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  • 94
    Bio-Rad immunoblot
    In vitro and in vivo superactivation of the PI3K–Akt pathway in an EGF-dependent and AP-2α null–dependent fashion. (A) Primary WT and KO keratinocytes were cultured ± the EGFR-specific protein kinase inhibitor AG1478 without the addition of fibroblast feeder cells after the original plating. Growth curves represents three independent experiments performed in duplicate (see Separation of epidermis…transfections). Error bars represent SD for these experiments. (B) Keratinocytes were serum-starved for 24 h and at t = 0, 50 ng/ml EGF was added. At the times indicated, cell extracts were prepared, proteins were resolved by SDS-PAGE, and <t>immunoblot</t> analyses were conducted with the antibodies indicated at right. The antibodies against the active forms of Erk1/2 and Akt (p-Erk and p-Akt, respectively) are phosphospecific antibodies and do not recognize the inactive states of the kinases. β-tubulin antibodies are used as a loading control, along with ponseau red staining of the blots (not depicted). Note EGF-dependent superactivation of Akt and Erk1/2 in AP-2α –null keratinocytes. (C) Same experiment as in B, except 10× increments of EGF concentrations (0–50 ng/ml) were used, and extracts were harvested at t = 2 min. (D) Same experiment as in B, but insulin growth factor 1 (IGF1) was added at 50 ng/ml. Note that in contrast to EGF, IGF1 did not generate enhanced activated Akt in KO relative to WT cells. (E) Same experiment as in B, but the PI3K-specific inhibitor LY294002 was added at 20 μM. Note that the activation of Akt, but not Erk1/2, is dependent on PI3K activation. (F) Antiphospho-Akt staining of frozen sections (8 μm) from (top) lesional KO and WT thoracic skins and (bottom) TPA-treated KO and WT back skins. Note the superactivation of Akt in KO skin regions that are lesional and that correlate with elevated EGFR ligand expression. De, dermis; epi, epidermis; hf, hair follicle. Dotted lines denote dermo–epidermal borders. Bars, 20 μm.
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    93
    Cell Signaling Technology Inc immunoblot
    <t>Immunoblot</t> analyis of γH2AX and p21 . γH2AX-expression measured immunhistochemically 2 hours after HIT and p21 expression in KHOS-24OS, A-204 and HFOB1.19 treated with vehicle control (C), 0,5 (A-204)-1 μM (KHOS-24OS and HFOB1.19) SAHA and HIT (cell specific doses, see Figure 9) or the combination of SAHA and HIT 24 h after HIT.
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    92
    EUROIMMUN immunoblot
    <t>Immunoblot</t> analyis of γH2AX and p21 . γH2AX-expression measured immunhistochemically 2 hours after HIT and p21 expression in KHOS-24OS, A-204 and HFOB1.19 treated with vehicle control (C), 0,5 (A-204)-1 μM (KHOS-24OS and HFOB1.19) SAHA and HIT (cell specific doses, see Figure 9) or the combination of SAHA and HIT 24 h after HIT.
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    92
    Millipore immunoblot
    AQP6 protein expression in mouse kidney homogenate. <t>Immunoblot</t> of mouse kidney total homogenate was performed as described (see Methods). Two bands of about 55 and 36 kDa were observed. Similar results were obtained from at least four separate experiments. MW, molecular weight markers.
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    93
    Santa Cruz Biotechnology immunoblot
    Dominant negative effect of H3.3R26K or H3.3K27R in oocytes accelerates oogenesis. (A) Scheme of HISRainbow; Cre oocytes with four copies of the chromosomes that express 1-2 alleles of WT and/or H3.3 mutants. (B) Confocal (full projection) and DIC images of MII oocytes isolated from 8- to 12-week-old HISRainbow; Figla -iCre mice. Oocytes expressed H3.3 eGFP (G), H3.3R26K eCFP (B), or H3.3K27R mCherry (R), from one or two alleles. Scale bar: 50 µm. (C) <t>Immunoblot</t> analysis of HISRainbow; Figla -iCre GV oocytes with indicated genotypes for H3.3 expression. 25 oocytes were collected for each group: WT, H3.3 eGFP (GG), H3.3K27R mCherry (RR) and H3.3R26K eCFP ) in: oocytes in HISRainbow; Figla -iCre P2 ovaries (6 replicates); MII oocytes from 8- to 12-week-old HISRainbow; Figla -iCre (7 replicates, 191 oocytes); and HISRainbow; ZP3 -Cre mice (8 replicates, 193 oocytes). Percentage of each subpopulation was normalized to the percentage of oocytes expressing H3.3 eGFP , which was set to 1. Mean±s.e.m. (E) Quantification of histone modifications in HISRainbow; Figla -iCre oocytes. Mean±s.e.m of n ≥6.; * p
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    Valiant immunoblot
    Dominant negative effect of H3.3R26K or H3.3K27R in oocytes accelerates oogenesis. (A) Scheme of HISRainbow; Cre oocytes with four copies of the chromosomes that express 1-2 alleles of WT and/or H3.3 mutants. (B) Confocal (full projection) and DIC images of MII oocytes isolated from 8- to 12-week-old HISRainbow; Figla -iCre mice. Oocytes expressed H3.3 eGFP (G), H3.3R26K eCFP (B), or H3.3K27R mCherry (R), from one or two alleles. Scale bar: 50 µm. (C) <t>Immunoblot</t> analysis of HISRainbow; Figla -iCre GV oocytes with indicated genotypes for H3.3 expression. 25 oocytes were collected for each group: WT, H3.3 eGFP (GG), H3.3K27R mCherry (RR) and H3.3R26K eCFP ) in: oocytes in HISRainbow; Figla -iCre P2 ovaries (6 replicates); MII oocytes from 8- to 12-week-old HISRainbow; Figla -iCre (7 replicates, 191 oocytes); and HISRainbow; ZP3 -Cre mice (8 replicates, 193 oocytes). Percentage of each subpopulation was normalized to the percentage of oocytes expressing H3.3 eGFP , which was set to 1. Mean±s.e.m. (E) Quantification of histone modifications in HISRainbow; Figla -iCre oocytes. Mean±s.e.m of n ≥6.; * p
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    92
    MIKROGEN immunoblot
    Analysis of Arthrobacter mysorens . A) Randomly amplified polymorphic DNA (RAPD) analysis of the clinical isolate (lane 1) and the soil isolate (lane 2) using primer P1 [5'-GGTGCGGGAA-3'] and primer P5 [5'-AACGCGCAAC-3']. The analyses were done with the Immunoblot analysis of the patient's serum using a whole cell extract of A. mysorens clinical isolate. Arrow heads indicate the two reacting proteins of ~53-kDa and ~32-kDa, respectively. " width="250" height="auto" />
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    93
    Becton Dickinson immunoblot
    Levels of ROCK protein in ROCKII −/− mice. Brain and spinal cord tissue from ROCKII −/− and wild-type mice was examined by <t>immunoblot</t> for ROCKII and ROCKI protein. A representative blotfor each antigen is shown from one pair
    Immunoblot, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 360 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim immunoblot
    Levels of ROCK protein in ROCKII −/− mice. Brain and spinal cord tissue from ROCKII −/− and wild-type mice was examined by <t>immunoblot</t> for ROCKII and ROCKI protein. A representative blotfor each antigen is shown from one pair
    Immunoblot, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Epitomics immunoblot
    A : representative gel images of <t>immunoblot</t> analyses using antibodies against the free fatty acid receptor 1 (FFAR1) and FFAR4 using total protein prepared from human ( i ) and guinea pig ( ii ) tissues: human brain cerebral cortex (50 μg), freshly
    Immunoblot, supplied by Epitomics, used in various techniques. Bioz Stars score: 92/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    BioLegend immunoblot
    A : representative gel images of <t>immunoblot</t> analyses using antibodies against the free fatty acid receptor 1 (FFAR1) and FFAR4 using total protein prepared from human ( i ) and guinea pig ( ii ) tissues: human brain cerebral cortex (50 μg), freshly
    Immunoblot, supplied by BioLegend, used in various techniques. Bioz Stars score: 91/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Torrey Pines Biolabs immunoblot
    A : representative gel images of <t>immunoblot</t> analyses using antibodies against the free fatty acid receptor 1 (FFAR1) and FFAR4 using total protein prepared from human ( i ) and guinea pig ( ii ) tissues: human brain cerebral cortex (50 μg), freshly
    Immunoblot, supplied by Torrey Pines Biolabs, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    ATUM immunoblotting
    and protein levels on chromatin were analyzed by <t>immunoblotting.</t>
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    93
    Bio-Rad immunoblotting
    LKB1 contributes to lovastatin‐induced AMPK, p38MAPK and p53 phosphorylation in MCF‐7 cells. A, Cells were transfected with negative control siRNA or LKB1 siRNA for 48 h. After transfection, cells were treated with vehicle or lovastatin (30 μmol/L) for another 1 h. The extent of LKB1 and phosphorylation status of AMPK, p38MAPK or p53 was determined by <t>immunoblotting.</t> The compiled results of AMPK (B), p38MAPK (C) and p53 (D) phosphorylations are shown. Each column represents the mean ± SEM of six independent experiments (Statistically significant differences were determined using the Mann‐Whitney test. * P
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    99
    Cell Signaling Technology Inc immunoblotting
    Temporal transcriptome of IMR90 fibroblasts inducibly expressing MCPyV ST. A) IMR90 fibroblasts containing dox-inducible MCPyV ST or GFP vectors were treated with dox and harvested every 8 hours for RNA extraction. Each time point represents three biological replicas. B) Mean ST transcript levels and C) <t>immunoblotting</t> for ST, GFP and vinculin from cells collected every 8 hours for 96 hours following dox treatment. D) Hierarchical clustering and fold change between MCPyV ST and GFP following dox induction for 96 hours. Each bar represents an average of three experiments for each time point. The enrichment of “Cancer Hallmark” gene sets are represented relative to the ST-differentially expressed clusters, including epithelial to mesenchymal transition (EMT), tumor necrosis factor-α (TNFA signaling via NF-κB), hypoxia, mTORC1, oxidative phosphorylation, glycolysis, MYC, and several cell cycle clusters including E2F targets, G2M checkpoint and mitotic spindle. The color bar indicates statistical significance, yellow p
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    92
    Covance immunoblotting
    Heparitinase treatment and blockade of sulfation diminish responses of cultured cells to BMP2. (A) Smad phosphorylation and p38 MAPK activation in mouse C2C12 and rat PC12 cells. Cells maintained in serum-free medium were treated with human recombinant BMP2 at 5 ng/ml for 1 h. Where indicated, cultures also were treated with 25 mIU/ml heparitinase 1 h before BMP addition and throughout the remainder of the assay. Cells lysates were subjected to <t>immunoblotting</t> for phospho-Smad1/5/8 (P-Smad) and active p38. Total Smad, total p38, and β-tubulin served as loading controls. (B and C) Kinetic profiles of BMP-induced Smad phosphorylation. C2C12 (B) or PC12 cells (C) were treated with heparitinase for 1 h, and BMP2 (5 ng/ml) was added. Cell lysates were collected at indicated time points and subjected to immunoblotting for P-Smad. Data are normalized to loading controls. (D and E) Exogenous heparin does not rescue cells from the effect of heparitinase treatment. C2C12 cells were treated with heparitinase for 1 h, and BMP2 (5 ng/ml), or BMP2 and heparin (3–100 μg/ml) were added for a subsequent hour. Cell lysates were subjected to immunoblotting for active p38 (D) or P-Smad (E), and band intensities were quantified. Data are from duplicate cultures for each condition and are normalized to loading controls. Both activation of p38 and Smad in response to BMP2 were substantially lower in heparitinase-treated cells than in untreated cells (*p
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    Millipore immunoblotting
    NDR1 regulates localization of kindlin-3 and Rab8. (A) Association of NDR1 and kindlin-3. 293T cells transfected with Myc–kindlin-3 and FLAG-NDR1 as indicated were subjected to immunoprecipitation (IP) of FLAG-NDR1 followed by <t>immunoblotting</t> for Myc–kindlin-3. (B) Primary T cells stimulated with anti-CD3 or left unstimulated were analyzed for association of NDR1 with kindlin-3. IP with anti-NDR Ab was immunoblotted for kindlin-3. Total and precipitated proteins are indicated. The arrowhead indicates the predicted molecular mass of kindlin-3 (75.6 kDa). (C) Mislocalized kindlin-3 in NDR1 knockdown T cell blasts. Representative views at the contact plane and 3D images ( z ) of NDR1 knockdown (sh NDR) and control (sh Ctrl) OT-II T cells. Distances of peak intensities from the contact plane of kindlin-3 are shown. Scale bars, 2.5 μm. ***, P
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    97
    Santa Cruz Biotechnology immunoblotting
    The 1 st type 1 repeat is required for TSP1/CD148-mediated cell growth inhibition. (A) The trimeric TSP1 fragment (ΔType1-R1) that contains the procollagen domain and the 2 nd and 3 rd , but not the 1 st , type 1 repeat was prepared using HEK293E cells. Upper panel shows a schematic representation of the trimeric TSP1 fragment that lacks the 1 st type 1 repeat. Amino acid residues (aa 374–429) of the 1 st type 1 repeat were deleted. Lower panel shows colloidal blue staining of the purified ΔType1-R1 fragment. Five micrograms of protein were separated on a 10% polyacrylamide gel in reducing (+DTT) and non-reducing (-DTT) conditions and stained with colloidal blue to assess size, purity, and trimerization. The expected size of protein is also shown. (B) A431D/CD148wt or A431D/CD36 (stably expressing CD36) cells were treated with 12 nM of trimeric TSP1 fragments that lack or contain the 1 st type 1 repeat or whole TSP1 protein. Cell density was measured at the indicated time points. The data show mean ± SEM of quadruplicate determinations. Representative data of four independent experiments is shown. Note: The ΔType1-R1 fragment shows no growth inhibitory activity in A431D/CD148wt cells, while it inhibits cell proliferation in A431D/CD36 cells. (C) A431D/CD36 cells were treated with trimeric TSP1 fragments (12 nM) that lacked or contained the 1 st type 1 repeat or whole TSP1 protein (12 nM) for 18 h. Tyrosine phosphorylation of p38 and cleaved caspase 3 was assessed by <t>immunoblotting</t> using the phopho-specific p38 (pThr180+Tyr182) or cleaved caspase 3 antibodies. The membranes were reprobed with antibodies to total p38 or γ-tubulin. Representative data of four independent experiments is shown. (D) A series of monomeric TSP1 fragments were prepared from the regions containing the procollagen domain and type 1 repeats as shown in a schema on right side. Each fragment (17 nM) was incubated with either 44 pmol of CD148-Fc or control Fc (Fc alone), and Fc-proteins were pulled down with protein-G beads. Bound TSP1 fragments were assessed by anti-Myc immunoblotting (upper panel). Half of each sample was subjected to anti-CD148 immunoblotting to confirm the pull down of CD148-Fc (lower panel). Representative data of five independent experiments is shown. Note: TSP1 fragments that contain the 1 st type 1 repeat bind to CD148-Fc. (E) A431D/CD148wt cells were treated with or without indicated TSP1 fragments (36 nM) for 1 h, then a trimeric TSP1 fragment (12nM) containing the procollagen domain and type 1 repeats was added to the medium. Cell proliferation was assessed at day 2. The data show mean ± SEM of quadruplicate determinations. Representative data of five independent experiments is shown. ** P
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    Signalway Antibody immunoblotting
    The 1 st type 1 repeat is required for TSP1/CD148-mediated cell growth inhibition. (A) The trimeric TSP1 fragment (ΔType1-R1) that contains the procollagen domain and the 2 nd and 3 rd , but not the 1 st , type 1 repeat was prepared using HEK293E cells. Upper panel shows a schematic representation of the trimeric TSP1 fragment that lacks the 1 st type 1 repeat. Amino acid residues (aa 374–429) of the 1 st type 1 repeat were deleted. Lower panel shows colloidal blue staining of the purified ΔType1-R1 fragment. Five micrograms of protein were separated on a 10% polyacrylamide gel in reducing (+DTT) and non-reducing (-DTT) conditions and stained with colloidal blue to assess size, purity, and trimerization. The expected size of protein is also shown. (B) A431D/CD148wt or A431D/CD36 (stably expressing CD36) cells were treated with 12 nM of trimeric TSP1 fragments that lack or contain the 1 st type 1 repeat or whole TSP1 protein. Cell density was measured at the indicated time points. The data show mean ± SEM of quadruplicate determinations. Representative data of four independent experiments is shown. Note: The ΔType1-R1 fragment shows no growth inhibitory activity in A431D/CD148wt cells, while it inhibits cell proliferation in A431D/CD36 cells. (C) A431D/CD36 cells were treated with trimeric TSP1 fragments (12 nM) that lacked or contained the 1 st type 1 repeat or whole TSP1 protein (12 nM) for 18 h. Tyrosine phosphorylation of p38 and cleaved caspase 3 was assessed by <t>immunoblotting</t> using the phopho-specific p38 (pThr180+Tyr182) or cleaved caspase 3 antibodies. The membranes were reprobed with antibodies to total p38 or γ-tubulin. Representative data of four independent experiments is shown. (D) A series of monomeric TSP1 fragments were prepared from the regions containing the procollagen domain and type 1 repeats as shown in a schema on right side. Each fragment (17 nM) was incubated with either 44 pmol of CD148-Fc or control Fc (Fc alone), and Fc-proteins were pulled down with protein-G beads. Bound TSP1 fragments were assessed by anti-Myc immunoblotting (upper panel). Half of each sample was subjected to anti-CD148 immunoblotting to confirm the pull down of CD148-Fc (lower panel). Representative data of five independent experiments is shown. Note: TSP1 fragments that contain the 1 st type 1 repeat bind to CD148-Fc. (E) A431D/CD148wt cells were treated with or without indicated TSP1 fragments (36 nM) for 1 h, then a trimeric TSP1 fragment (12nM) containing the procollagen domain and type 1 repeats was added to the medium. Cell proliferation was assessed at day 2. The data show mean ± SEM of quadruplicate determinations. Representative data of five independent experiments is shown. ** P
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    92
    Thermo Fisher immunoblotting
    Knockdown of SIRT2 suppresses the tumorigenicity of GB2 cells Kaplan–Meier overall survival curves of mice transplanted with GB2 cells (left panel), GB13 cells (Middle panel) or GB16 cells (right panel) infected with the indicated lentivirus (1 × 10 4 cells, 7 mice per group). The y ‐axis indicates the percent survival. Mice were transplanted with the indicated number of GB2 cells infected with a control (empty) or shSIRT2‐expressing (shS2 #1) lentivirus. Six weeks after transplantation, mice (3 or 4 animals, see number of dots) were sacrificed and the expression levels of human β‐actin mRNA were quantified by qRT–PCR. H E staining of tumors that were developed in mice implanted with GB16 cells that had been infected with a control (empty) or shSIRT2‐expressing (shS2#1) lentivirus. Scale bar, 1 mm. An image with higher magnification is shown on the right (scale bar, 50 μm). The effect of AK7 on the deacetylation of acetyl‐tubulin (left panel) and the proliferation of GB2 cells (right panel). (Left panel) <t>Immunoblotting</t> analysis of the effect of AK7 (20 μM) on deacetylation of acetyl‐tubulin/tubulin was performed on day 3 in right panel. Bars indicate mean ± SD ( n = 3). Ten days after intracranial transplantation of GB2 cells (1.0 × 10 4 cells), AK7 was intraperitoneally administrated for 4 weeks (15 mg/kg, twice/week). After 8 weeks, mice (4 or 5 animals, see number of dots) were sacrificed and the expression levels of human β‐actin mRNA were quantified by qRT–PCR. Data information: Statistical significance was evaluated using the log‐rank test (for panel A) or unpaired two‐tailed t ‐test. ** P
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    Bio-Rad immunoblot analysis
    Western blot analysis of Xenopus oocytes expressing human DMT1 and FPN1-c-Myc Xenopus oocytes were injected with 10 ng of cRNA for FPN1, DMT1A , both FPN1 and DMT1 , or H 2 O and incubated for 3 days. For <t>immunoblot</t> detection, membrane-enriched fractions were subject to SDS-PAGE. Blots were probed with primary antibodies against DMT1, c-Myc, and actin, and detected with horseradish peroxidase linked secondary antibodies. For DMT1 analysis (A), 10 µg of protein was used in each lane, and for FPN1 analysis 40 µg was used (B).
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    92
    rdi research diagnostics immunoblot analysis
    Deletion of p53 identifies the novel mitochrondria -associated senescence domain (MASD) between amino acids 64 and 209 of p53. ( A ) Various deletions of FLAG –tagged p53 were used to determine involvement in mitochrondria –associated senescence programs in EJ-p53 cells. The following abbreviations were used to characterize individual domains within p53 as, AD1, activation domain 1; AD2, activation domain 2; PRD, proline rich domain; DBD-NLS-TD-NES, combined DNA binding domain-nuclear localization signal-transactivation domain-nuclear export signal; BD, basic domain. Following the transient transfection of individual p53 constructs into the uninduced EJ-p53 cells, expression of p53 protein levels were normalized versus cell number to measure the level of SA -β galactosidase activity by staining with Xgal. ELISA analysis was then performed using antisera against human prohibitin (Research Diagnostics, Inc.). Colormetric analysis was then used to measure the amount of SA-β galactosidase activity ELISA was performed to measure prohibitin levels (fg/ml lysate) and normalized by the amount of immunoprecipitated FLAG-tagged p53 protein used as input from the ELISA assay. ( B ) <t>Immunoblot</t> analysis was then performed with anti –prohibitin nitrocellulose filter was reused to immunoblot with an anti- β actin polyclonal antisera (Sigma-Aldrich). ( C ) Electron micrograph (10,000X) of EJ carcinoma cells transfected with the different FLAG-tagged and truncated variants of human p53 and stained with the anti-FLAG monoclonal antibody (Sigma-Aldrich). Region corresponding to the outline of the mitochrondria is indicated. ( D ) Interaction of Sirt3 with the MASD region of p53. Using FLAG-tagged variants of the deleted p53 cDNAs expressed by transient transfections of p53 shown (left) were used to identify specific interactions with endogenous Sirt3 by immunoprecipitation with M2 agarose (Sigma-Aldrich) followed by standard immunoblotting protocols.
    Immunoblot Analysis, supplied by rdi research diagnostics, used in various techniques. Bioz Stars score: 92/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare immunoblot detection
    Increased tyrosine phosphorylation of 53 kDa by K15 expression. A total of 2 × 10 6 Bjab cells were stimulated with either PBS or OKT8 (anti-CD8) antibody for 5 min and the cell extracts were subjected to a western blot with antiphospho tyrosine <t>immunoblot.</t> At the bottom, the cell surface expression of CD8 chimera proteins were measured by anti CD8-PE antibody.
    Immunoblot Detection, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 283 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Bio-Rad immunoblot development
    Increased tyrosine phosphorylation of 53 kDa by K15 expression. A total of 2 × 10 6 Bjab cells were stimulated with either PBS or OKT8 (anti-CD8) antibody for 5 min and the cell extracts were subjected to a western blot with antiphospho tyrosine <t>immunoblot.</t> At the bottom, the cell surface expression of CD8 chimera proteins were measured by anti CD8-PE antibody.
    Immunoblot Development, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    In vitro and in vivo superactivation of the PI3K–Akt pathway in an EGF-dependent and AP-2α null–dependent fashion. (A) Primary WT and KO keratinocytes were cultured ± the EGFR-specific protein kinase inhibitor AG1478 without the addition of fibroblast feeder cells after the original plating. Growth curves represents three independent experiments performed in duplicate (see Separation of epidermis…transfections). Error bars represent SD for these experiments. (B) Keratinocytes were serum-starved for 24 h and at t = 0, 50 ng/ml EGF was added. At the times indicated, cell extracts were prepared, proteins were resolved by SDS-PAGE, and immunoblot analyses were conducted with the antibodies indicated at right. The antibodies against the active forms of Erk1/2 and Akt (p-Erk and p-Akt, respectively) are phosphospecific antibodies and do not recognize the inactive states of the kinases. β-tubulin antibodies are used as a loading control, along with ponseau red staining of the blots (not depicted). Note EGF-dependent superactivation of Akt and Erk1/2 in AP-2α –null keratinocytes. (C) Same experiment as in B, except 10× increments of EGF concentrations (0–50 ng/ml) were used, and extracts were harvested at t = 2 min. (D) Same experiment as in B, but insulin growth factor 1 (IGF1) was added at 50 ng/ml. Note that in contrast to EGF, IGF1 did not generate enhanced activated Akt in KO relative to WT cells. (E) Same experiment as in B, but the PI3K-specific inhibitor LY294002 was added at 20 μM. Note that the activation of Akt, but not Erk1/2, is dependent on PI3K activation. (F) Antiphospho-Akt staining of frozen sections (8 μm) from (top) lesional KO and WT thoracic skins and (bottom) TPA-treated KO and WT back skins. Note the superactivation of Akt in KO skin regions that are lesional and that correlate with elevated EGFR ligand expression. De, dermis; epi, epidermis; hf, hair follicle. Dotted lines denote dermo–epidermal borders. Bars, 20 μm.

    Journal: The Journal of Cell Biology

    Article Title: AP-2?: a regulator of EGF receptor signaling and proliferation in skin epidermis

    doi: 10.1083/jcb.200510002

    Figure Lengend Snippet: In vitro and in vivo superactivation of the PI3K–Akt pathway in an EGF-dependent and AP-2α null–dependent fashion. (A) Primary WT and KO keratinocytes were cultured ± the EGFR-specific protein kinase inhibitor AG1478 without the addition of fibroblast feeder cells after the original plating. Growth curves represents three independent experiments performed in duplicate (see Separation of epidermis…transfections). Error bars represent SD for these experiments. (B) Keratinocytes were serum-starved for 24 h and at t = 0, 50 ng/ml EGF was added. At the times indicated, cell extracts were prepared, proteins were resolved by SDS-PAGE, and immunoblot analyses were conducted with the antibodies indicated at right. The antibodies against the active forms of Erk1/2 and Akt (p-Erk and p-Akt, respectively) are phosphospecific antibodies and do not recognize the inactive states of the kinases. β-tubulin antibodies are used as a loading control, along with ponseau red staining of the blots (not depicted). Note EGF-dependent superactivation of Akt and Erk1/2 in AP-2α –null keratinocytes. (C) Same experiment as in B, except 10× increments of EGF concentrations (0–50 ng/ml) were used, and extracts were harvested at t = 2 min. (D) Same experiment as in B, but insulin growth factor 1 (IGF1) was added at 50 ng/ml. Note that in contrast to EGF, IGF1 did not generate enhanced activated Akt in KO relative to WT cells. (E) Same experiment as in B, but the PI3K-specific inhibitor LY294002 was added at 20 μM. Note that the activation of Akt, but not Erk1/2, is dependent on PI3K activation. (F) Antiphospho-Akt staining of frozen sections (8 μm) from (top) lesional KO and WT thoracic skins and (bottom) TPA-treated KO and WT back skins. Note the superactivation of Akt in KO skin regions that are lesional and that correlate with elevated EGFR ligand expression. De, dermis; epi, epidermis; hf, hair follicle. Dotted lines denote dermo–epidermal borders. Bars, 20 μm.

    Article Snippet: The immunoblot and PCR gel images were acquired with an Alphaimager (Innotech), and Quantity One software (Bio-Rad Laboratories) was used for contrast and brightness adjustment.

    Techniques: In Vitro, In Vivo, Cell Culture, SDS Page, Staining, Activation Assay, Expressing

    EGFR levels are elevated in nonproliferative regions of adult AP-2α –null skin, but EGFR signaling is silent unless externally stimulated. (A) H E and immunofluorescence of sections of nonproliferative cKO skins and WT counterparts from P6 mice. Antibodies are against EGFR, phosphorylated (active) EGFR, and K6. Note elevated EGFR but not phospho-EGFR or K6 in P6 cKO skin. (B) H E and anti-EGFR/antiphospho-EGFR immunofluorescence of sections of nonproliferative cKO and WT adult back skins. (C) Immunoblot analyses of epidermal proteins isolated from adult back skin from WT and AP-2α conditionally null mice. Blots were probed with antibodies against the proteins indicated at right. Note elevated EGFR but not phospho-EGFR, which is normally weak in thin adult skin. (D) mRNAs were isolated from WT and cKO adult back skin or neonatal epidermis and subjected to real-time PCR using a primer set specific for TGFα mRNA. Controls shown are for the reaction involving the primer set alone in the absence of added mRNA. Error bars represent SD. (E) Back skins of adult AP-2α –null and control littermates were treated with TPA, a procedure known to elevate the expression of EGFR ligands ( Kiguchi et al., 1998 ). After treatment, skins were processed for H E and EGFR/phospho-EGFR immunofluorescence microscopy. No papillomas were observed even after 4 mo of treatment. Epidermal thickening was accompanied by EGFR activation, both of which were more robust in the TPA-treated cKO animals. Epi, epidermis; de, dermis; hf, hair follicle. Dotted lines denote dermo–epidermal borders. Bars, 20 μm.

    Journal: The Journal of Cell Biology

    Article Title: AP-2?: a regulator of EGF receptor signaling and proliferation in skin epidermis

    doi: 10.1083/jcb.200510002

    Figure Lengend Snippet: EGFR levels are elevated in nonproliferative regions of adult AP-2α –null skin, but EGFR signaling is silent unless externally stimulated. (A) H E and immunofluorescence of sections of nonproliferative cKO skins and WT counterparts from P6 mice. Antibodies are against EGFR, phosphorylated (active) EGFR, and K6. Note elevated EGFR but not phospho-EGFR or K6 in P6 cKO skin. (B) H E and anti-EGFR/antiphospho-EGFR immunofluorescence of sections of nonproliferative cKO and WT adult back skins. (C) Immunoblot analyses of epidermal proteins isolated from adult back skin from WT and AP-2α conditionally null mice. Blots were probed with antibodies against the proteins indicated at right. Note elevated EGFR but not phospho-EGFR, which is normally weak in thin adult skin. (D) mRNAs were isolated from WT and cKO adult back skin or neonatal epidermis and subjected to real-time PCR using a primer set specific for TGFα mRNA. Controls shown are for the reaction involving the primer set alone in the absence of added mRNA. Error bars represent SD. (E) Back skins of adult AP-2α –null and control littermates were treated with TPA, a procedure known to elevate the expression of EGFR ligands ( Kiguchi et al., 1998 ). After treatment, skins were processed for H E and EGFR/phospho-EGFR immunofluorescence microscopy. No papillomas were observed even after 4 mo of treatment. Epidermal thickening was accompanied by EGFR activation, both of which were more robust in the TPA-treated cKO animals. Epi, epidermis; de, dermis; hf, hair follicle. Dotted lines denote dermo–epidermal borders. Bars, 20 μm.

    Article Snippet: The immunoblot and PCR gel images were acquired with an Alphaimager (Innotech), and Quantity One software (Bio-Rad Laboratories) was used for contrast and brightness adjustment.

    Techniques: Immunofluorescence, Mouse Assay, Isolation, Real-time Polymerase Chain Reaction, Expressing, Microscopy, Activation Assay

    Intact epidermal barrier but elevated EGFR expression and signaling in hyperproliferative AP-2α –null skin. (A) Barrier function assay. Newborn litters were placed in blue dye to test for the presence of an intact epidermal barrier. Note tail cut for genotyping and umbilicus show areas of blue staining, indicating dye penetration in areas where there is loss of barrier. (B) Semiquantitative RT-PCR of neonatal epidermal mRNAs. Epidermis was separated from the rest of the skin (including hair follicles) by enzymatic treatment with dispase. After mRNA isolation, semiquantitative RT-PCR was conducted using primer sets specific for EGFR (test) and GAPDH (control) mRNAs. All bands were of the expected size and were generated only in the presence of reverse transcriptase (RT). (C) Anti-EGFR and antiphospho-EGFR immunoblot analyses of epidermal proteins isolated from neonatal mice in which the cKO epidermis is hyperproliferative. Anti-tubulin is used as a control. (D) EGFR immunofluorescence of sections of hyperproliferative cKO skins and WT counterparts from P0 mice. Inset shows a high magnification confocal image of the colocalization of phospho-EGFR and E-cadherin at the membrane. Arrows denote suprabasal phospho-EGFR staining. (E) Same as in D, but on adult thoracic skins. Epi, epidermis; de, dermis; hf, hair follicle. Dotted lines denote dermo–epidermal borders. Solid white lines denote skin surface. (F) Same as in C, but on adult epidermis. Normalizations are to anti-tubulin. Bars, 20 μm.

    Journal: The Journal of Cell Biology

    Article Title: AP-2?: a regulator of EGF receptor signaling and proliferation in skin epidermis

    doi: 10.1083/jcb.200510002

    Figure Lengend Snippet: Intact epidermal barrier but elevated EGFR expression and signaling in hyperproliferative AP-2α –null skin. (A) Barrier function assay. Newborn litters were placed in blue dye to test for the presence of an intact epidermal barrier. Note tail cut for genotyping and umbilicus show areas of blue staining, indicating dye penetration in areas where there is loss of barrier. (B) Semiquantitative RT-PCR of neonatal epidermal mRNAs. Epidermis was separated from the rest of the skin (including hair follicles) by enzymatic treatment with dispase. After mRNA isolation, semiquantitative RT-PCR was conducted using primer sets specific for EGFR (test) and GAPDH (control) mRNAs. All bands were of the expected size and were generated only in the presence of reverse transcriptase (RT). (C) Anti-EGFR and antiphospho-EGFR immunoblot analyses of epidermal proteins isolated from neonatal mice in which the cKO epidermis is hyperproliferative. Anti-tubulin is used as a control. (D) EGFR immunofluorescence of sections of hyperproliferative cKO skins and WT counterparts from P0 mice. Inset shows a high magnification confocal image of the colocalization of phospho-EGFR and E-cadherin at the membrane. Arrows denote suprabasal phospho-EGFR staining. (E) Same as in D, but on adult thoracic skins. Epi, epidermis; de, dermis; hf, hair follicle. Dotted lines denote dermo–epidermal borders. Solid white lines denote skin surface. (F) Same as in C, but on adult epidermis. Normalizations are to anti-tubulin. Bars, 20 μm.

    Article Snippet: The immunoblot and PCR gel images were acquired with an Alphaimager (Innotech), and Quantity One software (Bio-Rad Laboratories) was used for contrast and brightness adjustment.

    Techniques: Expressing, Functional Assay, Staining, Reverse Transcription Polymerase Chain Reaction, Isolation, Generated, Mouse Assay, Immunofluorescence

    Targeted ablation of AP-2α gene expression in mouse epidermis. K14-Cre:AP-2α lox/lox conditional knockout (cKO) mice were generated and compared with their control wild-type (WT) littermates. (A) PCR genotype analysis. Bands were of the expected sizes. (B) Real-time PCR on P0 epidermal mRNAs. Primers are specific for the five known AP-2 family members. Error bars represent SD. (C) Immunoblot analyses of P0 epidermal extracts probed with antibodies to AP-2α and AP-2γ, the two most abundantly expressed members. (D) Immunofluorescence microscopy. Color coding is for secondary antibodies used in detection. Nuclei are counterstained with DAPI. Note that in WT skin, the basal epidermal layer displayed some cells with strong anti–AP-2α labeling (white arrows) and others with weaker labeling (yellow arrows). In the inner spinous layers, cells were typically strongly labeled. Anti-AP2α labeling was uniformly absent in cKO skin. De, dermis; epi, epidermis; hf, hair follicle. Dotted white lines denote dermo–epidermal boundaries. Solid white lines denote skin surface. Bars, 20 μm.

    Journal: The Journal of Cell Biology

    Article Title: AP-2?: a regulator of EGF receptor signaling and proliferation in skin epidermis

    doi: 10.1083/jcb.200510002

    Figure Lengend Snippet: Targeted ablation of AP-2α gene expression in mouse epidermis. K14-Cre:AP-2α lox/lox conditional knockout (cKO) mice were generated and compared with their control wild-type (WT) littermates. (A) PCR genotype analysis. Bands were of the expected sizes. (B) Real-time PCR on P0 epidermal mRNAs. Primers are specific for the five known AP-2 family members. Error bars represent SD. (C) Immunoblot analyses of P0 epidermal extracts probed with antibodies to AP-2α and AP-2γ, the two most abundantly expressed members. (D) Immunofluorescence microscopy. Color coding is for secondary antibodies used in detection. Nuclei are counterstained with DAPI. Note that in WT skin, the basal epidermal layer displayed some cells with strong anti–AP-2α labeling (white arrows) and others with weaker labeling (yellow arrows). In the inner spinous layers, cells were typically strongly labeled. Anti-AP2α labeling was uniformly absent in cKO skin. De, dermis; epi, epidermis; hf, hair follicle. Dotted white lines denote dermo–epidermal boundaries. Solid white lines denote skin surface. Bars, 20 μm.

    Article Snippet: The immunoblot and PCR gel images were acquired with an Alphaimager (Innotech), and Quantity One software (Bio-Rad Laboratories) was used for contrast and brightness adjustment.

    Techniques: Expressing, Knock-Out, Mouse Assay, Generated, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Immunofluorescence, Microscopy, Labeling

    Regulation of EGFR gene expression by AP-2α. (A) Primary WT and AP-2α –null keratinocytes were cultured in rich medium (15% serum, growth factors, high density, and with feeder layer). When indicated, 1.5 mM Ca 2+ was added to induce terminal differentiation. Protein lysates and RNA were prepared after removal of the feeder cells by treatment with 0.1 mM EDTA. Immunoblots of keratinocyte proteins probed for AP-2α and AP-2γ are shown, along with real-time PCR of keratinocyte mRNAs tested for K1 and involucrin expression. Real-time data are normalized to 0.05 mM calcium (= 1). (B) Immunoblot analysis and real-time PCR of keratinocyte protein and mRNAs tested for EGFR expression. (C) Chromatin immunoprecipitation (ChIP). (top) Within 1.2 kb of sequence 5′ from the putative transcriptional initiation site of the mouse EGFR gene are four consensus AP-2–binding sites (ovals), two of which are conserved (in red). ChIP, WT, and KO keratinocytes were either treated or not treated with formaldehyde to transiently cross-link proteins to chromatin. Both cross-linked and noncross-linked chromatin was isolated and sheared to ∼400-bp fragments. Immunoprecipitations were then performed using anti-AP2α or no antibody or control IgG antisera. PCR analyses on the immunoprecipitated fragments were performed using primers encompassing the putative AP-2–binding sites located within the EGFR promoter (marked on the diagram). Note that only the combination of formaldehyde cross-linking and the application of anti–AP-2α antibody specifically immunoprecipitated AP-2α site-containing EGFR promoter sequences in WT and not in KO keratinocytes. (D) Luciferase assays of EGFR promoter activity in primary WT or KO mouse keratinocytes ± infection with the AP-2α–expressing retroviral vector (see Separation of epidermis…transfections). 1.2 kb of sequence 5′ from the transcription initiation site of the EGFR promoter (see C) was used to drive firefly luciferase expression (pEGFRpr-Luc). pMutEGFRpr-luc was engineered by introducing point mutations in the three AP-2 sites within the parent vector. Fold increases represent EGFRpr firefly luciferase activity divided by the basal level of p-Luc firefly luciferase activity, with cytomegalo virus– Renilla luciferase activity as the standardized internal control for transfection efficiency. The graph represents three independent experiments performed in duplicate. Error bars represent SD for these experiments. Asterisk denotes a significant difference from WT cells (P

    Journal: The Journal of Cell Biology

    Article Title: AP-2?: a regulator of EGF receptor signaling and proliferation in skin epidermis

    doi: 10.1083/jcb.200510002

    Figure Lengend Snippet: Regulation of EGFR gene expression by AP-2α. (A) Primary WT and AP-2α –null keratinocytes were cultured in rich medium (15% serum, growth factors, high density, and with feeder layer). When indicated, 1.5 mM Ca 2+ was added to induce terminal differentiation. Protein lysates and RNA were prepared after removal of the feeder cells by treatment with 0.1 mM EDTA. Immunoblots of keratinocyte proteins probed for AP-2α and AP-2γ are shown, along with real-time PCR of keratinocyte mRNAs tested for K1 and involucrin expression. Real-time data are normalized to 0.05 mM calcium (= 1). (B) Immunoblot analysis and real-time PCR of keratinocyte protein and mRNAs tested for EGFR expression. (C) Chromatin immunoprecipitation (ChIP). (top) Within 1.2 kb of sequence 5′ from the putative transcriptional initiation site of the mouse EGFR gene are four consensus AP-2–binding sites (ovals), two of which are conserved (in red). ChIP, WT, and KO keratinocytes were either treated or not treated with formaldehyde to transiently cross-link proteins to chromatin. Both cross-linked and noncross-linked chromatin was isolated and sheared to ∼400-bp fragments. Immunoprecipitations were then performed using anti-AP2α or no antibody or control IgG antisera. PCR analyses on the immunoprecipitated fragments were performed using primers encompassing the putative AP-2–binding sites located within the EGFR promoter (marked on the diagram). Note that only the combination of formaldehyde cross-linking and the application of anti–AP-2α antibody specifically immunoprecipitated AP-2α site-containing EGFR promoter sequences in WT and not in KO keratinocytes. (D) Luciferase assays of EGFR promoter activity in primary WT or KO mouse keratinocytes ± infection with the AP-2α–expressing retroviral vector (see Separation of epidermis…transfections). 1.2 kb of sequence 5′ from the transcription initiation site of the EGFR promoter (see C) was used to drive firefly luciferase expression (pEGFRpr-Luc). pMutEGFRpr-luc was engineered by introducing point mutations in the three AP-2 sites within the parent vector. Fold increases represent EGFRpr firefly luciferase activity divided by the basal level of p-Luc firefly luciferase activity, with cytomegalo virus– Renilla luciferase activity as the standardized internal control for transfection efficiency. The graph represents three independent experiments performed in duplicate. Error bars represent SD for these experiments. Asterisk denotes a significant difference from WT cells (P

    Article Snippet: The immunoblot and PCR gel images were acquired with an Alphaimager (Innotech), and Quantity One software (Bio-Rad Laboratories) was used for contrast and brightness adjustment.

    Techniques: Expressing, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Sequencing, Binding Assay, Isolation, Polymerase Chain Reaction, Immunoprecipitation, Luciferase, Activity Assay, Infection, Plasmid Preparation, Transfection

    Immunoblot analyis of γH2AX and p21 . γH2AX-expression measured immunhistochemically 2 hours after HIT and p21 expression in KHOS-24OS, A-204 and HFOB1.19 treated with vehicle control (C), 0,5 (A-204)-1 μM (KHOS-24OS and HFOB1.19) SAHA and HIT (cell specific doses, see Figure 9) or the combination of SAHA and HIT 24 h after HIT.

    Journal: Radiation Oncology (London, England)

    Article Title: Combination of suberoylanilide hydroxamic acid with heavy ion therapy shows promising effects in infantile sarcoma cell lines

    doi: 10.1186/1748-717X-6-119

    Figure Lengend Snippet: Immunoblot analyis of γH2AX and p21 . γH2AX-expression measured immunhistochemically 2 hours after HIT and p21 expression in KHOS-24OS, A-204 and HFOB1.19 treated with vehicle control (C), 0,5 (A-204)-1 μM (KHOS-24OS and HFOB1.19) SAHA and HIT (cell specific doses, see Figure 9) or the combination of SAHA and HIT 24 h after HIT.

    Article Snippet: Primary monoclonal mouse antibodies against ß-actin as well as a secondary antibody for immunoblot experiments were purchased from CellSignaling Technology (Danvers, MA, USA).

    Techniques: Expressing

    AQP6 protein expression in mouse kidney homogenate. Immunoblot of mouse kidney total homogenate was performed as described (see Methods). Two bands of about 55 and 36 kDa were observed. Similar results were obtained from at least four separate experiments. MW, molecular weight markers.

    Journal: Journal of Biomedicine and Biotechnology

    Article Title: Aquaporin-6 Expression in the Cochlear Sensory Epithelium Is Downregulated by Salicylates

    doi: 10.1155/2010/264704

    Figure Lengend Snippet: AQP6 protein expression in mouse kidney homogenate. Immunoblot of mouse kidney total homogenate was performed as described (see Methods). Two bands of about 55 and 36 kDa were observed. Similar results were obtained from at least four separate experiments. MW, molecular weight markers.

    Article Snippet: ChemiBlot Molecular Weight Markers were used to accurately estimate the molecular weight and as a positive control for the immunoblot (Chemicon International, Inc., CA, USA).

    Techniques: Expressing, Molecular Weight

    Dominant negative effect of H3.3R26K or H3.3K27R in oocytes accelerates oogenesis. (A) Scheme of HISRainbow; Cre oocytes with four copies of the chromosomes that express 1-2 alleles of WT and/or H3.3 mutants. (B) Confocal (full projection) and DIC images of MII oocytes isolated from 8- to 12-week-old HISRainbow; Figla -iCre mice. Oocytes expressed H3.3 eGFP (G), H3.3R26K eCFP (B), or H3.3K27R mCherry (R), from one or two alleles. Scale bar: 50 µm. (C) Immunoblot analysis of HISRainbow; Figla -iCre GV oocytes with indicated genotypes for H3.3 expression. 25 oocytes were collected for each group: WT, H3.3 eGFP (GG), H3.3K27R mCherry (RR) and H3.3R26K eCFP ) in: oocytes in HISRainbow; Figla -iCre P2 ovaries (6 replicates); MII oocytes from 8- to 12-week-old HISRainbow; Figla -iCre (7 replicates, 191 oocytes); and HISRainbow; ZP3 -Cre mice (8 replicates, 193 oocytes). Percentage of each subpopulation was normalized to the percentage of oocytes expressing H3.3 eGFP , which was set to 1. Mean±s.e.m. (E) Quantification of histone modifications in HISRainbow; Figla -iCre oocytes. Mean±s.e.m of n ≥6.; * p

    Journal: Development (Cambridge, England)

    Article Title: Genetic mosaics and time-lapse imaging identify functions of histone H3.3 residues in mouse oocytes and embryos

    doi: 10.1242/dev.141390

    Figure Lengend Snippet: Dominant negative effect of H3.3R26K or H3.3K27R in oocytes accelerates oogenesis. (A) Scheme of HISRainbow; Cre oocytes with four copies of the chromosomes that express 1-2 alleles of WT and/or H3.3 mutants. (B) Confocal (full projection) and DIC images of MII oocytes isolated from 8- to 12-week-old HISRainbow; Figla -iCre mice. Oocytes expressed H3.3 eGFP (G), H3.3R26K eCFP (B), or H3.3K27R mCherry (R), from one or two alleles. Scale bar: 50 µm. (C) Immunoblot analysis of HISRainbow; Figla -iCre GV oocytes with indicated genotypes for H3.3 expression. 25 oocytes were collected for each group: WT, H3.3 eGFP (GG), H3.3K27R mCherry (RR) and H3.3R26K eCFP ) in: oocytes in HISRainbow; Figla -iCre P2 ovaries (6 replicates); MII oocytes from 8- to 12-week-old HISRainbow; Figla -iCre (7 replicates, 191 oocytes); and HISRainbow; ZP3 -Cre mice (8 replicates, 193 oocytes). Percentage of each subpopulation was normalized to the percentage of oocytes expressing H3.3 eGFP , which was set to 1. Mean±s.e.m. (E) Quantification of histone modifications in HISRainbow; Figla -iCre oocytes. Mean±s.e.m of n ≥6.; * p

    Article Snippet: Secondary antibodies for immunofluorescence (Thermo Fisher Scientific) or immunoblot (Santa Cruz) were used according to the manufacturer's instructions.

    Techniques: Dominant Negative Mutation, Isolation, Mouse Assay, Expressing

    Analysis of Arthrobacter mysorens . A) Randomly amplified polymorphic DNA (RAPD) analysis of the clinical isolate (lane 1) and the soil isolate (lane 2) using primer P1 [5'-GGTGCGGGAA-3'] and primer P5 [5'-AACGCGCAAC-3']. The analyses were done with the

    Journal: BMC Infectious Diseases

    Article Title: Erythema caused by a localised skin infection with Arthrobacter mysorens

    doi: 10.1186/1471-2334-10-352

    Figure Lengend Snippet: Analysis of Arthrobacter mysorens . A) Randomly amplified polymorphic DNA (RAPD) analysis of the clinical isolate (lane 1) and the soil isolate (lane 2) using primer P1 [5'-GGTGCGGGAA-3'] and primer P5 [5'-AACGCGCAAC-3']. The analyses were done with the "Ready To Go RAPD analysis beads" test kit from Pharmacia (Fribourg, Germany). B) Yellow-pigmented A. mysorens clinical isolates grown on sheep blood agar plates after an incubation period of 72 hours at room temperature. C) Scanning electron microscopy of A.mys orens clinical isolates. Magnification: 30,000 ×. D) Immunoblot analysis of the patient's serum using a whole cell extract of A. mysorens clinical isolate. Arrow heads indicate the two reacting proteins of ~53-kDa and ~32-kDa, respectively.

    Article Snippet: An immunoblot (Recomblot Borrelia, Mikrogen, Germany) with the patient's serum revealed a B. burgdorferi sensu lato -specific, IgG antibody response to p100, p41, BmpA, OspC (weakly positive), p41, and p18 but no IgM-specific antibody response could be detected.

    Techniques: Amplification, Incubation, Electron Microscopy

    Levels of ROCK protein in ROCKII −/− mice. Brain and spinal cord tissue from ROCKII −/− and wild-type mice was examined by immunoblot for ROCKII and ROCKI protein. A representative blotfor each antigen is shown from one pair

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Rho-Associated Kinase II (ROCKII) Limits Axonal Growth after Trauma within the Adult Mouse Spinal Cord

    doi: 10.1523/JNEUROSCI.4650-09.2009

    Figure Lengend Snippet: Levels of ROCK protein in ROCKII −/− mice. Brain and spinal cord tissue from ROCKII −/− and wild-type mice was examined by immunoblot for ROCKII and ROCKI protein. A representative blotfor each antigen is shown from one pair

    Article Snippet: Total homogenates of adult mouse brain and spinal cord were assessed by immunoblot using the following primary antibodies: anti-ROCKII, mouse monoclonal antibody directed against amino acids 906–1012 (BD Transduction Laboratories); anti-ROCKI, mouse monoclonal antibody directed against amino acids 567–718 (BD Transduction Laboratories); and anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH), rabbit polyclonal antibody (Santa Cruz Biotechnology).

    Techniques: Mouse Assay

    A : representative gel images of immunoblot analyses using antibodies against the free fatty acid receptor 1 (FFAR1) and FFAR4 using total protein prepared from human ( i ) and guinea pig ( ii ) tissues: human brain cerebral cortex (50 μg), freshly

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Novel identification of the free fatty acid receptor FFAR1 that promotes contraction in airway smooth muscle

    doi: 10.1152/ajplung.00041.2015

    Figure Lengend Snippet: A : representative gel images of immunoblot analyses using antibodies against the free fatty acid receptor 1 (FFAR1) and FFAR4 using total protein prepared from human ( i ) and guinea pig ( ii ) tissues: human brain cerebral cortex (50 μg), freshly

    Article Snippet: Different antibodies were used to detect the FFAR1 protein in immunoblot and immunohistochemistry experiments because 1 ) the antibody used for immunoblot (3393-1; Epitomics) is not recommended for immunohistochemistry in paraffin-embedded tissues, and 2 ) the antibody used for immunohistochemistry (sc-32905; Santa Cruz Biotechnology) has been used previously for immunohistochemistry in paraffin-embedded tissues ( ).

    Techniques:

    Involvement of FFAR1 and FFAR4 in transient [Ca 2+ ] i increases induced by long-chain free fatty acids (oleic acid or linoleic acid) in HASM cells. A and B : representative gel images of immunoblot analyses of FFAR1 ( A ) and FFAR4 ( B ) protein expression in

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Novel identification of the free fatty acid receptor FFAR1 that promotes contraction in airway smooth muscle

    doi: 10.1152/ajplung.00041.2015

    Figure Lengend Snippet: Involvement of FFAR1 and FFAR4 in transient [Ca 2+ ] i increases induced by long-chain free fatty acids (oleic acid or linoleic acid) in HASM cells. A and B : representative gel images of immunoblot analyses of FFAR1 ( A ) and FFAR4 ( B ) protein expression in

    Article Snippet: Different antibodies were used to detect the FFAR1 protein in immunoblot and immunohistochemistry experiments because 1 ) the antibody used for immunoblot (3393-1; Epitomics) is not recommended for immunohistochemistry in paraffin-embedded tissues, and 2 ) the antibody used for immunohistochemistry (sc-32905; Santa Cruz Biotechnology) has been used previously for immunohistochemistry in paraffin-embedded tissues ( ).

    Techniques: Expressing

    and protein levels on chromatin were analyzed by immunoblotting.

    Journal: Cell cycle (Georgetown, Tex.)

    Article Title: Xenopus DNA2 is a helicase/nuclease that is found in complexes with replication proteins And-1/Ctf4 and Mcm10 and DSB response proteins Nbs1 and ATM

    doi:

    Figure Lengend Snippet: and protein levels on chromatin were analyzed by immunoblotting.

    Article Snippet: Only hits with an ion score above 500 are listed. (B) Immunoprecipitations from interphase extract were performed with control or anti-And-1 antibodies, and immunoprecipitates were analyzed by immunoblotting. (C) Control IgG (Mock) and anti-Dna2 antibodies were used for immunoprecipitations from interphase extracts, and samples were analyzed by immunoblotting. (D) Dna2 was immunoprecipitated in interphase extract using anti-Dna2 antibodies, and isolates were analyzed by immunoblotting. (E) Control and anti-Mcm10 antibodies were used to immunoprecipitate proteins from interphase extract.

    Techniques:

    The electrophoretic mobility of 35 S-Chk1 was monitored by autoradiography after a 100 min incubation in extract containing pA/T70, and 35 Dna2 levels in nuclei were assessed by immunoblotting, while 35 S-Chk1 electrophoretic mobility was assessed by SDS-PAGE and autoradiography.

    Journal: Cell cycle (Georgetown, Tex.)

    Article Title: Xenopus DNA2 is a helicase/nuclease that is found in complexes with replication proteins And-1/Ctf4 and Mcm10 and DSB response proteins Nbs1 and ATM

    doi:

    Figure Lengend Snippet: The electrophoretic mobility of 35 S-Chk1 was monitored by autoradiography after a 100 min incubation in extract containing pA/T70, and 35 Dna2 levels in nuclei were assessed by immunoblotting, while 35 S-Chk1 electrophoretic mobility was assessed by SDS-PAGE and autoradiography.

    Article Snippet: Only hits with an ion score above 500 are listed. (B) Immunoprecipitations from interphase extract were performed with control or anti-And-1 antibodies, and immunoprecipitates were analyzed by immunoblotting. (C) Control IgG (Mock) and anti-Dna2 antibodies were used for immunoprecipitations from interphase extracts, and samples were analyzed by immunoblotting. (D) Dna2 was immunoprecipitated in interphase extract using anti-Dna2 antibodies, and isolates were analyzed by immunoblotting. (E) Control and anti-Mcm10 antibodies were used to immunoprecipitate proteins from interphase extract.

    Techniques: Autoradiography, Incubation, SDS Page

    Dna2 and MRN at DNA ends. (A) Effect of Dna2 depletion on processing of DNA ends. Interphase extracts were untreated, mock or Dna2-depleted, and incubated with the appropriate beads for 15 or 30 min. Beads were isolated and protein binding was assessed by immunoblotting. (B) DNA end binding of proteins in Nbs1-depleted extract. Extracts were untreated, mock-depleted, or Nbs1-depleted, which depletes the whole MRN complex, and incubated with the appropriate beads for 15 or 30 min. Beads were isolated, and protein binding to the beads was analyzed by immunoblotting. (C) Mirin was used to inhibit the nuclease activity of Mre11. Mirin or DMSO was incubated in extracts with the appropriate beads. Beads were isolated at the indicated times and protein levels were analyzed by immunoblotting.

    Journal: Cell cycle (Georgetown, Tex.)

    Article Title: Xenopus DNA2 is a helicase/nuclease that is found in complexes with replication proteins And-1/Ctf4 and Mcm10 and DSB response proteins Nbs1 and ATM

    doi:

    Figure Lengend Snippet: Dna2 and MRN at DNA ends. (A) Effect of Dna2 depletion on processing of DNA ends. Interphase extracts were untreated, mock or Dna2-depleted, and incubated with the appropriate beads for 15 or 30 min. Beads were isolated and protein binding was assessed by immunoblotting. (B) DNA end binding of proteins in Nbs1-depleted extract. Extracts were untreated, mock-depleted, or Nbs1-depleted, which depletes the whole MRN complex, and incubated with the appropriate beads for 15 or 30 min. Beads were isolated, and protein binding to the beads was analyzed by immunoblotting. (C) Mirin was used to inhibit the nuclease activity of Mre11. Mirin or DMSO was incubated in extracts with the appropriate beads. Beads were isolated at the indicated times and protein levels were analyzed by immunoblotting.

    Article Snippet: Only hits with an ion score above 500 are listed. (B) Immunoprecipitations from interphase extract were performed with control or anti-And-1 antibodies, and immunoprecipitates were analyzed by immunoblotting. (C) Control IgG (Mock) and anti-Dna2 antibodies were used for immunoprecipitations from interphase extracts, and samples were analyzed by immunoblotting. (D) Dna2 was immunoprecipitated in interphase extract using anti-Dna2 antibodies, and isolates were analyzed by immunoblotting. (E) Control and anti-Mcm10 antibodies were used to immunoprecipitate proteins from interphase extract.

    Techniques: Incubation, Isolation, Protein Binding, Binding Assay, Activity Assay

    and chromatin-associated proteins were analyzed by immunoblotting.

    Journal: Cell cycle (Georgetown, Tex.)

    Article Title: Xenopus DNA2 is a helicase/nuclease that is found in complexes with replication proteins And-1/Ctf4 and Mcm10 and DSB response proteins Nbs1 and ATM

    doi:

    Figure Lengend Snippet: and chromatin-associated proteins were analyzed by immunoblotting.

    Article Snippet: Only hits with an ion score above 500 are listed. (B) Immunoprecipitations from interphase extract were performed with control or anti-And-1 antibodies, and immunoprecipitates were analyzed by immunoblotting. (C) Control IgG (Mock) and anti-Dna2 antibodies were used for immunoprecipitations from interphase extracts, and samples were analyzed by immunoblotting. (D) Dna2 was immunoprecipitated in interphase extract using anti-Dna2 antibodies, and isolates were analyzed by immunoblotting. (E) Control and anti-Mcm10 antibodies were used to immunoprecipitate proteins from interphase extract.

    Techniques:

    Dna2 at DNA ends. (A) Schematic of beads used for experiments. pBluescriptIIKS-was linearized and biotinylated on one or both ends, and bound to streptavidin beads. These beads simulated unbroken DNA or DNA with a DSB. (B) Time-course of binding of DSB repair and checkpoint proteins to DNA ends. Beads were incubated in interphase extract, isolated at indicated time-points, and the relative amounts of Dna2, ATM, Nbs1, RPA70 and ATR bound to the beads were analyzed by immunoblotting. (C) Time-course of binding of DSB proteins to DNA ends in CSF extract. Experiment was performed as described for (B) of this figure, except in CSF, not interphase, extract.

    Journal: Cell cycle (Georgetown, Tex.)

    Article Title: Xenopus DNA2 is a helicase/nuclease that is found in complexes with replication proteins And-1/Ctf4 and Mcm10 and DSB response proteins Nbs1 and ATM

    doi:

    Figure Lengend Snippet: Dna2 at DNA ends. (A) Schematic of beads used for experiments. pBluescriptIIKS-was linearized and biotinylated on one or both ends, and bound to streptavidin beads. These beads simulated unbroken DNA or DNA with a DSB. (B) Time-course of binding of DSB repair and checkpoint proteins to DNA ends. Beads were incubated in interphase extract, isolated at indicated time-points, and the relative amounts of Dna2, ATM, Nbs1, RPA70 and ATR bound to the beads were analyzed by immunoblotting. (C) Time-course of binding of DSB proteins to DNA ends in CSF extract. Experiment was performed as described for (B) of this figure, except in CSF, not interphase, extract.

    Article Snippet: Only hits with an ion score above 500 are listed. (B) Immunoprecipitations from interphase extract were performed with control or anti-And-1 antibodies, and immunoprecipitates were analyzed by immunoblotting. (C) Control IgG (Mock) and anti-Dna2 antibodies were used for immunoprecipitations from interphase extracts, and samples were analyzed by immunoblotting. (D) Dna2 was immunoprecipitated in interphase extract using anti-Dna2 antibodies, and isolates were analyzed by immunoblotting. (E) Control and anti-Mcm10 antibodies were used to immunoprecipitate proteins from interphase extract.

    Techniques: Binding Assay, Incubation, Isolation

    LKB1 contributes to lovastatin‐induced AMPK, p38MAPK and p53 phosphorylation in MCF‐7 cells. A, Cells were transfected with negative control siRNA or LKB1 siRNA for 48 h. After transfection, cells were treated with vehicle or lovastatin (30 μmol/L) for another 1 h. The extent of LKB1 and phosphorylation status of AMPK, p38MAPK or p53 was determined by immunoblotting. The compiled results of AMPK (B), p38MAPK (C) and p53 (D) phosphorylations are shown. Each column represents the mean ± SEM of six independent experiments (Statistically significant differences were determined using the Mann‐Whitney test. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Lovastatin‐mediated MCF‐7 cancer cell death involves LKB1‐AMPK‐p38MAPK‐p53‐survivin signalling cascade. Lovastatin‐mediated MCF‐7 cancer cell death involves LKB1‐AMPK‐p38MAPK‐p53‐survivin signalling cascade

    doi: 10.1111/jcmm.14879

    Figure Lengend Snippet: LKB1 contributes to lovastatin‐induced AMPK, p38MAPK and p53 phosphorylation in MCF‐7 cells. A, Cells were transfected with negative control siRNA or LKB1 siRNA for 48 h. After transfection, cells were treated with vehicle or lovastatin (30 μmol/L) for another 1 h. The extent of LKB1 and phosphorylation status of AMPK, p38MAPK or p53 was determined by immunoblotting. The compiled results of AMPK (B), p38MAPK (C) and p53 (D) phosphorylations are shown. Each column represents the mean ± SEM of six independent experiments (Statistically significant differences were determined using the Mann‐Whitney test. * P

    Article Snippet: All materials for immunoblotting were purchased from Bio‐Rad.

    Techniques: Transfection, Negative Control, MANN-WHITNEY

    AMPK mediates lovastatin‐induced p38MAPK and p53 phosphorylation in MCF‐7 cells. Cells were treated with vehicle or lovastatin at 30 μmol/L for indicated periods. The extent of LKB1 (MW 54 kD) (A) or AMPK (MW 62 kD) (B) phosphorylation was determined by immunoblotting. Each column represents the mean ± SEM of six independent experiments (Statistically significant differences were determined using the Kruskal‐Wallis test. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Lovastatin‐mediated MCF‐7 cancer cell death involves LKB1‐AMPK‐p38MAPK‐p53‐survivin signalling cascade. Lovastatin‐mediated MCF‐7 cancer cell death involves LKB1‐AMPK‐p38MAPK‐p53‐survivin signalling cascade

    doi: 10.1111/jcmm.14879

    Figure Lengend Snippet: AMPK mediates lovastatin‐induced p38MAPK and p53 phosphorylation in MCF‐7 cells. Cells were treated with vehicle or lovastatin at 30 μmol/L for indicated periods. The extent of LKB1 (MW 54 kD) (A) or AMPK (MW 62 kD) (B) phosphorylation was determined by immunoblotting. Each column represents the mean ± SEM of six independent experiments (Statistically significant differences were determined using the Kruskal‐Wallis test. * P

    Article Snippet: All materials for immunoblotting were purchased from Bio‐Rad.

    Techniques:

    p38MAPK contributes to lovastatin‐induced p53 activation, p21 elevation and survivin reduction in MCF‐7 cells. A, Cells were treated with vehicle or lovastatin at 30 μmol/L for indicated periods. The extent of p38MAPK phosphorylation (MW 38 kD) was examined by immunoblotting. Each column represents the mean ± SEM of six independent experiments (Statistically significant differences were determined using the Kruskal‐Wallis test. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Lovastatin‐mediated MCF‐7 cancer cell death involves LKB1‐AMPK‐p38MAPK‐p53‐survivin signalling cascade. Lovastatin‐mediated MCF‐7 cancer cell death involves LKB1‐AMPK‐p38MAPK‐p53‐survivin signalling cascade

    doi: 10.1111/jcmm.14879

    Figure Lengend Snippet: p38MAPK contributes to lovastatin‐induced p53 activation, p21 elevation and survivin reduction in MCF‐7 cells. A, Cells were treated with vehicle or lovastatin at 30 μmol/L for indicated periods. The extent of p38MAPK phosphorylation (MW 38 kD) was examined by immunoblotting. Each column represents the mean ± SEM of six independent experiments (Statistically significant differences were determined using the Kruskal‐Wallis test. * P

    Article Snippet: All materials for immunoblotting were purchased from Bio‐Rad.

    Techniques: Activation Assay

    Lovastatin caused p53 activation in MCF‐7 cells. A, MCF‐7 cells were treated with vehicle or lovastatin at 30 μmol/L for indicated periods. The phosphorylation or acetylation status of p53 (MW 53 kD) was determined by immunoblotting. Each column represents the mean ± SEM of seven independent experiments (Statistically significant differences were determined using the Kruskal‐Wallis test. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Lovastatin‐mediated MCF‐7 cancer cell death involves LKB1‐AMPK‐p38MAPK‐p53‐survivin signalling cascade. Lovastatin‐mediated MCF‐7 cancer cell death involves LKB1‐AMPK‐p38MAPK‐p53‐survivin signalling cascade

    doi: 10.1111/jcmm.14879

    Figure Lengend Snippet: Lovastatin caused p53 activation in MCF‐7 cells. A, MCF‐7 cells were treated with vehicle or lovastatin at 30 μmol/L for indicated periods. The phosphorylation or acetylation status of p53 (MW 53 kD) was determined by immunoblotting. Each column represents the mean ± SEM of seven independent experiments (Statistically significant differences were determined using the Kruskal‐Wallis test. * P

    Article Snippet: All materials for immunoblotting were purchased from Bio‐Rad.

    Techniques: Activation Assay

    Temporal transcriptome of IMR90 fibroblasts inducibly expressing MCPyV ST. A) IMR90 fibroblasts containing dox-inducible MCPyV ST or GFP vectors were treated with dox and harvested every 8 hours for RNA extraction. Each time point represents three biological replicas. B) Mean ST transcript levels and C) immunoblotting for ST, GFP and vinculin from cells collected every 8 hours for 96 hours following dox treatment. D) Hierarchical clustering and fold change between MCPyV ST and GFP following dox induction for 96 hours. Each bar represents an average of three experiments for each time point. The enrichment of “Cancer Hallmark” gene sets are represented relative to the ST-differentially expressed clusters, including epithelial to mesenchymal transition (EMT), tumor necrosis factor-α (TNFA signaling via NF-κB), hypoxia, mTORC1, oxidative phosphorylation, glycolysis, MYC, and several cell cycle clusters including E2F targets, G2M checkpoint and mitotic spindle. The color bar indicates statistical significance, yellow p

    Journal: PLoS Pathogens

    Article Title: Merkel Cell Polyomavirus Small T Antigen Promotes Pro-Glycolytic Metabolic Perturbations Required for Transformation

    doi: 10.1371/journal.ppat.1006020

    Figure Lengend Snippet: Temporal transcriptome of IMR90 fibroblasts inducibly expressing MCPyV ST. A) IMR90 fibroblasts containing dox-inducible MCPyV ST or GFP vectors were treated with dox and harvested every 8 hours for RNA extraction. Each time point represents three biological replicas. B) Mean ST transcript levels and C) immunoblotting for ST, GFP and vinculin from cells collected every 8 hours for 96 hours following dox treatment. D) Hierarchical clustering and fold change between MCPyV ST and GFP following dox induction for 96 hours. Each bar represents an average of three experiments for each time point. The enrichment of “Cancer Hallmark” gene sets are represented relative to the ST-differentially expressed clusters, including epithelial to mesenchymal transition (EMT), tumor necrosis factor-α (TNFA signaling via NF-κB), hypoxia, mTORC1, oxidative phosphorylation, glycolysis, MYC, and several cell cycle clusters including E2F targets, G2M checkpoint and mitotic spindle. The color bar indicates statistical significance, yellow p

    Article Snippet: Immunoblotting The following antibodies were used: MCPyV Ab5 [ , ]; GFP (D5.1, Cell Signaling); vinculin (H-10, Santa Cruz); actin (D6A8, Cell Signaling); HK2 (C64G5, Cell Signaling); LDHA (EP1565Y, Abcam); MCT1 (A1512, NeoBiolab); LAT1 (5347, Cell Signaling); RelA (D14E12, Cell Signaling); IκBα (L35A5, Cell Signaling); MYC (9E10, Santa Cruz); MYCN (9405, Cell Signaling); MYCL (AF4050, R & D Systems).

    Techniques: Expressing, RNA Extraction

    Heparitinase treatment and blockade of sulfation diminish responses of cultured cells to BMP2. (A) Smad phosphorylation and p38 MAPK activation in mouse C2C12 and rat PC12 cells. Cells maintained in serum-free medium were treated with human recombinant BMP2 at 5 ng/ml for 1 h. Where indicated, cultures also were treated with 25 mIU/ml heparitinase 1 h before BMP addition and throughout the remainder of the assay. Cells lysates were subjected to immunoblotting for phospho-Smad1/5/8 (P-Smad) and active p38. Total Smad, total p38, and β-tubulin served as loading controls. (B and C) Kinetic profiles of BMP-induced Smad phosphorylation. C2C12 (B) or PC12 cells (C) were treated with heparitinase for 1 h, and BMP2 (5 ng/ml) was added. Cell lysates were collected at indicated time points and subjected to immunoblotting for P-Smad. Data are normalized to loading controls. (D and E) Exogenous heparin does not rescue cells from the effect of heparitinase treatment. C2C12 cells were treated with heparitinase for 1 h, and BMP2 (5 ng/ml), or BMP2 and heparin (3–100 μg/ml) were added for a subsequent hour. Cell lysates were subjected to immunoblotting for active p38 (D) or P-Smad (E), and band intensities were quantified. Data are from duplicate cultures for each condition and are normalized to loading controls. Both activation of p38 and Smad in response to BMP2 were substantially lower in heparitinase-treated cells than in untreated cells (*p

    Journal: Molecular Biology of the Cell

    Article Title: Heparan Sulfate Acts as a Bone Morphogenetic Protein Coreceptor by Facilitating Ligand-induced Receptor Hetero-oligomerization

    doi: 10.1091/mbc.E10-04-0348

    Figure Lengend Snippet: Heparitinase treatment and blockade of sulfation diminish responses of cultured cells to BMP2. (A) Smad phosphorylation and p38 MAPK activation in mouse C2C12 and rat PC12 cells. Cells maintained in serum-free medium were treated with human recombinant BMP2 at 5 ng/ml for 1 h. Where indicated, cultures also were treated with 25 mIU/ml heparitinase 1 h before BMP addition and throughout the remainder of the assay. Cells lysates were subjected to immunoblotting for phospho-Smad1/5/8 (P-Smad) and active p38. Total Smad, total p38, and β-tubulin served as loading controls. (B and C) Kinetic profiles of BMP-induced Smad phosphorylation. C2C12 (B) or PC12 cells (C) were treated with heparitinase for 1 h, and BMP2 (5 ng/ml) was added. Cell lysates were collected at indicated time points and subjected to immunoblotting for P-Smad. Data are normalized to loading controls. (D and E) Exogenous heparin does not rescue cells from the effect of heparitinase treatment. C2C12 cells were treated with heparitinase for 1 h, and BMP2 (5 ng/ml), or BMP2 and heparin (3–100 μg/ml) were added for a subsequent hour. Cell lysates were subjected to immunoblotting for active p38 (D) or P-Smad (E), and band intensities were quantified. Data are from duplicate cultures for each condition and are normalized to loading controls. Both activation of p38 and Smad in response to BMP2 were substantially lower in heparitinase-treated cells than in untreated cells (*p

    Article Snippet: After washing twice with 50 mM Tris-HCl, pH 7.5, with 150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 5 μg/ml aprotinin, 1 μg/ml pepstatin A, 1 μg/ml leupeptin 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS, and boiling in 2× SDS-PAGE sample buffer for 10 min, samples were subjected to 8% SDS-PAGE and immunoblotting using anti-myc antibodies (Covance Research Products).

    Techniques: Cell Culture, Activation Assay, Recombinant

    BMP binding to type II, but not type I, receptor subunits depends on HS. (A) Binding of 125 I-BMP4 to BMPRIA. C2C12 cells were transiently transfected with HA-tagged BMPRIA, treated with heparitinase (1 h at 37°C), incubated with 125 I-BMP4 (20 ng/ml, 2 h at room temperature), and cross-linked with BS 3 . Lysates were immunoprecipitated with anti-HA antibodies, and precipitates subjected to SDS-PAGE and autoradiography. Locations of molecular sizes corresponding to BMP monomer (18 kDa; arrow 1), cross-linked BMP dimer (36 kDa, arrow 2), and cross-linked BMPRIA-BMP complexes (78 kDa; arrow 3) are shown. An arrow marked RI shows the location of uncrosslinked HA-tagged BMPRIA (60 kDa; as determined separately by immunoblotting). (B) Binding of 125 I-BMP4 to BMPRII. C2C12 cells were transfected stably with myc-tagged BMPRII (lanes labeled RII), or stably with BMPRII-myc and transiently with BMPRIA-HA (lanes labeled RI RII). Mock-transfected cells were transiently transfected with vector (pcDNA3.1) only. 125 I-BMP4 binding was carried out as in A. Lysates were immunoprecipitated with anti-myc antibodies, and precipitates subjected to SDS-PAGE and autoradiography. Locations of molecular sizes corresponding to BMP monomer (18 kDa; arrow 1), cross-linked BMPRIA-BMP complexes (78 kDa, arrow 2), cross-linked BMPRII-BMP complexes (93 kDa, arrow 3), and higher order complexes (∼153 kDa, arrow 4) are shown. Arrows RI and RII mark the locations of uncrosslinked BMPRIA-HA receptor (60 kDa) and uncrosslinked BMPRII-myc (75 kDa), as determined separately by immunoblotting. (C and D) Quantification of B. Results from cells transfected with BMPRII alone are shown in C, whereas those from cells transfected with both BMPRII and BMPRIA are in D (data are mean values ± SD of band intensities). Black bars quantify binding to cells not treated with heparitinase, whereas gray bars quantify binding to heparitinase-treated cells. The categories RI-BMP, RII-BMP, and higher-order complexes refer to the intensities of bands at arrows 2, 3, and 4, respectively, in B. Statistical significance of heparitinase effects was calculated by t test (*p

    Journal: Molecular Biology of the Cell

    Article Title: Heparan Sulfate Acts as a Bone Morphogenetic Protein Coreceptor by Facilitating Ligand-induced Receptor Hetero-oligomerization

    doi: 10.1091/mbc.E10-04-0348

    Figure Lengend Snippet: BMP binding to type II, but not type I, receptor subunits depends on HS. (A) Binding of 125 I-BMP4 to BMPRIA. C2C12 cells were transiently transfected with HA-tagged BMPRIA, treated with heparitinase (1 h at 37°C), incubated with 125 I-BMP4 (20 ng/ml, 2 h at room temperature), and cross-linked with BS 3 . Lysates were immunoprecipitated with anti-HA antibodies, and precipitates subjected to SDS-PAGE and autoradiography. Locations of molecular sizes corresponding to BMP monomer (18 kDa; arrow 1), cross-linked BMP dimer (36 kDa, arrow 2), and cross-linked BMPRIA-BMP complexes (78 kDa; arrow 3) are shown. An arrow marked RI shows the location of uncrosslinked HA-tagged BMPRIA (60 kDa; as determined separately by immunoblotting). (B) Binding of 125 I-BMP4 to BMPRII. C2C12 cells were transfected stably with myc-tagged BMPRII (lanes labeled RII), or stably with BMPRII-myc and transiently with BMPRIA-HA (lanes labeled RI RII). Mock-transfected cells were transiently transfected with vector (pcDNA3.1) only. 125 I-BMP4 binding was carried out as in A. Lysates were immunoprecipitated with anti-myc antibodies, and precipitates subjected to SDS-PAGE and autoradiography. Locations of molecular sizes corresponding to BMP monomer (18 kDa; arrow 1), cross-linked BMPRIA-BMP complexes (78 kDa, arrow 2), cross-linked BMPRII-BMP complexes (93 kDa, arrow 3), and higher order complexes (∼153 kDa, arrow 4) are shown. Arrows RI and RII mark the locations of uncrosslinked BMPRIA-HA receptor (60 kDa) and uncrosslinked BMPRII-myc (75 kDa), as determined separately by immunoblotting. (C and D) Quantification of B. Results from cells transfected with BMPRII alone are shown in C, whereas those from cells transfected with both BMPRII and BMPRIA are in D (data are mean values ± SD of band intensities). Black bars quantify binding to cells not treated with heparitinase, whereas gray bars quantify binding to heparitinase-treated cells. The categories RI-BMP, RII-BMP, and higher-order complexes refer to the intensities of bands at arrows 2, 3, and 4, respectively, in B. Statistical significance of heparitinase effects was calculated by t test (*p

    Article Snippet: After washing twice with 50 mM Tris-HCl, pH 7.5, with 150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 5 μg/ml aprotinin, 1 μg/ml pepstatin A, 1 μg/ml leupeptin 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS, and boiling in 2× SDS-PAGE sample buffer for 10 min, samples were subjected to 8% SDS-PAGE and immunoblotting using anti-myc antibodies (Covance Research Products).

    Techniques: Binding Assay, Transfection, Incubation, Immunoprecipitation, SDS Page, Autoradiography, Stable Transfection, Labeling, Plasmid Preparation

    Assembly of heteromeric receptor complexes is HS-dependent. (A) C2C12 cells stably expressing BMPRII-myc were transiently transfected with BMPRIA-HA. After treatment with or without heparitinase for 1 h, BMP2 (10 ng/ml) was added for 2 h at room temperature and cross-linked for 30 min with BS 3 . Cell lysates were immunoprecipitated with anti-HA antibodies and precipitates were subjected to SDS-PAGE and immunoblotting with anti-myc antibodies. The arrow shows the location of BMPRII-myc (75 kDa), as readily visualized in the blot of total cell lysates. (B) Long exposure of the blot in panel A. Arrow 1 shows the location of BMPRII. Arrow 2 marks bands with molecular weight corresponding to cross-linked BMPRII-BMP complexes (93 kDa). Larger bands, consistent with complexes containing BMPRI and BMPRII are also indicated (bracket 3).

    Journal: Molecular Biology of the Cell

    Article Title: Heparan Sulfate Acts as a Bone Morphogenetic Protein Coreceptor by Facilitating Ligand-induced Receptor Hetero-oligomerization

    doi: 10.1091/mbc.E10-04-0348

    Figure Lengend Snippet: Assembly of heteromeric receptor complexes is HS-dependent. (A) C2C12 cells stably expressing BMPRII-myc were transiently transfected with BMPRIA-HA. After treatment with or without heparitinase for 1 h, BMP2 (10 ng/ml) was added for 2 h at room temperature and cross-linked for 30 min with BS 3 . Cell lysates were immunoprecipitated with anti-HA antibodies and precipitates were subjected to SDS-PAGE and immunoblotting with anti-myc antibodies. The arrow shows the location of BMPRII-myc (75 kDa), as readily visualized in the blot of total cell lysates. (B) Long exposure of the blot in panel A. Arrow 1 shows the location of BMPRII. Arrow 2 marks bands with molecular weight corresponding to cross-linked BMPRII-BMP complexes (93 kDa). Larger bands, consistent with complexes containing BMPRI and BMPRII are also indicated (bracket 3).

    Article Snippet: After washing twice with 50 mM Tris-HCl, pH 7.5, with 150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 5 μg/ml aprotinin, 1 μg/ml pepstatin A, 1 μg/ml leupeptin 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS, and boiling in 2× SDS-PAGE sample buffer for 10 min, samples were subjected to 8% SDS-PAGE and immunoblotting using anti-myc antibodies (Covance Research Products).

    Techniques: Stable Transfection, Expressing, Transfection, Immunoprecipitation, SDS Page, Molecular Weight

    A non-heparin binding BMP2 variant is partially resistant to chlorate treatment. (A) Smad phosphorylation and p38 activation were assessed by immunoblotting in C2C12 and PC12 cells pretreated with various concentrations of chlorate (as indicated) for 48 h, and stimulated with either BMP2 (labeled B) or EHBMP2 (labeled E), at 5 ng/ml for 1 h. Total Smad, total p38 and β-tubulin served as loading controls. The data (mean values normalized to loading controls ± SD) are quantified in panels B (P-Smad in C2C12 cells), C (P-Smad in PC12 cells) and D (active p38 in PC12 cells). Effects of heparitinase were statistically significant for both BMP2 (black bars) and EHBMP2 (gray bars), but weaker for EHBMP2, particularly when cells were treated with intermediate chlorate levels (*, # p

    Journal: Molecular Biology of the Cell

    Article Title: Heparan Sulfate Acts as a Bone Morphogenetic Protein Coreceptor by Facilitating Ligand-induced Receptor Hetero-oligomerization

    doi: 10.1091/mbc.E10-04-0348

    Figure Lengend Snippet: A non-heparin binding BMP2 variant is partially resistant to chlorate treatment. (A) Smad phosphorylation and p38 activation were assessed by immunoblotting in C2C12 and PC12 cells pretreated with various concentrations of chlorate (as indicated) for 48 h, and stimulated with either BMP2 (labeled B) or EHBMP2 (labeled E), at 5 ng/ml for 1 h. Total Smad, total p38 and β-tubulin served as loading controls. The data (mean values normalized to loading controls ± SD) are quantified in panels B (P-Smad in C2C12 cells), C (P-Smad in PC12 cells) and D (active p38 in PC12 cells). Effects of heparitinase were statistically significant for both BMP2 (black bars) and EHBMP2 (gray bars), but weaker for EHBMP2, particularly when cells were treated with intermediate chlorate levels (*, # p

    Article Snippet: After washing twice with 50 mM Tris-HCl, pH 7.5, with 150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 5 μg/ml aprotinin, 1 μg/ml pepstatin A, 1 μg/ml leupeptin 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS, and boiling in 2× SDS-PAGE sample buffer for 10 min, samples were subjected to 8% SDS-PAGE and immunoblotting using anti-myc antibodies (Covance Research Products).

    Techniques: Binding Assay, Variant Assay, Activation Assay, Labeling

    A non-heparin binding BMP2 variant is resistant to heparitinase treatment. (A) Comparison of the N-terminal sequences of BMP2 and the engineered variant EH-BMP2, in which the first 12 amino acids have been replaced ( Ruppert et al. , 1996 ). Cationic residues in BMP2 are labeled with dots. (B) BMP2 and EHBMP2 are equal in potency. C2C12 cells were stimulated for 1 h with either BMP2 (circles) or EHBMP2 (triangles) at the indicated concentrations, lysed and subjected to immunoblotting for P-Smad. (C–E) Effect of heparitinase on p38 activation and Smad phosphorylation in BMP2- or EHBMP2-stimulated C2C12 cells. Cells were treated with heparitinase for 1 h and stimulated with either BMP2 or EHBMP2 for 1 h before sample preparation. Cell lysates were subjected to immunoblotting (panel C) for active p38 or P-Smad, with total p38 and β-tubulin serving as loading controls, and osmotic shock (sorbitol; 250 mM) as a positive control for active p38 (lane labeled “PC”). Band intensities were quantified and normalized to loading controls. In D and E, these data are plotted as mean values ± SD for each of the duplicate determinations shown in C. Significant effects of heparitinase on p38 activation and Smad phosphorylation are seen for BMP2-treated cells (*p

    Journal: Molecular Biology of the Cell

    Article Title: Heparan Sulfate Acts as a Bone Morphogenetic Protein Coreceptor by Facilitating Ligand-induced Receptor Hetero-oligomerization

    doi: 10.1091/mbc.E10-04-0348

    Figure Lengend Snippet: A non-heparin binding BMP2 variant is resistant to heparitinase treatment. (A) Comparison of the N-terminal sequences of BMP2 and the engineered variant EH-BMP2, in which the first 12 amino acids have been replaced ( Ruppert et al. , 1996 ). Cationic residues in BMP2 are labeled with dots. (B) BMP2 and EHBMP2 are equal in potency. C2C12 cells were stimulated for 1 h with either BMP2 (circles) or EHBMP2 (triangles) at the indicated concentrations, lysed and subjected to immunoblotting for P-Smad. (C–E) Effect of heparitinase on p38 activation and Smad phosphorylation in BMP2- or EHBMP2-stimulated C2C12 cells. Cells were treated with heparitinase for 1 h and stimulated with either BMP2 or EHBMP2 for 1 h before sample preparation. Cell lysates were subjected to immunoblotting (panel C) for active p38 or P-Smad, with total p38 and β-tubulin serving as loading controls, and osmotic shock (sorbitol; 250 mM) as a positive control for active p38 (lane labeled “PC”). Band intensities were quantified and normalized to loading controls. In D and E, these data are plotted as mean values ± SD for each of the duplicate determinations shown in C. Significant effects of heparitinase on p38 activation and Smad phosphorylation are seen for BMP2-treated cells (*p

    Article Snippet: After washing twice with 50 mM Tris-HCl, pH 7.5, with 150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 5 μg/ml aprotinin, 1 μg/ml pepstatin A, 1 μg/ml leupeptin 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS, and boiling in 2× SDS-PAGE sample buffer for 10 min, samples were subjected to 8% SDS-PAGE and immunoblotting using anti-myc antibodies (Covance Research Products).

    Techniques: Binding Assay, Variant Assay, Labeling, Activation Assay, Sample Prep, Positive Control

    NDR1 regulates localization of kindlin-3 and Rab8. (A) Association of NDR1 and kindlin-3. 293T cells transfected with Myc–kindlin-3 and FLAG-NDR1 as indicated were subjected to immunoprecipitation (IP) of FLAG-NDR1 followed by immunoblotting for Myc–kindlin-3. (B) Primary T cells stimulated with anti-CD3 or left unstimulated were analyzed for association of NDR1 with kindlin-3. IP with anti-NDR Ab was immunoblotted for kindlin-3. Total and precipitated proteins are indicated. The arrowhead indicates the predicted molecular mass of kindlin-3 (75.6 kDa). (C) Mislocalized kindlin-3 in NDR1 knockdown T cell blasts. Representative views at the contact plane and 3D images ( z ) of NDR1 knockdown (sh NDR) and control (sh Ctrl) OT-II T cells. Distances of peak intensities from the contact plane of kindlin-3 are shown. Scale bars, 2.5 μm. ***, P

    Journal: Molecular and Cellular Biology

    Article Title: NDR1-Dependent Regulation of Kindlin-3 Controls High-Affinity LFA-1 Binding and Immune Synapse Organization

    doi: 10.1128/MCB.00424-16

    Figure Lengend Snippet: NDR1 regulates localization of kindlin-3 and Rab8. (A) Association of NDR1 and kindlin-3. 293T cells transfected with Myc–kindlin-3 and FLAG-NDR1 as indicated were subjected to immunoprecipitation (IP) of FLAG-NDR1 followed by immunoblotting for Myc–kindlin-3. (B) Primary T cells stimulated with anti-CD3 or left unstimulated were analyzed for association of NDR1 with kindlin-3. IP with anti-NDR Ab was immunoblotted for kindlin-3. Total and precipitated proteins are indicated. The arrowhead indicates the predicted molecular mass of kindlin-3 (75.6 kDa). (C) Mislocalized kindlin-3 in NDR1 knockdown T cell blasts. Representative views at the contact plane and 3D images ( z ) of NDR1 knockdown (sh NDR) and control (sh Ctrl) OT-II T cells. Distances of peak intensities from the contact plane of kindlin-3 are shown. Scale bars, 2.5 μm. ***, P

    Article Snippet: Anti-kindlin-3 Ab and anti-Mst1 Ab, used for immunoblotting, were purchased from Millipore.

    Techniques: Transfection, Immunoprecipitation

    The 1 st type 1 repeat is required for TSP1/CD148-mediated cell growth inhibition. (A) The trimeric TSP1 fragment (ΔType1-R1) that contains the procollagen domain and the 2 nd and 3 rd , but not the 1 st , type 1 repeat was prepared using HEK293E cells. Upper panel shows a schematic representation of the trimeric TSP1 fragment that lacks the 1 st type 1 repeat. Amino acid residues (aa 374–429) of the 1 st type 1 repeat were deleted. Lower panel shows colloidal blue staining of the purified ΔType1-R1 fragment. Five micrograms of protein were separated on a 10% polyacrylamide gel in reducing (+DTT) and non-reducing (-DTT) conditions and stained with colloidal blue to assess size, purity, and trimerization. The expected size of protein is also shown. (B) A431D/CD148wt or A431D/CD36 (stably expressing CD36) cells were treated with 12 nM of trimeric TSP1 fragments that lack or contain the 1 st type 1 repeat or whole TSP1 protein. Cell density was measured at the indicated time points. The data show mean ± SEM of quadruplicate determinations. Representative data of four independent experiments is shown. Note: The ΔType1-R1 fragment shows no growth inhibitory activity in A431D/CD148wt cells, while it inhibits cell proliferation in A431D/CD36 cells. (C) A431D/CD36 cells were treated with trimeric TSP1 fragments (12 nM) that lacked or contained the 1 st type 1 repeat or whole TSP1 protein (12 nM) for 18 h. Tyrosine phosphorylation of p38 and cleaved caspase 3 was assessed by immunoblotting using the phopho-specific p38 (pThr180+Tyr182) or cleaved caspase 3 antibodies. The membranes were reprobed with antibodies to total p38 or γ-tubulin. Representative data of four independent experiments is shown. (D) A series of monomeric TSP1 fragments were prepared from the regions containing the procollagen domain and type 1 repeats as shown in a schema on right side. Each fragment (17 nM) was incubated with either 44 pmol of CD148-Fc or control Fc (Fc alone), and Fc-proteins were pulled down with protein-G beads. Bound TSP1 fragments were assessed by anti-Myc immunoblotting (upper panel). Half of each sample was subjected to anti-CD148 immunoblotting to confirm the pull down of CD148-Fc (lower panel). Representative data of five independent experiments is shown. Note: TSP1 fragments that contain the 1 st type 1 repeat bind to CD148-Fc. (E) A431D/CD148wt cells were treated with or without indicated TSP1 fragments (36 nM) for 1 h, then a trimeric TSP1 fragment (12nM) containing the procollagen domain and type 1 repeats was added to the medium. Cell proliferation was assessed at day 2. The data show mean ± SEM of quadruplicate determinations. Representative data of five independent experiments is shown. ** P

    Journal: PLoS ONE

    Article Title: Determination of the CD148-Interacting Region in Thrombospondin-1

    doi: 10.1371/journal.pone.0154916

    Figure Lengend Snippet: The 1 st type 1 repeat is required for TSP1/CD148-mediated cell growth inhibition. (A) The trimeric TSP1 fragment (ΔType1-R1) that contains the procollagen domain and the 2 nd and 3 rd , but not the 1 st , type 1 repeat was prepared using HEK293E cells. Upper panel shows a schematic representation of the trimeric TSP1 fragment that lacks the 1 st type 1 repeat. Amino acid residues (aa 374–429) of the 1 st type 1 repeat were deleted. Lower panel shows colloidal blue staining of the purified ΔType1-R1 fragment. Five micrograms of protein were separated on a 10% polyacrylamide gel in reducing (+DTT) and non-reducing (-DTT) conditions and stained with colloidal blue to assess size, purity, and trimerization. The expected size of protein is also shown. (B) A431D/CD148wt or A431D/CD36 (stably expressing CD36) cells were treated with 12 nM of trimeric TSP1 fragments that lack or contain the 1 st type 1 repeat or whole TSP1 protein. Cell density was measured at the indicated time points. The data show mean ± SEM of quadruplicate determinations. Representative data of four independent experiments is shown. Note: The ΔType1-R1 fragment shows no growth inhibitory activity in A431D/CD148wt cells, while it inhibits cell proliferation in A431D/CD36 cells. (C) A431D/CD36 cells were treated with trimeric TSP1 fragments (12 nM) that lacked or contained the 1 st type 1 repeat or whole TSP1 protein (12 nM) for 18 h. Tyrosine phosphorylation of p38 and cleaved caspase 3 was assessed by immunoblotting using the phopho-specific p38 (pThr180+Tyr182) or cleaved caspase 3 antibodies. The membranes were reprobed with antibodies to total p38 or γ-tubulin. Representative data of four independent experiments is shown. (D) A series of monomeric TSP1 fragments were prepared from the regions containing the procollagen domain and type 1 repeats as shown in a schema on right side. Each fragment (17 nM) was incubated with either 44 pmol of CD148-Fc or control Fc (Fc alone), and Fc-proteins were pulled down with protein-G beads. Bound TSP1 fragments were assessed by anti-Myc immunoblotting (upper panel). Half of each sample was subjected to anti-CD148 immunoblotting to confirm the pull down of CD148-Fc (lower panel). Representative data of five independent experiments is shown. Note: TSP1 fragments that contain the 1 st type 1 repeat bind to CD148-Fc. (E) A431D/CD148wt cells were treated with or without indicated TSP1 fragments (36 nM) for 1 h, then a trimeric TSP1 fragment (12nM) containing the procollagen domain and type 1 repeats was added to the medium. Cell proliferation was assessed at day 2. The data show mean ± SEM of quadruplicate determinations. Representative data of five independent experiments is shown. ** P

    Article Snippet: Antibodies The primary antibodies used for immunoblotting and immunoprecipitations: anti-CD148 (clone 143–41), anti-phospho-EGF receptor (Tyr 1173), anti-EGF receptor (1005), anti-CD36 (H-300), anti-β actin (C-2), and anti-γ tubulin (H-183) were from Santa Cruz Biotechnology (Dallas, TX).

    Techniques: Inhibition, Staining, Purification, Stable Transfection, Expressing, Activity Assay, Incubation

    Assessment of CD148-interacting region in TSP1. (A) Recombinant TSP1 fragments that correspond to the structural elements were prepared using HEK293E cells. Left panel shows a schematic representation of the TSP1 fragments. The number of amino acid residues includes the signal peptide sequence. Right panel shows colloidal blue stain of the purified TSP1 fragments. Twelve micrograms of protein were separated on a 10% polyacrylamide gel and stained with colloidal blue to assess size and purity. The expected size of protein is also shown. (B) TSP1 fragments (17 nM) were incubated with either 44 pmol of CD148-Fc or control Fc (Fc alone). Fc-proteins were pulled down with Protein-G beads and the binding of TSP1 fragments was assessed by immunoblotting using anti-Myc antibody (upper panel). The membrane was reprobed with anti-CD148 antibody to confirm the pull down of CD148-Fc (lower panel). Representative data of five independent experiments is shown. Note: The TSP1 fragment containing the procollagen domain and type 1 repeats binds to CD148-Fc. (C) Protein-A plates conjugated with CD148-Fc (11.3 nM) or equal molar of control Fc were incubated with AP-TSP1 or AP (12 nM) in the presence or absence of TSP1 fragments (25 nM) or whole TSP1 protein (25 nM). The bound AP-TSP1 was assessed by an AP activity assay. The data show mean ± SEM of quadruplicate determinations. Representative data of five independent experiments is shown. ** P

    Journal: PLoS ONE

    Article Title: Determination of the CD148-Interacting Region in Thrombospondin-1

    doi: 10.1371/journal.pone.0154916

    Figure Lengend Snippet: Assessment of CD148-interacting region in TSP1. (A) Recombinant TSP1 fragments that correspond to the structural elements were prepared using HEK293E cells. Left panel shows a schematic representation of the TSP1 fragments. The number of amino acid residues includes the signal peptide sequence. Right panel shows colloidal blue stain of the purified TSP1 fragments. Twelve micrograms of protein were separated on a 10% polyacrylamide gel and stained with colloidal blue to assess size and purity. The expected size of protein is also shown. (B) TSP1 fragments (17 nM) were incubated with either 44 pmol of CD148-Fc or control Fc (Fc alone). Fc-proteins were pulled down with Protein-G beads and the binding of TSP1 fragments was assessed by immunoblotting using anti-Myc antibody (upper panel). The membrane was reprobed with anti-CD148 antibody to confirm the pull down of CD148-Fc (lower panel). Representative data of five independent experiments is shown. Note: The TSP1 fragment containing the procollagen domain and type 1 repeats binds to CD148-Fc. (C) Protein-A plates conjugated with CD148-Fc (11.3 nM) or equal molar of control Fc were incubated with AP-TSP1 or AP (12 nM) in the presence or absence of TSP1 fragments (25 nM) or whole TSP1 protein (25 nM). The bound AP-TSP1 was assessed by an AP activity assay. The data show mean ± SEM of quadruplicate determinations. Representative data of five independent experiments is shown. ** P

    Article Snippet: Antibodies The primary antibodies used for immunoblotting and immunoprecipitations: anti-CD148 (clone 143–41), anti-phospho-EGF receptor (Tyr 1173), anti-EGF receptor (1005), anti-CD36 (H-300), anti-β actin (C-2), and anti-γ tubulin (H-183) were from Santa Cruz Biotechnology (Dallas, TX).

    Techniques: Recombinant, Sequencing, Staining, Purification, Incubation, Binding Assay, Activity Assay

    Trimeric TSP1 fragments that contain the 1 st type 1 repeat increase CD148 catalytic activity and reduce tyrosine phosphorylation of EGFR and ERK1/2 in A431D/CD148wt cells. (A) Left: A431D/CD148wt cells were treated with the indicated trimeric TSP1 fragments (12 nM) or whole TSP1 protein (12 nM) for 15 min. CD148 was immunoprecipitated using anti-CD148 antibody or class-matched control IgG. The washed immunocomplexes were subjected to a PTP activity assay with or without 1 mM sodium orthovanadate (VO 4 ). The amount of CD148 in the immunocomplexes was evaluated by immunoblotting using anti-CD148 antibody (lower panel). The data show mean ± SEM of quadruplicate determinations. Representative data of five independent experiments is shown. ** P

    Journal: PLoS ONE

    Article Title: Determination of the CD148-Interacting Region in Thrombospondin-1

    doi: 10.1371/journal.pone.0154916

    Figure Lengend Snippet: Trimeric TSP1 fragments that contain the 1 st type 1 repeat increase CD148 catalytic activity and reduce tyrosine phosphorylation of EGFR and ERK1/2 in A431D/CD148wt cells. (A) Left: A431D/CD148wt cells were treated with the indicated trimeric TSP1 fragments (12 nM) or whole TSP1 protein (12 nM) for 15 min. CD148 was immunoprecipitated using anti-CD148 antibody or class-matched control IgG. The washed immunocomplexes were subjected to a PTP activity assay with or without 1 mM sodium orthovanadate (VO 4 ). The amount of CD148 in the immunocomplexes was evaluated by immunoblotting using anti-CD148 antibody (lower panel). The data show mean ± SEM of quadruplicate determinations. Representative data of five independent experiments is shown. ** P

    Article Snippet: Antibodies The primary antibodies used for immunoblotting and immunoprecipitations: anti-CD148 (clone 143–41), anti-phospho-EGF receptor (Tyr 1173), anti-EGF receptor (1005), anti-CD36 (H-300), anti-β actin (C-2), and anti-γ tubulin (H-183) were from Santa Cruz Biotechnology (Dallas, TX).

    Techniques: Activity Assay, Immunoprecipitation

    Knockdown of SIRT2 suppresses the tumorigenicity of GB2 cells Kaplan–Meier overall survival curves of mice transplanted with GB2 cells (left panel), GB13 cells (Middle panel) or GB16 cells (right panel) infected with the indicated lentivirus (1 × 10 4 cells, 7 mice per group). The y ‐axis indicates the percent survival. Mice were transplanted with the indicated number of GB2 cells infected with a control (empty) or shSIRT2‐expressing (shS2 #1) lentivirus. Six weeks after transplantation, mice (3 or 4 animals, see number of dots) were sacrificed and the expression levels of human β‐actin mRNA were quantified by qRT–PCR. H E staining of tumors that were developed in mice implanted with GB16 cells that had been infected with a control (empty) or shSIRT2‐expressing (shS2#1) lentivirus. Scale bar, 1 mm. An image with higher magnification is shown on the right (scale bar, 50 μm). The effect of AK7 on the deacetylation of acetyl‐tubulin (left panel) and the proliferation of GB2 cells (right panel). (Left panel) Immunoblotting analysis of the effect of AK7 (20 μM) on deacetylation of acetyl‐tubulin/tubulin was performed on day 3 in right panel. Bars indicate mean ± SD ( n = 3). Ten days after intracranial transplantation of GB2 cells (1.0 × 10 4 cells), AK7 was intraperitoneally administrated for 4 weeks (15 mg/kg, twice/week). After 8 weeks, mice (4 or 5 animals, see number of dots) were sacrificed and the expression levels of human β‐actin mRNA were quantified by qRT–PCR. Data information: Statistical significance was evaluated using the log‐rank test (for panel A) or unpaired two‐tailed t ‐test. ** P

    Journal: EMBO Reports

    Article Title: SIRT2‐mediated inactivation of p73 is required for glioblastoma tumorigenicity

    doi: 10.15252/embr.201745587

    Figure Lengend Snippet: Knockdown of SIRT2 suppresses the tumorigenicity of GB2 cells Kaplan–Meier overall survival curves of mice transplanted with GB2 cells (left panel), GB13 cells (Middle panel) or GB16 cells (right panel) infected with the indicated lentivirus (1 × 10 4 cells, 7 mice per group). The y ‐axis indicates the percent survival. Mice were transplanted with the indicated number of GB2 cells infected with a control (empty) or shSIRT2‐expressing (shS2 #1) lentivirus. Six weeks after transplantation, mice (3 or 4 animals, see number of dots) were sacrificed and the expression levels of human β‐actin mRNA were quantified by qRT–PCR. H E staining of tumors that were developed in mice implanted with GB16 cells that had been infected with a control (empty) or shSIRT2‐expressing (shS2#1) lentivirus. Scale bar, 1 mm. An image with higher magnification is shown on the right (scale bar, 50 μm). The effect of AK7 on the deacetylation of acetyl‐tubulin (left panel) and the proliferation of GB2 cells (right panel). (Left panel) Immunoblotting analysis of the effect of AK7 (20 μM) on deacetylation of acetyl‐tubulin/tubulin was performed on day 3 in right panel. Bars indicate mean ± SD ( n = 3). Ten days after intracranial transplantation of GB2 cells (1.0 × 10 4 cells), AK7 was intraperitoneally administrated for 4 weeks (15 mg/kg, twice/week). After 8 weeks, mice (4 or 5 animals, see number of dots) were sacrificed and the expression levels of human β‐actin mRNA were quantified by qRT–PCR. Data information: Statistical significance was evaluated using the log‐rank test (for panel A) or unpaired two‐tailed t ‐test. ** P

    Article Snippet: Mouse mAb to p73α/β (Clone ER‐15) for immunoblotting was from Thermo Scientific.

    Techniques: Mouse Assay, Infection, Expressing, Transplantation Assay, Quantitative RT-PCR, Staining, Two Tailed Test

    p73 is required for SIRT2 knockdown‐induced suppression of glioblastoma proliferation Schematic representation of p73 isoforms. TAD, transactivation domain; DBD, DNA‐binding domain; OD, oligomerization domain; SAM, sterile alpha motif domain. Immunoblotting analysis of p73 in GB2 cells infected with a control (Empty) or shTP73‐expressing lentivirus for 96 h. Growth curve of GB16 cells infected with the indicated lentiviruses. Bars indicate mean ± SD ( n = 3). Ectopic expression of the ΔN isoform of p73β suppresses the expression levels of PUMA and GADD45 induced by SIRT2 knockdown in GB2 cells. Four days after lentiviral infection, the expression levels of PUMA and GADD45 mRNA were measured by qRT–PCR. Bars indicate mean ± SD ( n = 3). GB2 and GB4 cells [5 × 10 4 and 2 × 10 4 cells, respectively (indicated by the dashed lines)] were infected with the indicated lentiviruses. After 96 h, the number of viable cells was counted. Bars indicate mean ± SD ( n = 4). HEK293 cells were transfected with TAp73α along with ΔNp73β. PUMA mRNA was measured by qRT–PCR analyses. Bars indicate mean ± SD ( n = 3). GB2 cells (5 × 10 4 cells, dashed line) were infected with the indicated lentiviruses, respectively. After 96 h, the number of viable cells was counted. shTP73 corresponds to both α‐ and β‐isoforms of p73. Bars indicate mean ± SD ( n = 4). GB2 cells were transfected with TAp73α along with NLS‐SIRT2 (nuclear‐localizing mutant of SIRT2) or 3mut (deacetylase‐inactive, nuclear‐localizing mutant of SIRT2) and a reporter construct consisting of the promoter region of PUMA fused to a luciferase gene (left panel). Reporter activities were determined by dual‐luciferase assays (right panel). Bars indicate mean ± SD ( n = 3). GB2 cells were electroporated with TAp73α along with NLS‐SIRT2 (nuclear‐localizing mutant of SIRT2) or 3mut (deacetylase‐inactive, nuclear‐localizing mutant of SIRT2). The expression of PUMA and GADD45 was measured by qRT–PCR analysis. Bars indicate mean ± SD ( n = 3). Data information: Statistical significance was evaluated using unpaired two‐tailed t ‐test. * P

    Journal: EMBO Reports

    Article Title: SIRT2‐mediated inactivation of p73 is required for glioblastoma tumorigenicity

    doi: 10.15252/embr.201745587

    Figure Lengend Snippet: p73 is required for SIRT2 knockdown‐induced suppression of glioblastoma proliferation Schematic representation of p73 isoforms. TAD, transactivation domain; DBD, DNA‐binding domain; OD, oligomerization domain; SAM, sterile alpha motif domain. Immunoblotting analysis of p73 in GB2 cells infected with a control (Empty) or shTP73‐expressing lentivirus for 96 h. Growth curve of GB16 cells infected with the indicated lentiviruses. Bars indicate mean ± SD ( n = 3). Ectopic expression of the ΔN isoform of p73β suppresses the expression levels of PUMA and GADD45 induced by SIRT2 knockdown in GB2 cells. Four days after lentiviral infection, the expression levels of PUMA and GADD45 mRNA were measured by qRT–PCR. Bars indicate mean ± SD ( n = 3). GB2 and GB4 cells [5 × 10 4 and 2 × 10 4 cells, respectively (indicated by the dashed lines)] were infected with the indicated lentiviruses. After 96 h, the number of viable cells was counted. Bars indicate mean ± SD ( n = 4). HEK293 cells were transfected with TAp73α along with ΔNp73β. PUMA mRNA was measured by qRT–PCR analyses. Bars indicate mean ± SD ( n = 3). GB2 cells (5 × 10 4 cells, dashed line) were infected with the indicated lentiviruses, respectively. After 96 h, the number of viable cells was counted. shTP73 corresponds to both α‐ and β‐isoforms of p73. Bars indicate mean ± SD ( n = 4). GB2 cells were transfected with TAp73α along with NLS‐SIRT2 (nuclear‐localizing mutant of SIRT2) or 3mut (deacetylase‐inactive, nuclear‐localizing mutant of SIRT2) and a reporter construct consisting of the promoter region of PUMA fused to a luciferase gene (left panel). Reporter activities were determined by dual‐luciferase assays (right panel). Bars indicate mean ± SD ( n = 3). GB2 cells were electroporated with TAp73α along with NLS‐SIRT2 (nuclear‐localizing mutant of SIRT2) or 3mut (deacetylase‐inactive, nuclear‐localizing mutant of SIRT2). The expression of PUMA and GADD45 was measured by qRT–PCR analysis. Bars indicate mean ± SD ( n = 3). Data information: Statistical significance was evaluated using unpaired two‐tailed t ‐test. * P

    Article Snippet: Mouse mAb to p73α/β (Clone ER‐15) for immunoblotting was from Thermo Scientific.

    Techniques: Binding Assay, Infection, Expressing, Quantitative RT-PCR, Transfection, Mutagenesis, Histone Deacetylase Assay, Construct, Luciferase, Two Tailed Test

    SIRT2 deacetylates p73 and suppresses its transcriptional activity Cells transfected with FLAG‐tagged p73 isoforms were treated with 20 μM AGK2 or DMSO for 6 h and subjected to immunoprecipitation with anti‐FLAG antibody followed by immunoblotting with antibody against acetylated lysine or FLAG. Three conserved lysine residues are located in the most C‐terminal region of p73. Amino acid sequences of p73 of the indicated species and human p53 and p63 are aligned. *, conserved and acetylated lysine residues, black frame, highly conserved residues, gray frame, conserved residues. Schematic representation of TAp73α and K3R. K3R is a mutant TAp73α in which three lysine (K) residues in the C‐terminal region are replaced with arginine (R). Cells transfected with the indicated constructs were treated with 20 μM AGK2 or DMSO, respectively, for 6 h and subjected to immunoprecipitation with anti‐FLAG antibody followed by immunoblotting with antibody against acetylated lysine or FLAG. HEK293T cells transfected with FLAG‐tagged TAp73α were treated with 20 μM AGK2 (pre‐AGK2 + ) or DMSO (pre‐AGK2 − ) for 6 h. p73 was purified by immunoprecipitation and incubated with recombinant SIRT2 (10 U), NAD (1 mM), and/or AGK2 (3.5 μM) as indicated. qRT–PCR analysis of PUMA and GADD45 mRNA in GB2 cells infected with the indicated lentiviruses. Bars indicate mean ± SEM ( n = 4–5). Expression of p73 in (F) was determined by immunoblotting with anti‐p73 antibody. RFP was used as a control. GB glioblastoma neurospheres [5 × 10 4 cells (indicated by the dashed line)] were infected with the indicated lentiviruses. After 96 h, the number of viable cells was counted. Bars indicate mean ± SD ( n = 4). GB2 cells infected with the indicated lentiviruses were intracranially transplanted into immunocompromised mice. After 8 weeks, mice (3–5 animals, see number of dots) were sacrificed and the expression levels of human β‐actin mRNA were quantified by qRT–PCR. SIRT2‐mediated inactivation of p73 is critical for the proliferation and tumorigenicity of glioblastoma cells. SIRT2 regulates the transcriptional activity of the tumor suppressor p73 by deacetylating its C‐terminal lysine residues. Data information: Statistical significance was evaluated using unpaired two‐tailed t ‐test. * P

    Journal: EMBO Reports

    Article Title: SIRT2‐mediated inactivation of p73 is required for glioblastoma tumorigenicity

    doi: 10.15252/embr.201745587

    Figure Lengend Snippet: SIRT2 deacetylates p73 and suppresses its transcriptional activity Cells transfected with FLAG‐tagged p73 isoforms were treated with 20 μM AGK2 or DMSO for 6 h and subjected to immunoprecipitation with anti‐FLAG antibody followed by immunoblotting with antibody against acetylated lysine or FLAG. Three conserved lysine residues are located in the most C‐terminal region of p73. Amino acid sequences of p73 of the indicated species and human p53 and p63 are aligned. *, conserved and acetylated lysine residues, black frame, highly conserved residues, gray frame, conserved residues. Schematic representation of TAp73α and K3R. K3R is a mutant TAp73α in which three lysine (K) residues in the C‐terminal region are replaced with arginine (R). Cells transfected with the indicated constructs were treated with 20 μM AGK2 or DMSO, respectively, for 6 h and subjected to immunoprecipitation with anti‐FLAG antibody followed by immunoblotting with antibody against acetylated lysine or FLAG. HEK293T cells transfected with FLAG‐tagged TAp73α were treated with 20 μM AGK2 (pre‐AGK2 + ) or DMSO (pre‐AGK2 − ) for 6 h. p73 was purified by immunoprecipitation and incubated with recombinant SIRT2 (10 U), NAD (1 mM), and/or AGK2 (3.5 μM) as indicated. qRT–PCR analysis of PUMA and GADD45 mRNA in GB2 cells infected with the indicated lentiviruses. Bars indicate mean ± SEM ( n = 4–5). Expression of p73 in (F) was determined by immunoblotting with anti‐p73 antibody. RFP was used as a control. GB glioblastoma neurospheres [5 × 10 4 cells (indicated by the dashed line)] were infected with the indicated lentiviruses. After 96 h, the number of viable cells was counted. Bars indicate mean ± SD ( n = 4). GB2 cells infected with the indicated lentiviruses were intracranially transplanted into immunocompromised mice. After 8 weeks, mice (3–5 animals, see number of dots) were sacrificed and the expression levels of human β‐actin mRNA were quantified by qRT–PCR. SIRT2‐mediated inactivation of p73 is critical for the proliferation and tumorigenicity of glioblastoma cells. SIRT2 regulates the transcriptional activity of the tumor suppressor p73 by deacetylating its C‐terminal lysine residues. Data information: Statistical significance was evaluated using unpaired two‐tailed t ‐test. * P

    Article Snippet: Mouse mAb to p73α/β (Clone ER‐15) for immunoblotting was from Thermo Scientific.

    Techniques: Activity Assay, Transfection, Immunoprecipitation, Mutagenesis, Construct, Purification, Incubation, Recombinant, Quantitative RT-PCR, Infection, Expressing, Mouse Assay, Two Tailed Test

    Western blot analysis of Xenopus oocytes expressing human DMT1 and FPN1-c-Myc Xenopus oocytes were injected with 10 ng of cRNA for FPN1, DMT1A , both FPN1 and DMT1 , or H 2 O and incubated for 3 days. For immunoblot detection, membrane-enriched fractions were subject to SDS-PAGE. Blots were probed with primary antibodies against DMT1, c-Myc, and actin, and detected with horseradish peroxidase linked secondary antibodies. For DMT1 analysis (A), 10 µg of protein was used in each lane, and for FPN1 analysis 40 µg was used (B).

    Journal: Biochimica et biophysica acta

    Article Title: The iron transporter ferroportin can also function as a manganese exporter

    doi: 10.1016/j.bbamem.2011.12.002

    Figure Lengend Snippet: Western blot analysis of Xenopus oocytes expressing human DMT1 and FPN1-c-Myc Xenopus oocytes were injected with 10 ng of cRNA for FPN1, DMT1A , both FPN1 and DMT1 , or H 2 O and incubated for 3 days. For immunoblot detection, membrane-enriched fractions were subject to SDS-PAGE. Blots were probed with primary antibodies against DMT1, c-Myc, and actin, and detected with horseradish peroxidase linked secondary antibodies. For DMT1 analysis (A), 10 µg of protein was used in each lane, and for FPN1 analysis 40 µg was used (B).

    Article Snippet: For immunoblot analysis of oocyte membrane-enriched fractions, samples were dissolved in Laemmli buffer (1:1, Bio-Rad), and SDS-PAGE was performed on 4–20% TGX Gels (Bio-Rad).

    Techniques: Western Blot, Expressing, Injection, Incubation, SDS Page

    Deletion of p53 identifies the novel mitochrondria -associated senescence domain (MASD) between amino acids 64 and 209 of p53. ( A ) Various deletions of FLAG –tagged p53 were used to determine involvement in mitochrondria –associated senescence programs in EJ-p53 cells. The following abbreviations were used to characterize individual domains within p53 as, AD1, activation domain 1; AD2, activation domain 2; PRD, proline rich domain; DBD-NLS-TD-NES, combined DNA binding domain-nuclear localization signal-transactivation domain-nuclear export signal; BD, basic domain. Following the transient transfection of individual p53 constructs into the uninduced EJ-p53 cells, expression of p53 protein levels were normalized versus cell number to measure the level of SA -β galactosidase activity by staining with Xgal. ELISA analysis was then performed using antisera against human prohibitin (Research Diagnostics, Inc.). Colormetric analysis was then used to measure the amount of SA-β galactosidase activity ELISA was performed to measure prohibitin levels (fg/ml lysate) and normalized by the amount of immunoprecipitated FLAG-tagged p53 protein used as input from the ELISA assay. ( B ) Immunoblot analysis was then performed with anti –prohibitin nitrocellulose filter was reused to immunoblot with an anti- β actin polyclonal antisera (Sigma-Aldrich). ( C ) Electron micrograph (10,000X) of EJ carcinoma cells transfected with the different FLAG-tagged and truncated variants of human p53 and stained with the anti-FLAG monoclonal antibody (Sigma-Aldrich). Region corresponding to the outline of the mitochrondria is indicated. ( D ) Interaction of Sirt3 with the MASD region of p53. Using FLAG-tagged variants of the deleted p53 cDNAs expressed by transient transfections of p53 shown (left) were used to identify specific interactions with endogenous Sirt3 by immunoprecipitation with M2 agarose (Sigma-Aldrich) followed by standard immunoblotting protocols.

    Journal: PLoS ONE

    Article Title: p53-Induced Growth Arrest Is Regulated by the Mitochondrial SirT3 Deacetylase

    doi: 10.1371/journal.pone.0010486

    Figure Lengend Snippet: Deletion of p53 identifies the novel mitochrondria -associated senescence domain (MASD) between amino acids 64 and 209 of p53. ( A ) Various deletions of FLAG –tagged p53 were used to determine involvement in mitochrondria –associated senescence programs in EJ-p53 cells. The following abbreviations were used to characterize individual domains within p53 as, AD1, activation domain 1; AD2, activation domain 2; PRD, proline rich domain; DBD-NLS-TD-NES, combined DNA binding domain-nuclear localization signal-transactivation domain-nuclear export signal; BD, basic domain. Following the transient transfection of individual p53 constructs into the uninduced EJ-p53 cells, expression of p53 protein levels were normalized versus cell number to measure the level of SA -β galactosidase activity by staining with Xgal. ELISA analysis was then performed using antisera against human prohibitin (Research Diagnostics, Inc.). Colormetric analysis was then used to measure the amount of SA-β galactosidase activity ELISA was performed to measure prohibitin levels (fg/ml lysate) and normalized by the amount of immunoprecipitated FLAG-tagged p53 protein used as input from the ELISA assay. ( B ) Immunoblot analysis was then performed with anti –prohibitin nitrocellulose filter was reused to immunoblot with an anti- β actin polyclonal antisera (Sigma-Aldrich). ( C ) Electron micrograph (10,000X) of EJ carcinoma cells transfected with the different FLAG-tagged and truncated variants of human p53 and stained with the anti-FLAG monoclonal antibody (Sigma-Aldrich). Region corresponding to the outline of the mitochrondria is indicated. ( D ) Interaction of Sirt3 with the MASD region of p53. Using FLAG-tagged variants of the deleted p53 cDNAs expressed by transient transfections of p53 shown (left) were used to identify specific interactions with endogenous Sirt3 by immunoprecipitation with M2 agarose (Sigma-Aldrich) followed by standard immunoblotting protocols.

    Article Snippet: We have mapped domains of p53 relevant for premature growth arrest in EJ bladder carcinoma cells expression by measuring levels of senescence –associated (SA) β galatosidase activity and levels of prohibitin as having an counter-correlative relationship to growth arrest by ELISA and immunoblot analysis (Research Diagnostics, Inc.) from EJ-p53 cell lysates.

    Techniques: Activation Assay, Binding Assay, Transfection, Construct, Expressing, Activity Assay, Staining, Enzyme-linked Immunosorbent Assay, Immunoprecipitation

    Increased tyrosine phosphorylation of 53 kDa by K15 expression. A total of 2 × 10 6 Bjab cells were stimulated with either PBS or OKT8 (anti-CD8) antibody for 5 min and the cell extracts were subjected to a western blot with antiphospho tyrosine immunoblot. At the bottom, the cell surface expression of CD8 chimera proteins were measured by anti CD8-PE antibody.

    Journal: Experimental & Molecular Medicine

    Article Title: Multi-transmembrane protein K15 of Kaposi's sarcoma-associated herpesvirus targets Lyn kinase in the membrane raft and induces NFAT/AP1 activities

    doi: 10.3858/emm.2008.40.5.565

    Figure Lengend Snippet: Increased tyrosine phosphorylation of 53 kDa by K15 expression. A total of 2 × 10 6 Bjab cells were stimulated with either PBS or OKT8 (anti-CD8) antibody for 5 min and the cell extracts were subjected to a western blot with antiphospho tyrosine immunoblot. At the bottom, the cell surface expression of CD8 chimera proteins were measured by anti CD8-PE antibody.

    Article Snippet: Immunoblot detection was performed with a 1:1,000 dilution of primary antibody with an ECL kit (Amersham).

    Techniques: Expressing, Western Blot