Proteins by immunoprecipitation and purification of biotinylated biomolecules. Streptavidin Mag Sepharose(R) utilizes the strong interaction between biotin and streptavidin ligand, which is immobilized on magnetic beads. Magnetic beads simplify sample handling in small-scale purifications.The beads are available in two pack sizes: 2 x 1 mL 10% medium slurry and 5 x 1 mL 10% medium slurry. A milliliter of 10% medium slurry is the same as 100 muL sedimented medium and it is sufficient for 20 purification runs according to the recommended immunoprecipitation protocol.
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    Structured Review

    Millipore streptavidin agarose beads
    Streptavidin Mag Sepharose
    Streptavidin Mag Sepharose R is a magnetic bead for simple and efficient enrichment of target proteins by immunoprecipitation and purification of biotinylated biomolecules Streptavidin Mag Sepharose R utilizes the strong interaction between biotin and streptavidin ligand which is immobilized on magnetic beads Magnetic beads simplify sample handling in small scale purifications
    https://www.bioz.com/result/streptavidin agarose beads/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    streptavidin agarose beads - by Bioz Stars, 2021-09
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    Images

    1) Product Images from "Role of IGF-1R in ameliorating apoptosis of GNE deficient cells"

    Article Title: Role of IGF-1R in ameliorating apoptosis of GNE deficient cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-25510-9

    Determination of IGF-1R sialylation levels using Lectin affinity assay: ( A ) Sialylation status of IGF-1R: Equal amount of cell lysates from vector control cells were treated with 100 mU of C. perfringen s sialidase and untreated lysates from GNE knockdown cells were separated on 8% SDS-PAGE followed by immunoblotting with anti-α IGF-1R. ( B ) SNA Lectin affinity assay: Sialylated IGF-1R from GNE knockdown cells and scrambled shRNA vector control cell was pulled down using biotin labelled SNA/streptavidin-coupled agarose and immunoblotted with anti-α IGF-1R. ( C ) Sialylated IGF-1R complexes from GNE mutant and pcDNA3 transfected vector control cells were pulled down using biotin labelled SNA/streptavidin-coupled agarose and immunoblotted with anti-α IGF-1R. Sialylated IGF-1R complexes from 100 mU neuraminidase treated vector control cells that were pulled down using biotin labelled SNA/streptavidin-coupled agarose served as negative control in this experiment. Equal amount of protein was used as input lysates. ( D – F ) are showing representative densitometry graphs of ( A – C ), respectively, normalized to vector control.
    Figure Legend Snippet: Determination of IGF-1R sialylation levels using Lectin affinity assay: ( A ) Sialylation status of IGF-1R: Equal amount of cell lysates from vector control cells were treated with 100 mU of C. perfringen s sialidase and untreated lysates from GNE knockdown cells were separated on 8% SDS-PAGE followed by immunoblotting with anti-α IGF-1R. ( B ) SNA Lectin affinity assay: Sialylated IGF-1R from GNE knockdown cells and scrambled shRNA vector control cell was pulled down using biotin labelled SNA/streptavidin-coupled agarose and immunoblotted with anti-α IGF-1R. ( C ) Sialylated IGF-1R complexes from GNE mutant and pcDNA3 transfected vector control cells were pulled down using biotin labelled SNA/streptavidin-coupled agarose and immunoblotted with anti-α IGF-1R. Sialylated IGF-1R complexes from 100 mU neuraminidase treated vector control cells that were pulled down using biotin labelled SNA/streptavidin-coupled agarose served as negative control in this experiment. Equal amount of protein was used as input lysates. ( D – F ) are showing representative densitometry graphs of ( A – C ), respectively, normalized to vector control.

    Techniques Used: Plasmid Preparation, SDS Page, shRNA, Mutagenesis, Transfection, Negative Control

    2) Product Images from "TP53TG1 enhances cisplatin sensitivity of non-small cell lung cancer cells through regulating miR-18a/PTEN axis"

    Article Title: TP53TG1 enhances cisplatin sensitivity of non-small cell lung cancer cells through regulating miR-18a/PTEN axis

    Journal: Cell & Bioscience

    doi: 10.1186/s13578-018-0221-7

    TP53TG1 inhibited miR-18a expression in NSCLC cells. a Sequence alignment of miR-18a with the putative binding sites within the wild-type regions of TP53TG1. b Subcellular fractionation assay was performed to identify the subcellular location of TP53TG1 with GAPDH and U6 as internal references. c , d The luciferase activity was detected in A549 cells transfected with TP53TG1-WT or TP53TG1-MUT and miR-con, miR-18a mimics, anti-miR-con or anti-miR-18a. e Biotin-labeled TP53TG1 RNA was obtained and added to cell lysates with Streptavidin agarose beads, followed by the detection of miR-18a enrichment by RNA pull-down assay. f RIP assay was performed to evaluate the endogenous binding between TP53TG1 and miR-18a in A549 cells using specific antibody against Ago2, followed by detection of RNA levels by qRT-PCR. g qRT-PCR assay of miR-18a expression in A549 cells transfected with si-TP53TG1#1 or pcDNA-TP53TG1 for 48 h. h qRT-PCR assay of miR-18a expression in 40 pairs of NSCLC samples. i qRT-PCR assay of miR-18a expression in DDP-sensitive NSCLC tissues and DDP-resistant NSCLC samples. j The correlation between TP53TG1 and miR-18a expression was detected in NSCLC samples. All experiments are repeated three times. * P
    Figure Legend Snippet: TP53TG1 inhibited miR-18a expression in NSCLC cells. a Sequence alignment of miR-18a with the putative binding sites within the wild-type regions of TP53TG1. b Subcellular fractionation assay was performed to identify the subcellular location of TP53TG1 with GAPDH and U6 as internal references. c , d The luciferase activity was detected in A549 cells transfected with TP53TG1-WT or TP53TG1-MUT and miR-con, miR-18a mimics, anti-miR-con or anti-miR-18a. e Biotin-labeled TP53TG1 RNA was obtained and added to cell lysates with Streptavidin agarose beads, followed by the detection of miR-18a enrichment by RNA pull-down assay. f RIP assay was performed to evaluate the endogenous binding between TP53TG1 and miR-18a in A549 cells using specific antibody against Ago2, followed by detection of RNA levels by qRT-PCR. g qRT-PCR assay of miR-18a expression in A549 cells transfected with si-TP53TG1#1 or pcDNA-TP53TG1 for 48 h. h qRT-PCR assay of miR-18a expression in 40 pairs of NSCLC samples. i qRT-PCR assay of miR-18a expression in DDP-sensitive NSCLC tissues and DDP-resistant NSCLC samples. j The correlation between TP53TG1 and miR-18a expression was detected in NSCLC samples. All experiments are repeated three times. * P

    Techniques Used: Expressing, Sequencing, Binding Assay, Fractionation, Luciferase, Activity Assay, Transfection, Labeling, Pull Down Assay, Quantitative RT-PCR

    3) Product Images from "Glycosylation of BRI2 on asparagine 170 is involved in its trafficking to the cell surface but not in its processing by furin or ADAM10"

    Article Title: Glycosylation of BRI2 on asparagine 170 is involved in its trafficking to the cell surface but not in its processing by furin or ADAM10

    Journal: Glycobiology

    doi: 10.1093/glycob/cwr097

    The rate of cell surface expression/appearance/transport of BRI2 is reduced in the absence of N-glycosylation. Wild-type mycBRI2 or mycBRI2/N170A was expressed in HEK293 cells. The newly synthesized proteins were labeled with 35 S in radiolabeling medium for 2 h (pulse) at 16°C and then were incubated in non-radiolabeling medium for 0′, 20′, 40′ and 60′ (chase). ( A ) Cell surface proteins were labeled with biotin and precipitated with streptavidin beads. Precipitated cell surface proteins were eluted from the beads and immunoprecipitated with 9B11 antibody against the myc epitope before electrophoresis and autoradiography. ( B ) Immunoprecipitation of cell extracts with 9B11, electrophoresis and autoradiography were performed to verify the expression levels of BRI2.
    Figure Legend Snippet: The rate of cell surface expression/appearance/transport of BRI2 is reduced in the absence of N-glycosylation. Wild-type mycBRI2 or mycBRI2/N170A was expressed in HEK293 cells. The newly synthesized proteins were labeled with 35 S in radiolabeling medium for 2 h (pulse) at 16°C and then were incubated in non-radiolabeling medium for 0′, 20′, 40′ and 60′ (chase). ( A ) Cell surface proteins were labeled with biotin and precipitated with streptavidin beads. Precipitated cell surface proteins were eluted from the beads and immunoprecipitated with 9B11 antibody against the myc epitope before electrophoresis and autoradiography. ( B ) Immunoprecipitation of cell extracts with 9B11, electrophoresis and autoradiography were performed to verify the expression levels of BRI2.

    Techniques Used: Expressing, Synthesized, Labeling, Radioactivity, Incubation, Immunoprecipitation, Electrophoresis, Autoradiography

    Inhibition of N-glycosylation of BRI2 inhibits its expression at the cell surface. Wild-type mycBRI2 or mycBRI2/N170A was expressed in HEK293 cells. Cell surface proteins were labeled with biotin (lanes 1 and 2) or were not labeled (lanes 3 and 4), as a control for biotinylation specificity. ( A ) Cell extracts were precipitated with streptavidin beads and analyzed with western blot against myc with 9B11 antibody. ( B ) Cell extracts were directly analyzed with western blot as a control for protein expression. The two immunoreactive bands of BRI2 proteins correspond to the furin-cleaved and the non-cleaved wild-type mycBRI2 or mycBRI2/N170A.
    Figure Legend Snippet: Inhibition of N-glycosylation of BRI2 inhibits its expression at the cell surface. Wild-type mycBRI2 or mycBRI2/N170A was expressed in HEK293 cells. Cell surface proteins were labeled with biotin (lanes 1 and 2) or were not labeled (lanes 3 and 4), as a control for biotinylation specificity. ( A ) Cell extracts were precipitated with streptavidin beads and analyzed with western blot against myc with 9B11 antibody. ( B ) Cell extracts were directly analyzed with western blot as a control for protein expression. The two immunoreactive bands of BRI2 proteins correspond to the furin-cleaved and the non-cleaved wild-type mycBRI2 or mycBRI2/N170A.

    Techniques Used: Inhibition, Expressing, Labeling, Western Blot

    4) Product Images from "Identification of novel proteins associated with yeast snR30 small nucleolar RNA"

    Article Title: Identification of novel proteins associated with yeast snR30 small nucleolar RNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr659

    Selective purification of S1-tagged snR30 snoRNP. One-step (A and B) or two-step (C) purifications were done with extracts prepared from the S1-SNR30 strain grown in YPD. ( A ) 3′-End labeling analysis of recovered RNAs. Labeled RNAs from the extract (T), Gar1-TAP IP and S1-snR30 purification were analyzed. Radiolabeled DNA markers (M) were run in parallel. S1-snR30, H/ACA snoRNAs, 5.8S and 5S rRNAs, and tRNAs are indicated. ( B ) Northern blots of RNAs recovered from one-step purification: extract (T), streptavidin flow through (S) and eluate (P). ( C ) Northern blots of RNAs recovered from two-step purification: extract (T), IgG flow through (S1), IgG eluate (P1) and streptavidin eluate (P2). For B and C, snoRNAs analyzed are indicated on the right of each panel.
    Figure Legend Snippet: Selective purification of S1-tagged snR30 snoRNP. One-step (A and B) or two-step (C) purifications were done with extracts prepared from the S1-SNR30 strain grown in YPD. ( A ) 3′-End labeling analysis of recovered RNAs. Labeled RNAs from the extract (T), Gar1-TAP IP and S1-snR30 purification were analyzed. Radiolabeled DNA markers (M) were run in parallel. S1-snR30, H/ACA snoRNAs, 5.8S and 5S rRNAs, and tRNAs are indicated. ( B ) Northern blots of RNAs recovered from one-step purification: extract (T), streptavidin flow through (S) and eluate (P). ( C ) Northern blots of RNAs recovered from two-step purification: extract (T), IgG flow through (S1), IgG eluate (P1) and streptavidin eluate (P2). For B and C, snoRNAs analyzed are indicated on the right of each panel.

    Techniques Used: Purification, End Labeling, Labeling, Northern Blot, Flow Cytometry

    Purified S1-snR30 contains H/ACA proteins and a number of additional proteins. Proteins present in the extract (T), IgG eluate (P1) and streptavidin eluate (P2) were separated by SDS–PAGE and subjected to immunoblotting to detect Cbf5 and Nhp2 ( A ). In ( B ), the Criterion XT gel was silver-stained. Proteins identified by MS are indicated on the right. The molecular weights of protein markers (lane 1) are indicated on the left in kDa.
    Figure Legend Snippet: Purified S1-snR30 contains H/ACA proteins and a number of additional proteins. Proteins present in the extract (T), IgG eluate (P1) and streptavidin eluate (P2) were separated by SDS–PAGE and subjected to immunoblotting to detect Cbf5 and Nhp2 ( A ). In ( B ), the Criterion XT gel was silver-stained. Proteins identified by MS are indicated on the right. The molecular weights of protein markers (lane 1) are indicated on the left in kDa.

    Techniques Used: Purification, SDS Page, Staining, Mass Spectrometry

    5) Product Images from "Assembly and Trafficking of Homomeric and Heteromeric Kainate Receptors with Impaired Ligand Binding Sites"

    Article Title: Assembly and Trafficking of Homomeric and Heteromeric Kainate Receptors with Impaired Ligand Binding Sites

    Journal: Neurochemical Research

    doi: 10.1007/s11064-018-2654-0

    Thr/Val and Ala/Leu mutations reduce cell surface expression of GluK2 and GluK3 subunit proteins. a Transiently transfected HEK293 cells expressing wild type (GluK2-WT, GluK3-WT) or mutant subunits with impaired ligand binding sites (GluK2-T659V, GluK2-A487L, GluK3-T661V; red) or conversion mutants with functional ligand binding sites (GluK2-S689N/N690S, GluK2-A487T, GluK3-N691S; blue) were surface biotinylated 24 h after transfection. Following the solubilisation of membranes (total; T), biotin-labelled (cell surface exposed; S) proteins were separated from non-biotinylated (intracellular; I) proteins using streptavidin coated beads. The different subunit populations were analysed using immunoblotting with a GluK2/3 specific antibody. The β-actin content of the samples was analysed to confirm that biotinylation only labelled cell surface exposed proteins in transfected HEK293 cells. Due to differences in total expression levels of various KAR subunit mutants (Fig. 4 ), immunodetection of bands were optimised for the quantitative comparison of biotinylated and non-biotinylated (intracellular) bands for each construct. b Bar diagrams compare the biotinylated (surface) fractions of subunits expressed as % of total. Mutants with impaired LBDs (GluK2-T659V, GluK2-A487L and GluK3-T661V; red bars) show reduced cell surface expression (biotinylation) than the corresponding WT subunits (white bars). In contrast, LBD conversion mutants of GluK2 and GluK3 that retain ligand binding (GluK2-S689N/N690S, GluK2-A487T, GluK3-N691S; blue bars) show no significant change in cell-surface biotinylation compare to WT. Data are mean ± SEM ( n = 3), * p
    Figure Legend Snippet: Thr/Val and Ala/Leu mutations reduce cell surface expression of GluK2 and GluK3 subunit proteins. a Transiently transfected HEK293 cells expressing wild type (GluK2-WT, GluK3-WT) or mutant subunits with impaired ligand binding sites (GluK2-T659V, GluK2-A487L, GluK3-T661V; red) or conversion mutants with functional ligand binding sites (GluK2-S689N/N690S, GluK2-A487T, GluK3-N691S; blue) were surface biotinylated 24 h after transfection. Following the solubilisation of membranes (total; T), biotin-labelled (cell surface exposed; S) proteins were separated from non-biotinylated (intracellular; I) proteins using streptavidin coated beads. The different subunit populations were analysed using immunoblotting with a GluK2/3 specific antibody. The β-actin content of the samples was analysed to confirm that biotinylation only labelled cell surface exposed proteins in transfected HEK293 cells. Due to differences in total expression levels of various KAR subunit mutants (Fig. 4 ), immunodetection of bands were optimised for the quantitative comparison of biotinylated and non-biotinylated (intracellular) bands for each construct. b Bar diagrams compare the biotinylated (surface) fractions of subunits expressed as % of total. Mutants with impaired LBDs (GluK2-T659V, GluK2-A487L and GluK3-T661V; red bars) show reduced cell surface expression (biotinylation) than the corresponding WT subunits (white bars). In contrast, LBD conversion mutants of GluK2 and GluK3 that retain ligand binding (GluK2-S689N/N690S, GluK2-A487T, GluK3-N691S; blue bars) show no significant change in cell-surface biotinylation compare to WT. Data are mean ± SEM ( n = 3), * p

    Techniques Used: Expressing, Transfection, Mutagenesis, Ligand Binding Assay, Functional Assay, Immunodetection, Construct

    GluK5 co-expression restores the cell surface trafficking of GluK2 with Thr/Val or Ala/Leu mutations. a Wild type (WT) and mutant GluK2 subunits (GluK2-T659V, GluK2-A487L, GluK2-S689N/N690S, GluK2-A487T) were co-expressed with the GluK5 WT subunit in transiently transfected HEK293 cells. Following surface biotinylation, labelled proteins (S) were isolated using streptavidin coated beads and analysed using immunoblotting using anti-GluK2/3 and anti-GluK5 antibodies. T is 10% of total protein. The β-actin immunostaining confirmed that biotinylation only labelled cell surface exposed proteins. b Comparison of surface (biotinylated) GluK2 and GluK5 subunits (% of total) indicates no significant differences between the various LBD mutant and WT subunits (Data are mean ± SEM, n = 3, * p
    Figure Legend Snippet: GluK5 co-expression restores the cell surface trafficking of GluK2 with Thr/Val or Ala/Leu mutations. a Wild type (WT) and mutant GluK2 subunits (GluK2-T659V, GluK2-A487L, GluK2-S689N/N690S, GluK2-A487T) were co-expressed with the GluK5 WT subunit in transiently transfected HEK293 cells. Following surface biotinylation, labelled proteins (S) were isolated using streptavidin coated beads and analysed using immunoblotting using anti-GluK2/3 and anti-GluK5 antibodies. T is 10% of total protein. The β-actin immunostaining confirmed that biotinylation only labelled cell surface exposed proteins. b Comparison of surface (biotinylated) GluK2 and GluK5 subunits (% of total) indicates no significant differences between the various LBD mutant and WT subunits (Data are mean ± SEM, n = 3, * p

    Techniques Used: Expressing, Mutagenesis, Transfection, Isolation, Immunostaining

    6) Product Images from "Functional analysis of a nonstop mutation in MITF gene identified in a patient with Waardenburg syndrome type 2"

    Article Title: Functional analysis of a nonstop mutation in MITF gene identified in a patient with Waardenburg syndrome type 2

    Journal: Journal of Human Genetics

    doi: 10.1038/jhg.2017.30

    DNA-binding capacity of WS-associated MITF. ( a ) The melanoma UACC903 cells transfected with 1μg WT, X420Y plasmids were incubated with or without biotinylated double-stranded oligonucleotides of the MITF-binding region at the TYR promoter. The DNA/protein complex was pulled down with streptavidin agarose beads. The precipitated proteins were separated on SDS–PAGE in equal amounts of whole-cell lysates and analyzed by immunoblotting using anti-Flag M2 antibody. Actin was used as an internal control. DNA precipitation demonstrates that X420Y mutant retains the DNA-binding activity. As a negative control, WT and X420Y MITF protein did not bind to mutated double-stranded oligonucleotides. There were significant differences in the expression of WT and X420Y MITF protein binding with the TYR promoter. ( b ) The graph illustrates the quantification of WT and X420Y MITF by densitometry of experiments. (*** P
    Figure Legend Snippet: DNA-binding capacity of WS-associated MITF. ( a ) The melanoma UACC903 cells transfected with 1μg WT, X420Y plasmids were incubated with or without biotinylated double-stranded oligonucleotides of the MITF-binding region at the TYR promoter. The DNA/protein complex was pulled down with streptavidin agarose beads. The precipitated proteins were separated on SDS–PAGE in equal amounts of whole-cell lysates and analyzed by immunoblotting using anti-Flag M2 antibody. Actin was used as an internal control. DNA precipitation demonstrates that X420Y mutant retains the DNA-binding activity. As a negative control, WT and X420Y MITF protein did not bind to mutated double-stranded oligonucleotides. There were significant differences in the expression of WT and X420Y MITF protein binding with the TYR promoter. ( b ) The graph illustrates the quantification of WT and X420Y MITF by densitometry of experiments. (*** P

    Techniques Used: Binding Assay, Transfection, Incubation, SDS Page, Mutagenesis, Activity Assay, Negative Control, Expressing, Protein Binding

    7) Product Images from "H3K27me1 is essential for MMP-9-dependent H3N-terminal tail proteolysis during osteoclastogenesis"

    Article Title: H3K27me1 is essential for MMP-9-dependent H3N-terminal tail proteolysis during osteoclastogenesis

    Journal: Epigenetics & Chromatin

    doi: 10.1186/s13072-018-0193-1

    MMP-9 binding to H3K27me1 nucleosomes. a Schematic depiction of the domain structure of MMP-9. b Peptide pull-down assays with biotinylated H3 1–21 and 21–44 peptides and recombinant His-MMP-9 were analyzed by Western blotting with anti-His antibody. H3 peptides were unmodified, K18ac or K27me1 as indicated. Lane 1 represents 10% of the input MMP-9. c Nucleosomes were reconstituted on a 207-bp 601 nucleosome positioning sequence using unmodified or H3K27me1 histone octamers and immobilized on streptavidin beads. His-MMP-9 was incubated with immobilized nucleosomes, and its binding to nucleosomes was analyzed by Western blotting with anti-His antibody. Lane 1 contains 10% of the input MMP-9. d H3K27me1 nucleosomes were incubated with immobilized MMP-9 N-terminal (amino acids 112–447) and C-terminal (amino acids 448–730) domains. After extensive washing, the binding of H3K27me1 nucleosomes to MMP-9 domains was determined by Western blotting with anti-H3 antibody. Input corresponds to 10% of H3K27me1 nucleosomes used in the binding reactions. e After incubation with H3K27me1 nucleosomes, the binding of MMP-9N-terminal subregions to nucleosomes was determined by Western blotting with anti-His antibody. Input lanes 1–3 represent 10% of MMP-9 fragments used in the binding reactions. f OCP-induced cells were transfected with FLAG-H3 wild type (WT) or K27R mutant (K27R), and mononucleosomes were prepared by micrococcal nuclease digestion as summarized in Figure S3. Mononucleosomes containing ectopic H3 were immunoprecipitated from total mononucleosomes with FLAG antibody and analyzed by Western blotting with anti-MMP-9 antibody. g ]. Simulations were run with non-methylated H3. For context, H3K27 is shown monomethylated. h Nucleosome binding assays were conducted as in e , except that His-MMP-9 amino acids 384–447 carrying E402A mutation were used
    Figure Legend Snippet: MMP-9 binding to H3K27me1 nucleosomes. a Schematic depiction of the domain structure of MMP-9. b Peptide pull-down assays with biotinylated H3 1–21 and 21–44 peptides and recombinant His-MMP-9 were analyzed by Western blotting with anti-His antibody. H3 peptides were unmodified, K18ac or K27me1 as indicated. Lane 1 represents 10% of the input MMP-9. c Nucleosomes were reconstituted on a 207-bp 601 nucleosome positioning sequence using unmodified or H3K27me1 histone octamers and immobilized on streptavidin beads. His-MMP-9 was incubated with immobilized nucleosomes, and its binding to nucleosomes was analyzed by Western blotting with anti-His antibody. Lane 1 contains 10% of the input MMP-9. d H3K27me1 nucleosomes were incubated with immobilized MMP-9 N-terminal (amino acids 112–447) and C-terminal (amino acids 448–730) domains. After extensive washing, the binding of H3K27me1 nucleosomes to MMP-9 domains was determined by Western blotting with anti-H3 antibody. Input corresponds to 10% of H3K27me1 nucleosomes used in the binding reactions. e After incubation with H3K27me1 nucleosomes, the binding of MMP-9N-terminal subregions to nucleosomes was determined by Western blotting with anti-His antibody. Input lanes 1–3 represent 10% of MMP-9 fragments used in the binding reactions. f OCP-induced cells were transfected with FLAG-H3 wild type (WT) or K27R mutant (K27R), and mononucleosomes were prepared by micrococcal nuclease digestion as summarized in Figure S3. Mononucleosomes containing ectopic H3 were immunoprecipitated from total mononucleosomes with FLAG antibody and analyzed by Western blotting with anti-MMP-9 antibody. g ]. Simulations were run with non-methylated H3. For context, H3K27 is shown monomethylated. h Nucleosome binding assays were conducted as in e , except that His-MMP-9 amino acids 384–447 carrying E402A mutation were used

    Techniques Used: Binding Assay, Recombinant, Western Blot, Sequencing, Incubation, Transfection, Mutagenesis, Immunoprecipitation, Methylation

    8) Product Images from "Assembly with the NR1 Subunit Is Required for Surface Expression of NR3A-Containing NMDA Receptors"

    Article Title: Assembly with the NR1 Subunit Is Required for Surface Expression of NR3A-Containing NMDA Receptors

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.21-04-01228.2001

    NR3A is present at the cell surface only when coexpressed with the NR1-1a subunit. A , HEK293T cells were transfected with different combinations of NMDA receptor subunits and incubated for 15 min with sulfo-NHS-biotin. After solubilization, biotinylated protein was recovered by streptavidin precipitation. The streptavidin fractions ( lanes labeled 2 ), representing the membrane proteins, and aliquots of the lysate before ( lanes labeled 1 ) and after ( lanes labeled 3 ) streptavidin precipitation were analyzed by immunoblotting using anti-NR1, anti-NR2A/B, anti-NR3A, and anti-calreticulin antibodies. An excess amount of protein was loaded in the lanes labeled 2 to ensure detection of any NR3A or calreticulin at the cell surface. The subunit combinations used for transfection are indicated above each blot, and the positions of molecular size markers in kilodaltons are shown on the left . A representative experiment is shown; n = 3. B, C , Surface localization of GFP-tagged NR3A. B, Left , Schematic drawing of expected transmembrane ( TM ) topology of NR3A-GFP is shown. Right , Protein immunoblots of HEK293T cells transfected with NR3A or NR3AGFP and probed with anti-NR3A antibody show an increase in NR3A molecular weight that corresponds to the molecular mass of GFP (27 kDa). No lower molecular weight bands were observed. C , Cells transfected with GFP-tagged NR3A alone or in combination with the other NMDA receptor subunits were immunostained in nonpermeabilizing (NP) conditions with anti-GFP antibody followed by a Texas Red-conjugated secondary antibody and imaged with filters for GFP and Texas Red. All four panels show raw superimposed confocal images combining NP anti-GFP antibody staining ( red ) and native GFP fluorescence from NR3A-GFP ( green ). Yellow corresponds to the overlap of GFP immunostaining and GFP fluorescence and reflects NR3A-GFP expressed at the cell surface. Because the intensity of red immunostaining was brighter than was green GFP fluorescence, regions of overlapping can appear red-yellow . When expressed alone, NR3A-GFP exhibits a perinuclear and reticular fluorescence pattern, and no surface staining is observed. Cotransfection of NR1-1a/NR2A leads to the appearance of patches of fluorescence at the plasma membrane. Scale bar, 10 μm.
    Figure Legend Snippet: NR3A is present at the cell surface only when coexpressed with the NR1-1a subunit. A , HEK293T cells were transfected with different combinations of NMDA receptor subunits and incubated for 15 min with sulfo-NHS-biotin. After solubilization, biotinylated protein was recovered by streptavidin precipitation. The streptavidin fractions ( lanes labeled 2 ), representing the membrane proteins, and aliquots of the lysate before ( lanes labeled 1 ) and after ( lanes labeled 3 ) streptavidin precipitation were analyzed by immunoblotting using anti-NR1, anti-NR2A/B, anti-NR3A, and anti-calreticulin antibodies. An excess amount of protein was loaded in the lanes labeled 2 to ensure detection of any NR3A or calreticulin at the cell surface. The subunit combinations used for transfection are indicated above each blot, and the positions of molecular size markers in kilodaltons are shown on the left . A representative experiment is shown; n = 3. B, C , Surface localization of GFP-tagged NR3A. B, Left , Schematic drawing of expected transmembrane ( TM ) topology of NR3A-GFP is shown. Right , Protein immunoblots of HEK293T cells transfected with NR3A or NR3AGFP and probed with anti-NR3A antibody show an increase in NR3A molecular weight that corresponds to the molecular mass of GFP (27 kDa). No lower molecular weight bands were observed. C , Cells transfected with GFP-tagged NR3A alone or in combination with the other NMDA receptor subunits were immunostained in nonpermeabilizing (NP) conditions with anti-GFP antibody followed by a Texas Red-conjugated secondary antibody and imaged with filters for GFP and Texas Red. All four panels show raw superimposed confocal images combining NP anti-GFP antibody staining ( red ) and native GFP fluorescence from NR3A-GFP ( green ). Yellow corresponds to the overlap of GFP immunostaining and GFP fluorescence and reflects NR3A-GFP expressed at the cell surface. Because the intensity of red immunostaining was brighter than was green GFP fluorescence, regions of overlapping can appear red-yellow . When expressed alone, NR3A-GFP exhibits a perinuclear and reticular fluorescence pattern, and no surface staining is observed. Cotransfection of NR1-1a/NR2A leads to the appearance of patches of fluorescence at the plasma membrane. Scale bar, 10 μm.

    Techniques Used: Transfection, Incubation, Labeling, Western Blot, Molecular Weight, Staining, Fluorescence, Immunostaining, Cotransfection

    9) Product Images from "H3K27me1 is essential for MMP-9-dependent H3N-terminal tail proteolysis during osteoclastogenesis"

    Article Title: H3K27me1 is essential for MMP-9-dependent H3N-terminal tail proteolysis during osteoclastogenesis

    Journal: Epigenetics & Chromatin

    doi: 10.1186/s13072-018-0193-1

    MMP-9 binding to H3K27me1 nucleosomes. a Schematic depiction of the domain structure of MMP-9. b Peptide pull-down assays with biotinylated H3 1–21 and 21–44 peptides and recombinant His-MMP-9 were analyzed by Western blotting with anti-His antibody. H3 peptides were unmodified, K18ac or K27me1 as indicated. Lane 1 represents 10% of the input MMP-9. c Nucleosomes were reconstituted on a 207-bp 601 nucleosome positioning sequence using unmodified or H3K27me1 histone octamers and immobilized on streptavidin beads. His-MMP-9 was incubated with immobilized nucleosomes, and its binding to nucleosomes was analyzed by Western blotting with anti-His antibody. Lane 1 contains 10% of the input MMP-9. d H3K27me1 nucleosomes were incubated with immobilized MMP-9 N-terminal (amino acids 112–447) and C-terminal (amino acids 448–730) domains. After extensive washing, the binding of H3K27me1 nucleosomes to MMP-9 domains was determined by Western blotting with anti-H3 antibody. Input corresponds to 10% of H3K27me1 nucleosomes used in the binding reactions. e After incubation with H3K27me1 nucleosomes, the binding of MMP-9N-terminal subregions to nucleosomes was determined by Western blotting with anti-His antibody. Input lanes 1–3 represent 10% of MMP-9 fragments used in the binding reactions. f OCP-induced cells were transfected with FLAG-H3 wild type (WT) or K27R mutant (K27R), and mononucleosomes were prepared by micrococcal nuclease digestion as summarized in Figure S3. Mononucleosomes containing ectopic H3 were immunoprecipitated from total mononucleosomes with FLAG antibody and analyzed by Western blotting with anti-MMP-9 antibody. g Model of the MMP-9-H3K27me1 interaction. PDB entries 4h3x (mMMP-9) and 3avr (H3.1) were used in docking simulations using the program Cluspro 2.0 [ 28 – 30 ]. Simulations were run with non-methylated H3. For context, H3K27 is shown monomethylated. h Nucleosome binding assays were conducted as in e , except that His-MMP-9 amino acids 384–447 carrying E402A mutation were used
    Figure Legend Snippet: MMP-9 binding to H3K27me1 nucleosomes. a Schematic depiction of the domain structure of MMP-9. b Peptide pull-down assays with biotinylated H3 1–21 and 21–44 peptides and recombinant His-MMP-9 were analyzed by Western blotting with anti-His antibody. H3 peptides were unmodified, K18ac or K27me1 as indicated. Lane 1 represents 10% of the input MMP-9. c Nucleosomes were reconstituted on a 207-bp 601 nucleosome positioning sequence using unmodified or H3K27me1 histone octamers and immobilized on streptavidin beads. His-MMP-9 was incubated with immobilized nucleosomes, and its binding to nucleosomes was analyzed by Western blotting with anti-His antibody. Lane 1 contains 10% of the input MMP-9. d H3K27me1 nucleosomes were incubated with immobilized MMP-9 N-terminal (amino acids 112–447) and C-terminal (amino acids 448–730) domains. After extensive washing, the binding of H3K27me1 nucleosomes to MMP-9 domains was determined by Western blotting with anti-H3 antibody. Input corresponds to 10% of H3K27me1 nucleosomes used in the binding reactions. e After incubation with H3K27me1 nucleosomes, the binding of MMP-9N-terminal subregions to nucleosomes was determined by Western blotting with anti-His antibody. Input lanes 1–3 represent 10% of MMP-9 fragments used in the binding reactions. f OCP-induced cells were transfected with FLAG-H3 wild type (WT) or K27R mutant (K27R), and mononucleosomes were prepared by micrococcal nuclease digestion as summarized in Figure S3. Mononucleosomes containing ectopic H3 were immunoprecipitated from total mononucleosomes with FLAG antibody and analyzed by Western blotting with anti-MMP-9 antibody. g Model of the MMP-9-H3K27me1 interaction. PDB entries 4h3x (mMMP-9) and 3avr (H3.1) were used in docking simulations using the program Cluspro 2.0 [ 28 – 30 ]. Simulations were run with non-methylated H3. For context, H3K27 is shown monomethylated. h Nucleosome binding assays were conducted as in e , except that His-MMP-9 amino acids 384–447 carrying E402A mutation were used

    Techniques Used: Binding Assay, Recombinant, Western Blot, Sequencing, Incubation, Transfection, Mutagenesis, Immunoprecipitation, Methylation

    10) Product Images from "Activated AMPK inhibits PPAR-? and PPAR-? transcriptional activity in hepatoma cells"

    Article Title: Activated AMPK inhibits PPAR-? and PPAR-? transcriptional activity in hepatoma cells

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    doi: 10.1152/ajpgi.00432.2010

    H4IIEC3 cells were transfected with PPAR-α and treated with the AMPK activators for 24 h as labeled. Lysates were then incubated with biotinylated double-stranded PPRE oligonucleotides overnight, before using streptavidin beads to pull down the
    Figure Legend Snippet: H4IIEC3 cells were transfected with PPAR-α and treated with the AMPK activators for 24 h as labeled. Lysates were then incubated with biotinylated double-stranded PPRE oligonucleotides overnight, before using streptavidin beads to pull down the

    Techniques Used: Transfection, Labeling, Incubation

    11) Product Images from "RhoA S-nitrosylation as a regulatory mechanism influencing endothelial barrier function in response to G+-bacterial toxins"

    Article Title: RhoA S-nitrosylation as a regulatory mechanism influencing endothelial barrier function in response to G+-bacterial toxins

    Journal: Biochemical pharmacology

    doi: 10.1016/j.bcp.2016.12.014

    RhoA is a substrate for S-nitrosylation. (A) HLMVECs were treated with either vehicle or Cys-NO (100 μM) for 30 min, and the S-nitrosylation of proteins was determined by the biotin-switch assay in the presence of ascorbate and trace levels of copper. Biotinylated proteins were concentrated using streptavidin–agarose beads, and immunoblotted for RhoA (SNO-RhoA, top panel) versus total RhoA in cell lysates (total RhoA, bottom panel). (B) HLMVECs were treated with or without Cys-NO (100 μM) for 30 min, and S-nitrosylated proteins were selected using organomercury columns followed by immunoblotting for RhoA (SNO-RhoA, top panel) versus total RhoA in cell lysates (total RhoA, bottom panel). The relative densitometry of SNO-RhoA vs total-RhoA is expressed as means ± S.E., * P
    Figure Legend Snippet: RhoA is a substrate for S-nitrosylation. (A) HLMVECs were treated with either vehicle or Cys-NO (100 μM) for 30 min, and the S-nitrosylation of proteins was determined by the biotin-switch assay in the presence of ascorbate and trace levels of copper. Biotinylated proteins were concentrated using streptavidin–agarose beads, and immunoblotted for RhoA (SNO-RhoA, top panel) versus total RhoA in cell lysates (total RhoA, bottom panel). (B) HLMVECs were treated with or without Cys-NO (100 μM) for 30 min, and S-nitrosylated proteins were selected using organomercury columns followed by immunoblotting for RhoA (SNO-RhoA, top panel) versus total RhoA in cell lysates (total RhoA, bottom panel). The relative densitometry of SNO-RhoA vs total-RhoA is expressed as means ± S.E., * P

    Techniques Used: Biotin Switch Assay

    Mutation of RhoA on C16, 20, 159S reduces the eNOS-dependent S-nitrosylation of RhoA and protects RhoA from the inhibitory effects of NO. (A) COS-7 cells transfected with WT or mutant C16, 20, 159S RhoA constructs were treated with or without Cys-NO (100 μM) for 30 min. Cells were then lysed, the biotin-switch assay performed and biotinylated proteins concentrated using streptavidin agarose. Total S-nitrosylated proteins were identified using an anti-biotin antibody (top panel) and S-nitrosylated RhoA using a RhoA antibody (lower panel). (B) HEK293-eNOS cells were transfected with RhoA WT or the RhoAC16, 20, 159S mutant, and the degree of S-nitrosylation of RhoA was determined using the biotin-switch assay and immunoblotted for RhoA (SNO-RhoA, top panel) versus total RhoA in cell lysates (total RhoA, bottom panel). (C) COS-7 cells were transfected with WT or mutant C16, 20, 159S RhoA and exposed to the indicated concentrations of Cys-NO for 30 min. Cells were then lysed and RhoA activity determined using the G-LISA RhoA activation assay. Data are expressed as means ± S.E., * P
    Figure Legend Snippet: Mutation of RhoA on C16, 20, 159S reduces the eNOS-dependent S-nitrosylation of RhoA and protects RhoA from the inhibitory effects of NO. (A) COS-7 cells transfected with WT or mutant C16, 20, 159S RhoA constructs were treated with or without Cys-NO (100 μM) for 30 min. Cells were then lysed, the biotin-switch assay performed and biotinylated proteins concentrated using streptavidin agarose. Total S-nitrosylated proteins were identified using an anti-biotin antibody (top panel) and S-nitrosylated RhoA using a RhoA antibody (lower panel). (B) HEK293-eNOS cells were transfected with RhoA WT or the RhoAC16, 20, 159S mutant, and the degree of S-nitrosylation of RhoA was determined using the biotin-switch assay and immunoblotted for RhoA (SNO-RhoA, top panel) versus total RhoA in cell lysates (total RhoA, bottom panel). (C) COS-7 cells were transfected with WT or mutant C16, 20, 159S RhoA and exposed to the indicated concentrations of Cys-NO for 30 min. Cells were then lysed and RhoA activity determined using the G-LISA RhoA activation assay. Data are expressed as means ± S.E., * P

    Techniques Used: Mutagenesis, Transfection, Construct, Biotin Switch Assay, Activity Assay, Activation Assay

    12) Product Images from "Comprehensive Mutational Analysis of the Moloney Murine Leukemia Virus Envelope Protein"

    Article Title: Comprehensive Mutational Analysis of the Moloney Murine Leukemia Virus Envelope Protein

    Journal: Journal of Virology

    doi: 10.1128/JVI.75.23.11851-11862.2001

    Overview of genetic footprinting. (A) Selection and analysis. A pool of single insertion variants of a gene is subjected to a selection for gene function. The resulting pre- and postselection pools are analyzed by a PCR approach that generates a uniquely sized fragment for each mutant, reflecting the position of the insertion in the gene sequence. Polyacrylamide gel electrophoresis of the fragments generates a footprint, corresponding to an essential region of the gene in which insertions decrease function. Symbols: B→, biotinylated primer; ←✽, radiolabeled primer; SA, streptavidin-agarose. (B) Sequence of the insertion site. Duplication of the target sequence (XXXXX) results in the net in-frame insertion of 15 nucleotides. The identity of the inserted amino acids depends on the relative frame of the insert within the target gene sequence.
    Figure Legend Snippet: Overview of genetic footprinting. (A) Selection and analysis. A pool of single insertion variants of a gene is subjected to a selection for gene function. The resulting pre- and postselection pools are analyzed by a PCR approach that generates a uniquely sized fragment for each mutant, reflecting the position of the insertion in the gene sequence. Polyacrylamide gel electrophoresis of the fragments generates a footprint, corresponding to an essential region of the gene in which insertions decrease function. Symbols: B→, biotinylated primer; ←✽, radiolabeled primer; SA, streptavidin-agarose. (B) Sequence of the insertion site. Duplication of the target sequence (XXXXX) results in the net in-frame insertion of 15 nucleotides. The identity of the inserted amino acids depends on the relative frame of the insert within the target gene sequence.

    Techniques Used: Footprinting, Selection, Polymerase Chain Reaction, Mutagenesis, Sequencing, Polyacrylamide Gel Electrophoresis

    13) Product Images from "TRPM4 and TRPM5 Channels Share Crucial Amino Acid Residues for Ca2+ Sensitivity but Not Significance of PI(4,5)P2"

    Article Title: TRPM4 and TRPM5 Channels Share Crucial Amino Acid Residues for Ca2+ Sensitivity but Not Significance of PI(4,5)P2

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms20082012

    The amino acid residues of TRPM5 corresponding to those forming the Ca 2+ -binding site of TRPM4 were also necessary for the normal Ca 2+ -sensitivity of rTRPM5. ( A ) Numbers of corresponding amino acids of rTRPM5 are labeled in the illustration of the Ca 2+ -binding site of hTRPM4. ( B ) CRCs for the effect of Ca 2+ on the current densities mediated by WT rTRPM5 (black circles), D802A (red squares), N805D (dark red triangles), Q782A (green diamonds), D808E (dark yellow circles), E779D (dark yellow horizontal bars), E779Q (purple open diamonds), N805A (light green asterisks), D808N (brown crosses) and the empty vector (gray circles) ( n = 3 or 4 each). ( C ) A result of surface biotinylation assay. The proteins of rTRPM5 expressed in the plasma membrane were biotinylated and precipitated with streptavidin beads (Surface). Non-precipitated fractions contain intracellular proteins (Intracellular). ( D ) Signal ratios of the surface rTRPM5 to the intracellular rTRPM5 as an indication of the surface expression levels of rTRPM5. The mutants showed similar surface expression levels to WT rTRPM5 ( n = 3).
    Figure Legend Snippet: The amino acid residues of TRPM5 corresponding to those forming the Ca 2+ -binding site of TRPM4 were also necessary for the normal Ca 2+ -sensitivity of rTRPM5. ( A ) Numbers of corresponding amino acids of rTRPM5 are labeled in the illustration of the Ca 2+ -binding site of hTRPM4. ( B ) CRCs for the effect of Ca 2+ on the current densities mediated by WT rTRPM5 (black circles), D802A (red squares), N805D (dark red triangles), Q782A (green diamonds), D808E (dark yellow circles), E779D (dark yellow horizontal bars), E779Q (purple open diamonds), N805A (light green asterisks), D808N (brown crosses) and the empty vector (gray circles) ( n = 3 or 4 each). ( C ) A result of surface biotinylation assay. The proteins of rTRPM5 expressed in the plasma membrane were biotinylated and precipitated with streptavidin beads (Surface). Non-precipitated fractions contain intracellular proteins (Intracellular). ( D ) Signal ratios of the surface rTRPM5 to the intracellular rTRPM5 as an indication of the surface expression levels of rTRPM5. The mutants showed similar surface expression levels to WT rTRPM5 ( n = 3).

    Techniques Used: Binding Assay, Labeling, Plasmid Preparation, Surface Biotinylation Assay, Expressing

    The mutations of the negatively-charged amino acid residues near and in the TRP domain reduced the Ca 2+ -sensitivity of rTRPM5. ( A ) Positions of the acidic amino acid residues (Asp 987 (D987) and Glu 1000 (E1000)) which were mutated. (Upper) The predicted membrane topology of rTRPM5 and the position of TRP domain in the C-terminal tail. (Lower) An alignment of amino acid sequences around the TRP domain of rat TRPM (rTRPM) channels. The aspartate and the glutamate of rTRPM5 are conserved in rTRPM4 (GenBank #NP_001129701.1), rTRPM2 (NP_001011559.1) and rTRPM8 (NP_599198.2) but not in rTRPM1 (NP_001032823.1), rTRPM3 (NP_001178491.1), rTRPM6 (XP_006223728.1) nor rTRPM7 (NP_446157.2). ( B ) CRCs for the effect of Ca 2+ on the rTRPM5 current densities at +100 mV. EC 50 for Ca 2+ of WT rTRPM5 and D987N mutant were 3.25 and 196 µM, respectively. EC 50 for Ca 2+ of E1000Q was unable to be estimated but it seems to be at least more than 100 µM. ( C ) A result of a biotinylation assay in order to evaluate the surface expression level of rTRPM5. The proteins of rTRPM5 and EGFP were detected by Western blotting. The biotinylated rTRPM5 (biotin+, Surface) is indicative of rTRPM5 which was expressed in the plasma membrane. Expression levels of EGFP in the intracellular fractions indicate the transfection efficiencies, and no signal of EGFP in the surface fractions indicates that intracellular proteins were not biotinylated. In order to monitor nonspecific binding, lysates of cells which were not treated with biotin were also subjected to precipitations with the streptavidin-agarose beads (biotin−). ( D ) Signal ratios of the surface rTRPM5 to the intracellular rTRPM5 as an indication of the surface expression levels of rTRPM5. The expression levels of WT rTRPM5 and mutants did not differ significantly ( n = 3).
    Figure Legend Snippet: The mutations of the negatively-charged amino acid residues near and in the TRP domain reduced the Ca 2+ -sensitivity of rTRPM5. ( A ) Positions of the acidic amino acid residues (Asp 987 (D987) and Glu 1000 (E1000)) which were mutated. (Upper) The predicted membrane topology of rTRPM5 and the position of TRP domain in the C-terminal tail. (Lower) An alignment of amino acid sequences around the TRP domain of rat TRPM (rTRPM) channels. The aspartate and the glutamate of rTRPM5 are conserved in rTRPM4 (GenBank #NP_001129701.1), rTRPM2 (NP_001011559.1) and rTRPM8 (NP_599198.2) but not in rTRPM1 (NP_001032823.1), rTRPM3 (NP_001178491.1), rTRPM6 (XP_006223728.1) nor rTRPM7 (NP_446157.2). ( B ) CRCs for the effect of Ca 2+ on the rTRPM5 current densities at +100 mV. EC 50 for Ca 2+ of WT rTRPM5 and D987N mutant were 3.25 and 196 µM, respectively. EC 50 for Ca 2+ of E1000Q was unable to be estimated but it seems to be at least more than 100 µM. ( C ) A result of a biotinylation assay in order to evaluate the surface expression level of rTRPM5. The proteins of rTRPM5 and EGFP were detected by Western blotting. The biotinylated rTRPM5 (biotin+, Surface) is indicative of rTRPM5 which was expressed in the plasma membrane. Expression levels of EGFP in the intracellular fractions indicate the transfection efficiencies, and no signal of EGFP in the surface fractions indicates that intracellular proteins were not biotinylated. In order to monitor nonspecific binding, lysates of cells which were not treated with biotin were also subjected to precipitations with the streptavidin-agarose beads (biotin−). ( D ) Signal ratios of the surface rTRPM5 to the intracellular rTRPM5 as an indication of the surface expression levels of rTRPM5. The expression levels of WT rTRPM5 and mutants did not differ significantly ( n = 3).

    Techniques Used: Mutagenesis, Cell Surface Biotinylation Assay, Expressing, Western Blot, Transfection, Binding Assay

    14) Product Images from "The Mycobacterium tuberculosis Secreted Protein Rv0203 Transfers Heme to Membrane Proteins MmpL3 and MmpL11"

    Article Title: The Mycobacterium tuberculosis Secreted Protein Rv0203 Transfers Heme to Membrane Proteins MmpL3 and MmpL11

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.453076

    Heme transfer experiments. A, SDS-PAGE of the pulldown fractions for transfer analyses between Rv0203 and MmpL3-E1 and Rv0203 and MmpL11-E1. Lane 1, streptavidin beads after reaction with MmpL3-E1; lane 2, MmpL3-E1 flow-through; lane 3, streptavidin beads
    Figure Legend Snippet: Heme transfer experiments. A, SDS-PAGE of the pulldown fractions for transfer analyses between Rv0203 and MmpL3-E1 and Rv0203 and MmpL11-E1. Lane 1, streptavidin beads after reaction with MmpL3-E1; lane 2, MmpL3-E1 flow-through; lane 3, streptavidin beads

    Techniques Used: SDS Page, Flow Cytometry

    15) Product Images from "Receptor-Like Protein Tyrosine Phosphatase ? Homodimerizes on the Cell Surface"

    Article Title: Receptor-Like Protein Tyrosine Phosphatase ? Homodimerizes on the Cell Surface

    Journal: Molecular and Cellular Biology

    doi:

    RPTPα appears to exist on the cell surface predominantly as homodimers. (A) 293 cells transiently expressing FL were surface biotinylated or not biotinylated. Cells were lysed, and biotinylated proteins were precipitated with streptavidin beads and separated by SDS-PAGE, and the biotinylated FL was detected by immunoblotting using MAb 12CA5 followed by 125 I-labeled sheep anti-mouse IgG F(ab′) 2 . The blot was quantified using a PhosphorImager as described in Materials and Methods. Top and bottom panels are the immunoblot and quantification, respectively. The loading for each of the lanes was standardized using an equivalent amount of whole-cell lysate. Lane 7 is a longer exposure of lane 6. + or − biotin, labeled or not labeled with biotin; WCL, total whole-cell lysate; SN, whole-cell lysate supernatant after streptavidin bead precipitation; P, streptavidin precipitate. (B) BS 3 cross-linking was performed on 293 cells transiently transfected with either FL or FL.137C. Whole-cell lysates of cross-linked cells were separated by SDS-PAGE and probed using MAb 12CA5 followed by 125 I-labeled sheep anti-mouse IgG F(ab′) 2 ).
    Figure Legend Snippet: RPTPα appears to exist on the cell surface predominantly as homodimers. (A) 293 cells transiently expressing FL were surface biotinylated or not biotinylated. Cells were lysed, and biotinylated proteins were precipitated with streptavidin beads and separated by SDS-PAGE, and the biotinylated FL was detected by immunoblotting using MAb 12CA5 followed by 125 I-labeled sheep anti-mouse IgG F(ab′) 2 . The blot was quantified using a PhosphorImager as described in Materials and Methods. Top and bottom panels are the immunoblot and quantification, respectively. The loading for each of the lanes was standardized using an equivalent amount of whole-cell lysate. Lane 7 is a longer exposure of lane 6. + or − biotin, labeled or not labeled with biotin; WCL, total whole-cell lysate; SN, whole-cell lysate supernatant after streptavidin bead precipitation; P, streptavidin precipitate. (B) BS 3 cross-linking was performed on 293 cells transiently transfected with either FL or FL.137C. Whole-cell lysates of cross-linked cells were separated by SDS-PAGE and probed using MAb 12CA5 followed by 125 I-labeled sheep anti-mouse IgG F(ab′) 2 ).

    Techniques Used: Expressing, SDS Page, Labeling, Transfection

    Mutations in the wedge diminish but do not abolish RPTPα oligomerization. (A) A schematic of RPTPα wedge mutant constructs, including point mutants FL.P210L.P211L and FL.E234A and deletion mutant Δ224-235. (B) For the top panel, transiently transfected 293 cells were biotinylated. Whole-cell lysates were immunoprecipitated with MAb 12CA5 to isolate the total RPTPα proteins, which were then subjected to SDS-PAGE and probed with 125 I-labeled streptavidin to determine the levels of surface-expressed RPTPα protein. For the bottom panel, transiently transfected 293 cells were cross-linked with BS 3 . Whole-cell lysates were subjected to immunoblotting analysis with MAb 12CA5 followed by 125 I-labeled sheep anti-mouse IgG F(ab′) 2 to determine the levels of RPTPα dimers. The bands representing FL.P210L.P211L dimers and Δ224-235 dimers are faint but detectable by PhosphorImager analysis. Biotinylation and cross-linking were done on parallel dishes from the same transfection. All the constructs were expressed to a similar level on the cell surface. (C) Quantification of dimerization efficiency based on average of three replicates. The dimer/surface protein value is the ratio of the levels of RPTPα dimers over surface-expressed RPTPα, which were determined from the bottom and top portions of panel B, respectively, using a PhosphorImager. S, surface-expressed RPTPα (monomer).
    Figure Legend Snippet: Mutations in the wedge diminish but do not abolish RPTPα oligomerization. (A) A schematic of RPTPα wedge mutant constructs, including point mutants FL.P210L.P211L and FL.E234A and deletion mutant Δ224-235. (B) For the top panel, transiently transfected 293 cells were biotinylated. Whole-cell lysates were immunoprecipitated with MAb 12CA5 to isolate the total RPTPα proteins, which were then subjected to SDS-PAGE and probed with 125 I-labeled streptavidin to determine the levels of surface-expressed RPTPα protein. For the bottom panel, transiently transfected 293 cells were cross-linked with BS 3 . Whole-cell lysates were subjected to immunoblotting analysis with MAb 12CA5 followed by 125 I-labeled sheep anti-mouse IgG F(ab′) 2 to determine the levels of RPTPα dimers. The bands representing FL.P210L.P211L dimers and Δ224-235 dimers are faint but detectable by PhosphorImager analysis. Biotinylation and cross-linking were done on parallel dishes from the same transfection. All the constructs were expressed to a similar level on the cell surface. (C) Quantification of dimerization efficiency based on average of three replicates. The dimer/surface protein value is the ratio of the levels of RPTPα dimers over surface-expressed RPTPα, which were determined from the bottom and top portions of panel B, respectively, using a PhosphorImager. S, surface-expressed RPTPα (monomer).

    Techniques Used: Mutagenesis, Construct, Transfection, Immunoprecipitation, SDS Page, Labeling

    Deletion of D2 diminishes but does not abolish RPTPα oligomerization. (A) A schematic of the D2 deletion mutant construct. (B) 293 cells transiently expressing ΔD2 protein were cross-linked or not cross-linked with BS 3 . Shown are the results of an immunoblotting analysis with anti-HA tag MAb 12CA5 on whole-cell lysates using ECL detection. (C) Transiently transfected 293 cells were biotinylated. Whole-cell lysates were immunoprecipitated with MAb 12CA5 to isolate the total RPTPα proteins, which were then subjected to SDS-PAGE and probed with 125 I-labeled streptavidin to determine the levels of surface-expressed RPTPα protein. (D) Transiently transfected 293 cells were cross-linked with BS 3 . Whole-cell lysates were subjected to immunoblotting analysis using MAb 12CA5 followed by 125 I-labeled sheep anti-mouse IgG F(ab′) 2 to determine the levels of RPTPα dimers. Biotinylation (C) and cross-linking (D) were done on parallel dishes from the same transfection. Shown in panels C and D are images obtained via PhosphorImager analysis. S/M, surface-expressed monomeric proteins.
    Figure Legend Snippet: Deletion of D2 diminishes but does not abolish RPTPα oligomerization. (A) A schematic of the D2 deletion mutant construct. (B) 293 cells transiently expressing ΔD2 protein were cross-linked or not cross-linked with BS 3 . Shown are the results of an immunoblotting analysis with anti-HA tag MAb 12CA5 on whole-cell lysates using ECL detection. (C) Transiently transfected 293 cells were biotinylated. Whole-cell lysates were immunoprecipitated with MAb 12CA5 to isolate the total RPTPα proteins, which were then subjected to SDS-PAGE and probed with 125 I-labeled streptavidin to determine the levels of surface-expressed RPTPα protein. (D) Transiently transfected 293 cells were cross-linked with BS 3 . Whole-cell lysates were subjected to immunoblotting analysis using MAb 12CA5 followed by 125 I-labeled sheep anti-mouse IgG F(ab′) 2 to determine the levels of RPTPα dimers. Biotinylation (C) and cross-linking (D) were done on parallel dishes from the same transfection. Shown in panels C and D are images obtained via PhosphorImager analysis. S/M, surface-expressed monomeric proteins.

    Techniques Used: Mutagenesis, Construct, Expressing, Transfection, Immunoprecipitation, SDS Page, Labeling

    The ECD possesses relatively weak dimerization potential and is not required for the homodimerization of the full-length RPTPα. (A) A schematic of RPTPα constructs used in this figure. (B) Mock cross-linking and cross-linking on 293 cells transiently transfected with the construct ECD.GPI (lanes 1 to 4) or ephrin A1 (lanes 5 to 7). Shown are the results of immunoblotting analysis with MAb 12CA5 of whole-cell lysates using ECL detection. (C) In the left panel, transiently transfected 293 cells were biotinylated. Streptavidin-agarose beads were used to isolate the total biotinylated surface proteins, which were then subjected to immunoblotting analysis using MAb 12CA5 followed by 125 I-labeled sheep anti-mouse IgG F(ab′) 2 to determine the levels of surface-expressed RPTPα protein. In the right panel, transiently transfected 293 cells were cross-linked with BS 3 . Whole-cell lysates were subjected to immunoblotting analysis using MAb 12CA5 followed by 125 I-sheep anti-mouse IgG F(ab′) 2 to determine the levels of RPTPα dimers. n.s., nonspecific band. (D) Mock cross-linking and cross-linking on 293 cells transiently transfected with the construct ΔECD. Whole-cell lysates were subjected to immunoblotting analysis with anti-RPTPα serum 5478 using ECL detection.
    Figure Legend Snippet: The ECD possesses relatively weak dimerization potential and is not required for the homodimerization of the full-length RPTPα. (A) A schematic of RPTPα constructs used in this figure. (B) Mock cross-linking and cross-linking on 293 cells transiently transfected with the construct ECD.GPI (lanes 1 to 4) or ephrin A1 (lanes 5 to 7). Shown are the results of immunoblotting analysis with MAb 12CA5 of whole-cell lysates using ECL detection. (C) In the left panel, transiently transfected 293 cells were biotinylated. Streptavidin-agarose beads were used to isolate the total biotinylated surface proteins, which were then subjected to immunoblotting analysis using MAb 12CA5 followed by 125 I-labeled sheep anti-mouse IgG F(ab′) 2 to determine the levels of surface-expressed RPTPα protein. In the right panel, transiently transfected 293 cells were cross-linked with BS 3 . Whole-cell lysates were subjected to immunoblotting analysis using MAb 12CA5 followed by 125 I-sheep anti-mouse IgG F(ab′) 2 to determine the levels of RPTPα dimers. n.s., nonspecific band. (D) Mock cross-linking and cross-linking on 293 cells transiently transfected with the construct ΔECD. Whole-cell lysates were subjected to immunoblotting analysis with anti-RPTPα serum 5478 using ECL detection.

    Techniques Used: Construct, Transfection, Labeling

    ΔCyto homodimerizes on the cell surface with high efficiency. (A) A schematic of the construct ΔCyto lacking the entire cytoplasmic domain. (B) 293 cells transiently transfected with FL or ΔCyto were treated or not treated with tunicamycin at 200 ng/ml and subsequently cross-linked with BS 3 . Whole-cell lysates were subjected to immunoblotting analysis with MAb 12CA5 using ECL detection. (C) Transiently transfected 293 cells were biotinylated. Whole-cell lysates were precipitated with streptavidin beads to isolate the total RPTPα proteins, which were then subjected to SDS-PAGE and probed with 125 I-labeled streptavidin to determine the levels of surface-expressed RPTPα proteins. (D) Transiently transfected 293 cells were cross-linked with BS 3 . Whole-cell lysates were subjected to immunoblotting analysis using MAb 12CA5 followed by 125 I-labeled sheep anti-mouse IgG F(ab′) 2 to determine the levels of RPTPα dimers. Biotinylation (C) and cross-linking (D) were done on parallel dishes from the same transfection. Shown in panels C and D are images from PhosphorImager analysis. S/M, surface-expressed monomeric proteins. (E) Quantification of dimerization efficiency based on average of three replicates. The dimer/surface protein value is the ratio of the levels of RPTPα dimers over surface-expressed RPTPα, which were determined from panels C and D, respectively, using a PhosphorImager. n.s., nonspecific band.
    Figure Legend Snippet: ΔCyto homodimerizes on the cell surface with high efficiency. (A) A schematic of the construct ΔCyto lacking the entire cytoplasmic domain. (B) 293 cells transiently transfected with FL or ΔCyto were treated or not treated with tunicamycin at 200 ng/ml and subsequently cross-linked with BS 3 . Whole-cell lysates were subjected to immunoblotting analysis with MAb 12CA5 using ECL detection. (C) Transiently transfected 293 cells were biotinylated. Whole-cell lysates were precipitated with streptavidin beads to isolate the total RPTPα proteins, which were then subjected to SDS-PAGE and probed with 125 I-labeled streptavidin to determine the levels of surface-expressed RPTPα proteins. (D) Transiently transfected 293 cells were cross-linked with BS 3 . Whole-cell lysates were subjected to immunoblotting analysis using MAb 12CA5 followed by 125 I-labeled sheep anti-mouse IgG F(ab′) 2 to determine the levels of RPTPα dimers. Biotinylation (C) and cross-linking (D) were done on parallel dishes from the same transfection. Shown in panels C and D are images from PhosphorImager analysis. S/M, surface-expressed monomeric proteins. (E) Quantification of dimerization efficiency based on average of three replicates. The dimer/surface protein value is the ratio of the levels of RPTPα dimers over surface-expressed RPTPα, which were determined from panels C and D, respectively, using a PhosphorImager. n.s., nonspecific band.

    Techniques Used: Construct, Transfection, SDS Page, Labeling

    16) Product Images from "Identification of Plasmodium GAPDH epitopes for generation of antibodies that inhibit malaria infection"

    Article Title: Identification of Plasmodium GAPDH epitopes for generation of antibodies that inhibit malaria infection

    Journal: Life Science Alliance

    doi: 10.26508/lsa.201800111

    Three different mimotope peptides mimic different domains of the the P berghei GAPDH molecule. (A) The anti-P39 antibody specifically recognizes PbGAPDH in Western blots of P berghei sporozoite lysates. Ponceau stain and the anti-actin antibody were used as loading controls. “S”: lysates of sporozoites purified from infected A stephensi salivary glands; “M”: mock lysates obtained from uninfected A stephensi salivary glands. Anti-CSP antibody served as a positive control for the sporozoite lysate. A polyclonal anti-PbGAPDH antibody (red arrow) identifies a sporozoite GAPDH band with identical mobility to the anti-P39 band. (B) Antibodies against each of the mimotope peptides (P39, P61, and P52) recognize bands with identical mobility as PbGAPDH (red arrow), but not the pET tag protein (blue arrow). Each panel shows two lanes, the left containing the pET tag protein alone and the right the tagged recombinant PbGAPDH protein. The antibodies used to probe the blots are indicated at the bottom of each panel. The anti-tag and anti-KLH antibodies served as a positive and negative controls, respectively. The anti-mouse GAPDH antibody was used as a positive control for identification of the GAPDH protein. The position of the recombinant protein and tag proteins is indicated by arrows to the right. All data are representative of two independent experiments. (C) Each anti-peptide antibody specifically recognizes its own peptide. The biotinylated peptide indicated at the top of each panel was bound to the wells of streptavidin-coated ELISA plates and tested for binding by the antibodies denoted at the bottom of each panel. No evidence of cross-reaction was detected. All data are representative of two independent assays.
    Figure Legend Snippet: Three different mimotope peptides mimic different domains of the the P berghei GAPDH molecule. (A) The anti-P39 antibody specifically recognizes PbGAPDH in Western blots of P berghei sporozoite lysates. Ponceau stain and the anti-actin antibody were used as loading controls. “S”: lysates of sporozoites purified from infected A stephensi salivary glands; “M”: mock lysates obtained from uninfected A stephensi salivary glands. Anti-CSP antibody served as a positive control for the sporozoite lysate. A polyclonal anti-PbGAPDH antibody (red arrow) identifies a sporozoite GAPDH band with identical mobility to the anti-P39 band. (B) Antibodies against each of the mimotope peptides (P39, P61, and P52) recognize bands with identical mobility as PbGAPDH (red arrow), but not the pET tag protein (blue arrow). Each panel shows two lanes, the left containing the pET tag protein alone and the right the tagged recombinant PbGAPDH protein. The antibodies used to probe the blots are indicated at the bottom of each panel. The anti-tag and anti-KLH antibodies served as a positive and negative controls, respectively. The anti-mouse GAPDH antibody was used as a positive control for identification of the GAPDH protein. The position of the recombinant protein and tag proteins is indicated by arrows to the right. All data are representative of two independent experiments. (C) Each anti-peptide antibody specifically recognizes its own peptide. The biotinylated peptide indicated at the top of each panel was bound to the wells of streptavidin-coated ELISA plates and tested for binding by the antibodies denoted at the bottom of each panel. No evidence of cross-reaction was detected. All data are representative of two independent assays.

    Techniques Used: Western Blot, Staining, Purification, Infection, Positive Control, Positron Emission Tomography, Recombinant, Enzyme-linked Immunosorbent Assay, Binding Assay

    17) Product Images from "Vaccinia Virus A19 Protein Participates in the Transformation of Spherical Immature Particles to Barrel-Shaped Infectious Virions"

    Article Title: Vaccinia Virus A19 Protein Participates in the Transformation of Spherical Immature Particles to Barrel-Shaped Infectious Virions

    Journal: Journal of Virology

    doi: 10.1128/JVI.01258-13

    A19 phosphorylation. (A) Western blot analysis. BS-C-1 cells were infected with vFS-A11 and vFS-A19 in the presence of 100 μCi/ml 32 P i . After 18 h, the cells were lysed, and the soluble extract was bound to streptavidin-agarose beads. Bound proteins
    Figure Legend Snippet: A19 phosphorylation. (A) Western blot analysis. BS-C-1 cells were infected with vFS-A11 and vFS-A19 in the presence of 100 μCi/ml 32 P i . After 18 h, the cells were lysed, and the soluble extract was bound to streptavidin-agarose beads. Bound proteins

    Techniques Used: Western Blot, Infection

    Expression and characterization of the A19 protein. (A) Schematic representation of the DNA construct used for generating recombinant vFS-A19. The FLAG- and streptavidin-binding peptide tag fused at the N terminus of the A19 ORF (FS) is indicated. The
    Figure Legend Snippet: Expression and characterization of the A19 protein. (A) Schematic representation of the DNA construct used for generating recombinant vFS-A19. The FLAG- and streptavidin-binding peptide tag fused at the N terminus of the A19 ORF (FS) is indicated. The

    Techniques Used: Expressing, Construct, Recombinant, Binding Assay

    18) Product Images from "Bifunctional antisense oligonucleotides provide a trans-acting splicing enhancer that stimulates SMN2 gene expression in patient fibroblasts"

    Article Title: Bifunctional antisense oligonucleotides provide a trans-acting splicing enhancer that stimulates SMN2 gene expression in patient fibroblasts

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

    doi: 10.1073/pnas.0633863100

    Recruitment of SF2/ASF to SMN2 exon 7 by the 5′GGA oligonucleotide. Biotinylated RNA ( SMN2 exon 7 or β-globin, as indicated) was bound to streptavidin beads and incubated in nuclear extract. The proteins associated with the RNA were separated by SDS/PAGE, and SF2/ASF was detected by Western blotting. Lanes “+GGA” indicate that the RNA was incubated with the GGA oligonucleotide before addition to the beads.
    Figure Legend Snippet: Recruitment of SF2/ASF to SMN2 exon 7 by the 5′GGA oligonucleotide. Biotinylated RNA ( SMN2 exon 7 or β-globin, as indicated) was bound to streptavidin beads and incubated in nuclear extract. The proteins associated with the RNA were separated by SDS/PAGE, and SF2/ASF was detected by Western blotting. Lanes “+GGA” indicate that the RNA was incubated with the GGA oligonucleotide before addition to the beads.

    Techniques Used: Incubation, SDS Page, Western Blot

    19) Product Images from "LncRNA DANCR upregulates PI3K/AKT signaling through activating serine phosphorylation of RXRA"

    Article Title: LncRNA DANCR upregulates PI3K/AKT signaling through activating serine phosphorylation of RXRA

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-1220-7

    DANCR interacts with RXRA in TNBC cells. a Schematic diagram of putative RXRA binding sites in DANCR . b RIP-qPCR assay of the association of RXRA with DANCR in MDA-MB-231 and MDA-MB-468 cells. c Re-expression of shRNA-resistant DANCR wild type and RXRA-binding mutant types. d RIP-qPCR assay of effects of re-expression of shRNA-resistant DANCR wild type or mutant types on RXRA binding. e Biotinylated DANCR was incubated with nuclear extracts (MDA-MB-231 and MDA-MB-468 cells), targeted with streptavidin beads, and binding proteins were resolved in a gel. Western blotting assay of the specific binding of RXRA and DANCR . f , g RNAs corresponding to fragments in different regions of DANCR were treated as in ( e ), and binding RXRA was detected by western blotting assay. Error bars ± SD. * P
    Figure Legend Snippet: DANCR interacts with RXRA in TNBC cells. a Schematic diagram of putative RXRA binding sites in DANCR . b RIP-qPCR assay of the association of RXRA with DANCR in MDA-MB-231 and MDA-MB-468 cells. c Re-expression of shRNA-resistant DANCR wild type and RXRA-binding mutant types. d RIP-qPCR assay of effects of re-expression of shRNA-resistant DANCR wild type or mutant types on RXRA binding. e Biotinylated DANCR was incubated with nuclear extracts (MDA-MB-231 and MDA-MB-468 cells), targeted with streptavidin beads, and binding proteins were resolved in a gel. Western blotting assay of the specific binding of RXRA and DANCR . f , g RNAs corresponding to fragments in different regions of DANCR were treated as in ( e ), and binding RXRA was detected by western blotting assay. Error bars ± SD. * P

    Techniques Used: Binding Assay, Real-time Polymerase Chain Reaction, Multiple Displacement Amplification, Expressing, shRNA, Mutagenesis, Incubation, Western Blot

    20) Product Images from "Isoproterenol Acts as a Biased Agonist of the Alpha-1A-Adrenoceptor that Selectively Activates the MAPK/ERK Pathway"

    Article Title: Isoproterenol Acts as a Biased Agonist of the Alpha-1A-Adrenoceptor that Selectively Activates the MAPK/ERK Pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0115701

    Stimulation of α 1A -AR transduced HEK-293/EBNA with A-61603 and Iso leads to an increase in intracellular α 1A – AR. A . HEK293 cells were transiently transfected with α 1A – AR only (top panels), or co-transfected with α 1A – AR and Rab5 variant Q79L (middle panels) or Rab11 variant S25N (bottom panels). Following serum-deprivation, cells were stimulated with vehicle, 1μM A-61603 or 1mM ISO for 2h. Cells were then fixed and analyzed by confocal microscopy. B . HEK293 cells were transiently transfected with α 1A – AR. After serum deprivation for 24h, cells were pre-treated with a membrane impermeable, disulfide-cleavable biotin reagent to label plasma membrane α 1A – AR. Cells were then left untreated, or stimulated 1 μM A-61603 or 1mM ISO for 5, 30, or 60 min. After treatment, one dish of control cells was harvested without any further manipulations (C: total α 1A – AR). The remaining seven dishes were divided into one control (C+GSH), three treated with A-61603 (A-61603+GSH) and three treated with ISO (ISO+GSH). They were stripped of surface biotin label using a reducing agent, in order to reveal internalized, labeled α 1A – AR. Samples were then analyzed by immunoprecipitation (IP) with streptavidin followed by immunoblotting (IB) with an anti-FLAG antibody.
    Figure Legend Snippet: Stimulation of α 1A -AR transduced HEK-293/EBNA with A-61603 and Iso leads to an increase in intracellular α 1A – AR. A . HEK293 cells were transiently transfected with α 1A – AR only (top panels), or co-transfected with α 1A – AR and Rab5 variant Q79L (middle panels) or Rab11 variant S25N (bottom panels). Following serum-deprivation, cells were stimulated with vehicle, 1μM A-61603 or 1mM ISO for 2h. Cells were then fixed and analyzed by confocal microscopy. B . HEK293 cells were transiently transfected with α 1A – AR. After serum deprivation for 24h, cells were pre-treated with a membrane impermeable, disulfide-cleavable biotin reagent to label plasma membrane α 1A – AR. Cells were then left untreated, or stimulated 1 μM A-61603 or 1mM ISO for 5, 30, or 60 min. After treatment, one dish of control cells was harvested without any further manipulations (C: total α 1A – AR). The remaining seven dishes were divided into one control (C+GSH), three treated with A-61603 (A-61603+GSH) and three treated with ISO (ISO+GSH). They were stripped of surface biotin label using a reducing agent, in order to reveal internalized, labeled α 1A – AR. Samples were then analyzed by immunoprecipitation (IP) with streptavidin followed by immunoblotting (IB) with an anti-FLAG antibody.

    Techniques Used: Transfection, Variant Assay, Confocal Microscopy, Labeling, Immunoprecipitation

    21) Product Images from "MAGE-A Cancer/Testis Antigens Inhibit MDM2 Ubiquitylation Function and Promote Increased Levels of MDM4"

    Article Title: MAGE-A Cancer/Testis Antigens Inhibit MDM2 Ubiquitylation Function and Promote Increased Levels of MDM4

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0127713

    MAGE-A2 interacts with specific sites in MDM2. (A) Pepscan assays (pull-down experiments) were conducted in which a series of 15-mer biotinylated peptides (numbered 1–49) overlapping by 5 or more amino acids and representing the entire MDM2 amino acid sequence were coupled to streptavidin-sepharose beads and used to capture 35 S-labelled, in vitro—translated MAGE-A2. The co-precipitating MAGE-A2 was detected by SDS-PAGE followed by fluorography. The control pull-down (bottom left hand panel) was a peptide representing the BoxIV/V region of p53 which we previously established binds very tightly to MAGE-A2 [ 12 ]. (B) Schematic showing the regions represented by the interacting peptides in the pull-down assay. Strong and weak binding sites are indicated in the context of important functional domains within the MDM2 protein. The data are representative of three independent experiments. (C) The model shows a dimer of MDM2 RING fingers, based on the published 3D structure (Protein Data Bank accession number: 2HDP). The two identical subunits in the dimer are represented in grey and light blue respectively. The image on the right hand side was obtained by rotating the 3D structure by approximately 90° to the right in the horizontal plane. The location of the MAGE-A2 binding peptides are shown on one subunit: amino acids 456–470 are represented in red while amino acids 486–491 are shown in light orange.
    Figure Legend Snippet: MAGE-A2 interacts with specific sites in MDM2. (A) Pepscan assays (pull-down experiments) were conducted in which a series of 15-mer biotinylated peptides (numbered 1–49) overlapping by 5 or more amino acids and representing the entire MDM2 amino acid sequence were coupled to streptavidin-sepharose beads and used to capture 35 S-labelled, in vitro—translated MAGE-A2. The co-precipitating MAGE-A2 was detected by SDS-PAGE followed by fluorography. The control pull-down (bottom left hand panel) was a peptide representing the BoxIV/V region of p53 which we previously established binds very tightly to MAGE-A2 [ 12 ]. (B) Schematic showing the regions represented by the interacting peptides in the pull-down assay. Strong and weak binding sites are indicated in the context of important functional domains within the MDM2 protein. The data are representative of three independent experiments. (C) The model shows a dimer of MDM2 RING fingers, based on the published 3D structure (Protein Data Bank accession number: 2HDP). The two identical subunits in the dimer are represented in grey and light blue respectively. The image on the right hand side was obtained by rotating the 3D structure by approximately 90° to the right in the horizontal plane. The location of the MAGE-A2 binding peptides are shown on one subunit: amino acids 456–470 are represented in red while amino acids 486–491 are shown in light orange.

    Techniques Used: Sequencing, In Vitro, SDS Page, Pull Down Assay, Binding Assay, Functional Assay

    22) Product Images from "CARM1 methylates MED12 to regulate its RNA-binding ability"

    Article Title: CARM1 methylates MED12 to regulate its RNA-binding ability

    Journal: Life Science Alliance

    doi: 10.26508/lsa.201800117

    MED12 interacts with TDRD3 in a CARM1-dependent fashion. (A) Fluorograph (top panel) and Coomassie Brilliant Blue staining (bottom panel) of the peptides in vitro methylated by recombinant CARM1 in the presence of tritium-labeled AdoMet. (B) The peptides were used to pull down Tudor domains of the indicated proteins. The input samples and the eluted samples were immunoblotted with αGST antibody. Streptavidin HRP serves as a peptide loading control. (C) MCF-7-Tet-on-shCARM1 cells were untreated or treated with doxycycline (1 μg/ml) for 8 d. Nuclear extracts were subjected to IP with αTDRD3 antibody and the eluted samples were detected by Western blotting with αMED12 and αTDRD3. The input samples were immunoblotted with αTDRD3, αMED12, αmeMED12 a , and αCARM1. (D) HEK293T cells were transiently transfected with FLAG, FLAG-MED12 WT, FLAG-MED12-R1862K, FLAG-MED12-R1899K, and FLAG-MED12-R1862,1899,1912K. Total cell lysates were immunoprecipitated with αTDRD3 antibody and the eluted samples were subjected to Western analysis with αFLAG and αTDRD3 antibodies. The input samples were immunoblotted with αFLAG, αTDRD3, and αβ-actin.
    Figure Legend Snippet: MED12 interacts with TDRD3 in a CARM1-dependent fashion. (A) Fluorograph (top panel) and Coomassie Brilliant Blue staining (bottom panel) of the peptides in vitro methylated by recombinant CARM1 in the presence of tritium-labeled AdoMet. (B) The peptides were used to pull down Tudor domains of the indicated proteins. The input samples and the eluted samples were immunoblotted with αGST antibody. Streptavidin HRP serves as a peptide loading control. (C) MCF-7-Tet-on-shCARM1 cells were untreated or treated with doxycycline (1 μg/ml) for 8 d. Nuclear extracts were subjected to IP with αTDRD3 antibody and the eluted samples were detected by Western blotting with αMED12 and αTDRD3. The input samples were immunoblotted with αTDRD3, αMED12, αmeMED12 a , and αCARM1. (D) HEK293T cells were transiently transfected with FLAG, FLAG-MED12 WT, FLAG-MED12-R1862K, FLAG-MED12-R1899K, and FLAG-MED12-R1862,1899,1912K. Total cell lysates were immunoprecipitated with αTDRD3 antibody and the eluted samples were subjected to Western analysis with αFLAG and αTDRD3 antibodies. The input samples were immunoblotted with αFLAG, αTDRD3, and αβ-actin.

    Techniques Used: Staining, In Vitro, Methylation, Recombinant, Labeling, Western Blot, Transfection, Immunoprecipitation

    23) Product Images from "The Th17 immune response is controlled by the Rel-ROR?-ROR?T transcriptional axis"

    Article Title: The Th17 immune response is controlled by the Rel-ROR?-ROR?T transcriptional axis

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20110462

    c-Rel binds to and activates the Rorgt promoter through two Rel sites. (A) WT and Rel site-mutated Rorgt promoter constructs and the empty vector were analyzed in a luciferase reporter assay with c-Rel co-transfection. The “X” indicates the mutated (Mu) Rel site. Data are representative of three independent experiments. (B) Nuclear extracts were prepared from EL4 cells after stimulation for 6 h with PMA and ionomycin. Biotinylated Rorgt Rel oligonucleotides or their mutants were absorbed by streptavidin-agarose beads, and then added to the nuclear extracts. The amount of c-Rel proteins in the precipitates were assessed by immunoblotting with anti–c-Rel. (C) Purified CD4 + T cells from 6-wk-old WT mice ( n = 3) were cultured under Th17 differentiation condition as described in Materials and methods. After 17 h, cells were fixed, and ChIP was performed using anti–c-Rel or control IgG. Data are representative of two independent experiments. (D) Purified CD4 + T cells from 6-wk-old WT mice ( n = 3) were cultured under Th17 differentiation conditions, as described in Materials and methods. After 17 h, cells were fixed, and ChIP was performed using anti–c-Rel. Data are representative of two independent experiments.
    Figure Legend Snippet: c-Rel binds to and activates the Rorgt promoter through two Rel sites. (A) WT and Rel site-mutated Rorgt promoter constructs and the empty vector were analyzed in a luciferase reporter assay with c-Rel co-transfection. The “X” indicates the mutated (Mu) Rel site. Data are representative of three independent experiments. (B) Nuclear extracts were prepared from EL4 cells after stimulation for 6 h with PMA and ionomycin. Biotinylated Rorgt Rel oligonucleotides or their mutants were absorbed by streptavidin-agarose beads, and then added to the nuclear extracts. The amount of c-Rel proteins in the precipitates were assessed by immunoblotting with anti–c-Rel. (C) Purified CD4 + T cells from 6-wk-old WT mice ( n = 3) were cultured under Th17 differentiation condition as described in Materials and methods. After 17 h, cells were fixed, and ChIP was performed using anti–c-Rel or control IgG. Data are representative of two independent experiments. (D) Purified CD4 + T cells from 6-wk-old WT mice ( n = 3) were cultured under Th17 differentiation conditions, as described in Materials and methods. After 17 h, cells were fixed, and ChIP was performed using anti–c-Rel. Data are representative of two independent experiments.

    Techniques Used: Construct, Plasmid Preparation, Luciferase, Reporter Assay, Cotransfection, Purification, Mouse Assay, Cell Culture, Chromatin Immunoprecipitation

    c-Rel binds to and activates the Rorg promoter through two specific Rel sites. (A) EL4 cells were transiently transfected with murine Rorg promoter luciferase construct together with an expression vector for full-length c-Rel, p65, p50, or RelB, or the empty vector as indicated. After 24 h, cells were treated with PMA and ionomycin for 5 h, and the luciferase activities measured. The promoter activity is presented as fold increase over cells transfected with empty vector. To normalize the transfection efficiency across samples, the Renilla luciferase expression vector pRLTK was included as an internal control. (B) Deletion mutants of the Rorg promoter were analyzed in the luciferase reporter assay with or without c-Rel co-transfection. Putative binding sites for c-Rel (Rel1 and Rel2) and NFAT are indicated. (C) WT and Rel or NFAT site-mutated Rorg promoter constructs were analyzed in the luciferase reporter assay with or without c-Rel co-transfection. The Rel1 site was mutated to TGGGACTCG (−201 to −193), the Rel2 site to CTGAAGTGC (−288 to −280), and the NFAT site to TTGTCAC (−96 to −90). The “X” indicates the mutated site. (D) Nuclear extracts were prepared from EL4 cells after stimulation for 6 h with PMA and ionomycin. Biotinylated Rorg Rel oligonucleotides or their mutants were absorbed by streptavidin-agarose beads, and then added to the nuclear extracts. The amount of c-Rel proteins in the precipitates were assessed by immunoblotting with anti–c-Rel. (E) Purified CD4 + T cells from 6-wk-old WT mice ( n = 3) were cultured under Th17 differentiation conditions, as described in Materials and methods. After 17 h, cells were fixed, and ChIP was performed using anti–c-Rel or control IgG. *, P
    Figure Legend Snippet: c-Rel binds to and activates the Rorg promoter through two specific Rel sites. (A) EL4 cells were transiently transfected with murine Rorg promoter luciferase construct together with an expression vector for full-length c-Rel, p65, p50, or RelB, or the empty vector as indicated. After 24 h, cells were treated with PMA and ionomycin for 5 h, and the luciferase activities measured. The promoter activity is presented as fold increase over cells transfected with empty vector. To normalize the transfection efficiency across samples, the Renilla luciferase expression vector pRLTK was included as an internal control. (B) Deletion mutants of the Rorg promoter were analyzed in the luciferase reporter assay with or without c-Rel co-transfection. Putative binding sites for c-Rel (Rel1 and Rel2) and NFAT are indicated. (C) WT and Rel or NFAT site-mutated Rorg promoter constructs were analyzed in the luciferase reporter assay with or without c-Rel co-transfection. The Rel1 site was mutated to TGGGACTCG (−201 to −193), the Rel2 site to CTGAAGTGC (−288 to −280), and the NFAT site to TTGTCAC (−96 to −90). The “X” indicates the mutated site. (D) Nuclear extracts were prepared from EL4 cells after stimulation for 6 h with PMA and ionomycin. Biotinylated Rorg Rel oligonucleotides or their mutants were absorbed by streptavidin-agarose beads, and then added to the nuclear extracts. The amount of c-Rel proteins in the precipitates were assessed by immunoblotting with anti–c-Rel. (E) Purified CD4 + T cells from 6-wk-old WT mice ( n = 3) were cultured under Th17 differentiation conditions, as described in Materials and methods. After 17 h, cells were fixed, and ChIP was performed using anti–c-Rel or control IgG. *, P

    Techniques Used: Transfection, Luciferase, Construct, Expressing, Plasmid Preparation, Activity Assay, Reporter Assay, Cotransfection, Binding Assay, Purification, Mouse Assay, Cell Culture, Chromatin Immunoprecipitation

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    Chromatin Immunoprecipitation:

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    Determination of IGF-1R sialylation levels using Lectin affinity assay: ( A ) Sialylation status of IGF-1R: Equal amount of cell lysates from vector control cells were treated with 100 mU of C. perfringen s sialidase and untreated lysates from GNE knockdown cells were separated on 8% SDS-PAGE followed by immunoblotting with anti-α IGF-1R. ( B ) SNA Lectin affinity assay: Sialylated IGF-1R from GNE knockdown cells and scrambled shRNA vector control cell was pulled down using biotin labelled <t>SNA/streptavidin-coupled</t> agarose and immunoblotted with anti-α IGF-1R. ( C ) Sialylated IGF-1R complexes from GNE mutant and pcDNA3 transfected vector control cells were pulled down using biotin labelled SNA/streptavidin-coupled agarose and immunoblotted with anti-α IGF-1R. Sialylated IGF-1R complexes from 100 mU neuraminidase treated vector control cells that were pulled down using biotin labelled SNA/streptavidin-coupled agarose served as negative control in this experiment. Equal amount of protein was used as input lysates. ( D – F ) are showing representative densitometry graphs of ( A – C ), respectively, normalized to vector control.
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    Millipore streptavidin agarose from streptomyces avidinii
    P63 binds the PTEN promoter and enhances its activity. A. Schematic of the -1178/-1031 region of the PTEN promoter containing both p63-binding sites. Results of ChIP performed using an anti-p63 antibody and primers amplifying the -1178/-1031 region. The black arrow points to the target band. B. Sequences of two probes corresponding to the predicted p63-binding sites of the PTEN promoter shown relative to the initiating ATG. The bound complex was pulled down by <t>streptavidin-conjugated</t> agarose and subjected to western blot analysis. P63 was detected by anti-p63 antibodies. C and D. Mutated p63-binding sites are represented by crossed circles. Cells were transfected with wild-type (WT) and mutant reporter constructs for 24 h. D. vector or ΔNp63α was overexpressed before reporter being transfected. Relative fluorescence intensity was calculated and data (mean ± SD of nine separate experiments) are shown as fold change compared to control group. * P
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    Determination of IGF-1R sialylation levels using Lectin affinity assay: ( A ) Sialylation status of IGF-1R: Equal amount of cell lysates from vector control cells were treated with 100 mU of C. perfringen s sialidase and untreated lysates from GNE knockdown cells were separated on 8% SDS-PAGE followed by immunoblotting with anti-α IGF-1R. ( B ) SNA Lectin affinity assay: Sialylated IGF-1R from GNE knockdown cells and scrambled shRNA vector control cell was pulled down using biotin labelled SNA/streptavidin-coupled agarose and immunoblotted with anti-α IGF-1R. ( C ) Sialylated IGF-1R complexes from GNE mutant and pcDNA3 transfected vector control cells were pulled down using biotin labelled SNA/streptavidin-coupled agarose and immunoblotted with anti-α IGF-1R. Sialylated IGF-1R complexes from 100 mU neuraminidase treated vector control cells that were pulled down using biotin labelled SNA/streptavidin-coupled agarose served as negative control in this experiment. Equal amount of protein was used as input lysates. ( D – F ) are showing representative densitometry graphs of ( A – C ), respectively, normalized to vector control.

    Journal: Scientific Reports

    Article Title: Role of IGF-1R in ameliorating apoptosis of GNE deficient cells

    doi: 10.1038/s41598-018-25510-9

    Figure Lengend Snippet: Determination of IGF-1R sialylation levels using Lectin affinity assay: ( A ) Sialylation status of IGF-1R: Equal amount of cell lysates from vector control cells were treated with 100 mU of C. perfringen s sialidase and untreated lysates from GNE knockdown cells were separated on 8% SDS-PAGE followed by immunoblotting with anti-α IGF-1R. ( B ) SNA Lectin affinity assay: Sialylated IGF-1R from GNE knockdown cells and scrambled shRNA vector control cell was pulled down using biotin labelled SNA/streptavidin-coupled agarose and immunoblotted with anti-α IGF-1R. ( C ) Sialylated IGF-1R complexes from GNE mutant and pcDNA3 transfected vector control cells were pulled down using biotin labelled SNA/streptavidin-coupled agarose and immunoblotted with anti-α IGF-1R. Sialylated IGF-1R complexes from 100 mU neuraminidase treated vector control cells that were pulled down using biotin labelled SNA/streptavidin-coupled agarose served as negative control in this experiment. Equal amount of protein was used as input lysates. ( D – F ) are showing representative densitometry graphs of ( A – C ), respectively, normalized to vector control.

    Article Snippet: 20 μl of streptavidin-agarose beads (Sigma, USA) were added and then incubated for 4 h at 4 °C with rotation.

    Techniques: Plasmid Preparation, SDS Page, shRNA, Mutagenesis, Transfection, Negative Control

    TP53TG1 inhibited miR-18a expression in NSCLC cells. a Sequence alignment of miR-18a with the putative binding sites within the wild-type regions of TP53TG1. b Subcellular fractionation assay was performed to identify the subcellular location of TP53TG1 with GAPDH and U6 as internal references. c , d The luciferase activity was detected in A549 cells transfected with TP53TG1-WT or TP53TG1-MUT and miR-con, miR-18a mimics, anti-miR-con or anti-miR-18a. e Biotin-labeled TP53TG1 RNA was obtained and added to cell lysates with Streptavidin agarose beads, followed by the detection of miR-18a enrichment by RNA pull-down assay. f RIP assay was performed to evaluate the endogenous binding between TP53TG1 and miR-18a in A549 cells using specific antibody against Ago2, followed by detection of RNA levels by qRT-PCR. g qRT-PCR assay of miR-18a expression in A549 cells transfected with si-TP53TG1#1 or pcDNA-TP53TG1 for 48 h. h qRT-PCR assay of miR-18a expression in 40 pairs of NSCLC samples. i qRT-PCR assay of miR-18a expression in DDP-sensitive NSCLC tissues and DDP-resistant NSCLC samples. j The correlation between TP53TG1 and miR-18a expression was detected in NSCLC samples. All experiments are repeated three times. * P

    Journal: Cell & Bioscience

    Article Title: TP53TG1 enhances cisplatin sensitivity of non-small cell lung cancer cells through regulating miR-18a/PTEN axis

    doi: 10.1186/s13578-018-0221-7

    Figure Lengend Snippet: TP53TG1 inhibited miR-18a expression in NSCLC cells. a Sequence alignment of miR-18a with the putative binding sites within the wild-type regions of TP53TG1. b Subcellular fractionation assay was performed to identify the subcellular location of TP53TG1 with GAPDH and U6 as internal references. c , d The luciferase activity was detected in A549 cells transfected with TP53TG1-WT or TP53TG1-MUT and miR-con, miR-18a mimics, anti-miR-con or anti-miR-18a. e Biotin-labeled TP53TG1 RNA was obtained and added to cell lysates with Streptavidin agarose beads, followed by the detection of miR-18a enrichment by RNA pull-down assay. f RIP assay was performed to evaluate the endogenous binding between TP53TG1 and miR-18a in A549 cells using specific antibody against Ago2, followed by detection of RNA levels by qRT-PCR. g qRT-PCR assay of miR-18a expression in A549 cells transfected with si-TP53TG1#1 or pcDNA-TP53TG1 for 48 h. h qRT-PCR assay of miR-18a expression in 40 pairs of NSCLC samples. i qRT-PCR assay of miR-18a expression in DDP-sensitive NSCLC tissues and DDP-resistant NSCLC samples. j The correlation between TP53TG1 and miR-18a expression was detected in NSCLC samples. All experiments are repeated three times. * P

    Article Snippet: Then, cell lysates and Bio-TP53TG1-probe were incubated at room temperature for 60 min, and 50 μl of Streptavidin agarose beads (Sigma-Aldrich) were added into each binding reaction and further incubated for 60 min.

    Techniques: Expressing, Sequencing, Binding Assay, Fractionation, Luciferase, Activity Assay, Transfection, Labeling, Pull Down Assay, Quantitative RT-PCR

    The rate of cell surface expression/appearance/transport of BRI2 is reduced in the absence of N-glycosylation. Wild-type mycBRI2 or mycBRI2/N170A was expressed in HEK293 cells. The newly synthesized proteins were labeled with 35 S in radiolabeling medium for 2 h (pulse) at 16°C and then were incubated in non-radiolabeling medium for 0′, 20′, 40′ and 60′ (chase). ( A ) Cell surface proteins were labeled with biotin and precipitated with streptavidin beads. Precipitated cell surface proteins were eluted from the beads and immunoprecipitated with 9B11 antibody against the myc epitope before electrophoresis and autoradiography. ( B ) Immunoprecipitation of cell extracts with 9B11, electrophoresis and autoradiography were performed to verify the expression levels of BRI2.

    Journal: Glycobiology

    Article Title: Glycosylation of BRI2 on asparagine 170 is involved in its trafficking to the cell surface but not in its processing by furin or ADAM10

    doi: 10.1093/glycob/cwr097

    Figure Lengend Snippet: The rate of cell surface expression/appearance/transport of BRI2 is reduced in the absence of N-glycosylation. Wild-type mycBRI2 or mycBRI2/N170A was expressed in HEK293 cells. The newly synthesized proteins were labeled with 35 S in radiolabeling medium for 2 h (pulse) at 16°C and then were incubated in non-radiolabeling medium for 0′, 20′, 40′ and 60′ (chase). ( A ) Cell surface proteins were labeled with biotin and precipitated with streptavidin beads. Precipitated cell surface proteins were eluted from the beads and immunoprecipitated with 9B11 antibody against the myc epitope before electrophoresis and autoradiography. ( B ) Immunoprecipitation of cell extracts with 9B11, electrophoresis and autoradiography were performed to verify the expression levels of BRI2.

    Article Snippet: The cell extracts were centrifuged at 15,000 × g for 30 min and supernatants were incubated with 50 μL of streptavidin–agarose beads (Millipore) for 1 h at 4°C.

    Techniques: Expressing, Synthesized, Labeling, Radioactivity, Incubation, Immunoprecipitation, Electrophoresis, Autoradiography

    Inhibition of N-glycosylation of BRI2 inhibits its expression at the cell surface. Wild-type mycBRI2 or mycBRI2/N170A was expressed in HEK293 cells. Cell surface proteins were labeled with biotin (lanes 1 and 2) or were not labeled (lanes 3 and 4), as a control for biotinylation specificity. ( A ) Cell extracts were precipitated with streptavidin beads and analyzed with western blot against myc with 9B11 antibody. ( B ) Cell extracts were directly analyzed with western blot as a control for protein expression. The two immunoreactive bands of BRI2 proteins correspond to the furin-cleaved and the non-cleaved wild-type mycBRI2 or mycBRI2/N170A.

    Journal: Glycobiology

    Article Title: Glycosylation of BRI2 on asparagine 170 is involved in its trafficking to the cell surface but not in its processing by furin or ADAM10

    doi: 10.1093/glycob/cwr097

    Figure Lengend Snippet: Inhibition of N-glycosylation of BRI2 inhibits its expression at the cell surface. Wild-type mycBRI2 or mycBRI2/N170A was expressed in HEK293 cells. Cell surface proteins were labeled with biotin (lanes 1 and 2) or were not labeled (lanes 3 and 4), as a control for biotinylation specificity. ( A ) Cell extracts were precipitated with streptavidin beads and analyzed with western blot against myc with 9B11 antibody. ( B ) Cell extracts were directly analyzed with western blot as a control for protein expression. The two immunoreactive bands of BRI2 proteins correspond to the furin-cleaved and the non-cleaved wild-type mycBRI2 or mycBRI2/N170A.

    Article Snippet: The cell extracts were centrifuged at 15,000 × g for 30 min and supernatants were incubated with 50 μL of streptavidin–agarose beads (Millipore) for 1 h at 4°C.

    Techniques: Inhibition, Expressing, Labeling, Western Blot

    P63 binds the PTEN promoter and enhances its activity. A. Schematic of the -1178/-1031 region of the PTEN promoter containing both p63-binding sites. Results of ChIP performed using an anti-p63 antibody and primers amplifying the -1178/-1031 region. The black arrow points to the target band. B. Sequences of two probes corresponding to the predicted p63-binding sites of the PTEN promoter shown relative to the initiating ATG. The bound complex was pulled down by streptavidin-conjugated agarose and subjected to western blot analysis. P63 was detected by anti-p63 antibodies. C and D. Mutated p63-binding sites are represented by crossed circles. Cells were transfected with wild-type (WT) and mutant reporter constructs for 24 h. D. vector or ΔNp63α was overexpressed before reporter being transfected. Relative fluorescence intensity was calculated and data (mean ± SD of nine separate experiments) are shown as fold change compared to control group. * P

    Journal: American Journal of Translational Research

    Article Title: ΔNp63α promotes the expression and nuclear translocation of PTEN, leading to cisplatin resistance in oral cancer cells

    doi:

    Figure Lengend Snippet: P63 binds the PTEN promoter and enhances its activity. A. Schematic of the -1178/-1031 region of the PTEN promoter containing both p63-binding sites. Results of ChIP performed using an anti-p63 antibody and primers amplifying the -1178/-1031 region. The black arrow points to the target band. B. Sequences of two probes corresponding to the predicted p63-binding sites of the PTEN promoter shown relative to the initiating ATG. The bound complex was pulled down by streptavidin-conjugated agarose and subjected to western blot analysis. P63 was detected by anti-p63 antibodies. C and D. Mutated p63-binding sites are represented by crossed circles. Cells were transfected with wild-type (WT) and mutant reporter constructs for 24 h. D. vector or ΔNp63α was overexpressed before reporter being transfected. Relative fluorescence intensity was calculated and data (mean ± SD of nine separate experiments) are shown as fold change compared to control group. * P

    Article Snippet: The DNA protein complex was precipitated using 100 μL streptavidin-conjugated agarose beads (S1638, Sigma-Aldrich).

    Techniques: Activity Assay, Binding Assay, Chromatin Immunoprecipitation, Western Blot, Transfection, Mutagenesis, Construct, Plasmid Preparation, Fluorescence