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  • 92
    ATUM immunoblotting
    and protein levels on chromatin were analyzed by <t>immunoblotting.</t>
    Immunoblotting, supplied by ATUM, used in various techniques. Bioz Stars score: 92/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    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
    Immunoblotting, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 6484 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    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
    Immunoblotting, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 9083 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    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
    Immunoblotting, supplied by Covance, used in various techniques. Bioz Stars score: 93/100, based on 737 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore immunoblotting
    ( A , B ): Effect of Resveratrol and/or 5-FU on NF-κB activation and NF-κB-regulated gene end-products involved in apoptosis, metastasis induced by TNF-β in HCT116 and HCT116R in inflammatory microenvironment. Alginate cultures of HCT116 ( A ) and HCT116R ( B ) were treated for 10 days as described in detail in “ Section 2 ”. <t>Immunoblotting</t> with whole cell lysates was performed with antibodies against p65-NF-κB, CXCR4, MMP-9 and cleaved-caspase-3. Western blots shown are representative of three independent experiments. The housekeeping protein β-actin served as a positive loading control in all experiments. ( C , D ): Effect of resveratrol and/or 5-FU on TNF-β-induced epithelial-to-mesenchymal transition of CRC cells in tumor microenvironment cultures. Colorectal cancer cells in alginate culture were treated as described in Materials and Methods. After 10 days whole cell lysates of HCT116 ( C ) and HCT116R ( D ) were subjected to western blotting with antibodies against vimentin, E-cadherin and slug. Western blots shown are representative of three independent experiments. The housekeeping protein β-actin served as a positive loading control in all experiments.
    Immunoblotting, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 19734 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    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
    Immunoblotting, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 10088 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    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
    Immunoblotting, supplied by Signalway Antibody, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    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
    Immunoblotting, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 12426 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam immunoblotting
    Mutational Analysis of the A-ST Interface (A) Mutation of ST residues predicted to interact with PP2A Aα. Top: Expression of ST in whole cell lysates (WCL). Middle: Isolation of PP2A Aα complexes and <t>immunoblotting</t> with anti-PP2A Aα antibodies. Bottom: Isolation of PP2A Aα complexes and immunoblotting for ST. (B) Mutation of PP2A Aα residues predicted to interact with ST. Top: Isolation of PP2A Aα complexes and immunoblotting with anti-PP2A Aα. Bottom: Isolation of PP2A Aα complexes and immunoblotting for ST.
    Immunoblotting, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 5988 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    GE Healthcare immunoblotting
    Analysis of MDCK cells with reduced expression of GEF-H1. (A) MDCK cells transfected with a plasmid driving the expression of a control RNA duplex (control-RD c1) or RNA duplex targeting different regions of cGEF-H1 (GEF-RD-I c6 and GEF-RD-II c4) were harvested, and expression was analyzed by <t>immunoblotting</t> with mAb B4/7. ZO-1 and α-tubulin were immunoblotted as controls. The reduction of GEF-H1 expression in all clones used for the functional analysis was at least 50%. (B–D) MDCK cells were cultured on filters for 9 d and were then analyzed by immunofluorescence (B shows labelings with mAb B4/7 and a polyclonal anti–ZO-1 antibody; Bar, 8 μm), by measuring TER (C), and by determining paracellular permeability of 4 kD FITC-dextran (D). The values represent means ± SD of four clones for cells with reduced cGEF-H1 expression (GEF-RD) and two clones for control transfections (control-RD). The decrease in paracellular permeability of GEF-RD cells is significant (p
    Immunoblotting, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 6679 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Jackson Immuno immunoblotting
    STX1, STX4 and Syb are required for the fusion of secretory carriers with the plasma membrane. A) Clone 3 cells were mock transfected (TransFast only) or transfected with dsRNA targeting the indicated genes. After 96 hours, the cells were incubated with AP21998 at 25°C for 80 minutes and their mean fluorescence determined using flow cytometry. The amount of cargo remaining in the cells was calculated and plotted (Error bars show experimental range for six repeats). B) Clone 3 cells were mock transfected (TransFast only) or transfected with dsRNA targeting the indicated genes. After 96 hours, the cells were incubated with AP21998 at 25°C for 80 minutes and the media and cells harvested for <t>immunoblotting.</t> Solid arrowhead indicates unprocessed cargo and unfilled arrowhead furin processed cargo. The amount of processed GH in the media was quantified using densitometry and plotted in C (Error bars show experimental range for two repeats). *The apparent increase in STX1 and STX4 levels by immunoblotting when STX5 and Syb are depleted is not because of a change in total STX1 levels but is due to a change in its extractability from cells. No difference in STX1 or STX4 levels were observed when cells are directly prepared in Laemmli sample buffer ( S2 Fig ). D) Clone 3 cells were either mock transfected or transfected with dsRNA targeting STX1 and STX4. After 72 hours the cells were seeded onto coverslips. The next day, the cells were incubated with AP21998 at 25°C for 80 minutes. The cells were then fixed and stained for the Golgi marker GM130. Scale Bar 10μm.
    Immunoblotting, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 93/100, based on 588 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Roche immunoblotting
    Verapamil restores lysosomal degradation of autophagosomes in liver of obese mice 4 month-old C57BL/6 male mice kept on HFD for two months were subjected to daily administration of PBS (Con, n = 4) or verapamil (Ver, 25 mg/Kg body weight, i.p., n = 3) for 10 days. LFD-kept mice ( n = 5) of same age were used as a negative control. ( a , b ) Levels of LC3-II from 1% Triton X-100 insoluble fraction of livers were analyzed by <t>immunoblotting</t> ( a ) and quantified ( b ). ( c , d ) Frozen sections of indicated livers were subjected to LC3/LAMP1 immunostaining and DAPI counterstaining ( c ). Co-localization between LC3 and LAMP1 was quantified ( d ). Boxed areas in fluorescence images are magnified in right-most panels ( c ). Scale bars, 5 μm ( c ). All data are shown as mean ± s.e.m. *** P
    Immunoblotting, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 6342 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    3M Co immunoblotting
    Interaction of EphA4 with GHR. (A) Schemes of wild-type (WT) and a cytoplasmic deletion mutant of EphA4. The numbers represent the amino acid numbers counting from the one encoded by the initiation codon. TM, transmembrane domain; JM, juxtamembrane domain. Dotted blue rectangles indicate the deleted amino-acid regions. (B) Binding between the extracellular domains of EphA4 and GHR. EphA4(WT)-3Flag or EphA4(Δcyto)-3Flag was transiently co-expressed with GHR(WT)-6myc in HEK293T cells. These proteins were subjected to co-immunoprecipitation (IP), fractionation with SDS-PAGE, and detection by <t>immunoblotting</t> (IB) using the indicated antibodies. Arrowheads indicate the molecules shown in the figure. The other bands are regarded as non-specific bands, compared with the result without EphA4 expression (see also S1 Fig ). (C) Binding between the cytoplasmic domains of EphA4 and GHR. GHR(WT)-6myc and the chimeric protein in which the cytoplasmic domain of EphA4 was fused to the extracellular domain of ephrin-B2 (B2-A4-3Flag) were co-expressed in HEK293T cells and their interaction was examined by IB following IP and SDS-PAGE fractionation. The bands at approximately 100 kDa for GHR-6myc transfectants probably correspond to the GHR molecules modified with carbohydrates. (D) Control experiment for (C) showing no interaction between GHR and ephrin-B2. Ephrin-B2-3Flag was transiently co-expressed with GHR(WT)-6myc in HEK293T cells and their interaction was examined as described in (C). Some of the molecules that have extracellular domains (EphA4, ephrin-B2, and B2-A4) show broad bands, with the upper bands represented by the carbohydrate-modified proteins. All the IP and IB studies in this figure were repeated 3 times to confirm reproducibility, and representative results are shown.
    Immunoblotting, supplied by 3M Co, used in various techniques. Bioz Stars score: 91/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    R&D Systems immunoblotting
    ADAM9 reduction therapy decreased tumor volume by inhibiting cancer cell proliferation. (a) Schematic of ADAM9 knockdown therapy experiment. Mice were injected subcutaneously with PC3 cells in both flanks, and tumor size was measured twice a week until the size reached approximately 200 mm 3 . Mice then received injections of either PBS into both tumor sites (n = 5) or shGFP lentivirus into the left side tumor and shADAM9 into the right side (n = 10). Viruses were injected and tumors measured weekly for 6 consecutive weeks. (b) After shADAM9 therapy, tumor volumes did not increase compared to shGFP therapy or PBS controls (** p ≤0.01, Student’s t test). (c) <t>Immunoblotting</t> assay of ADAM9 expression confirms decreased ADAM9 expression after shADAM9 therapy. EF-1α was used as a loading control (* p ≤0.05; ** p ≤0.01, Student’s t test). (d) Statistical analysis of ki67 staining. shADAM9 therapy significantly decreased cell proliferation activities in PC3 tumors. PBS and shGFP therapy showed no difference in the proliferation index (** p ≤0.001, One Way ANOVA). (e) Statistical analysis of TUNEL staining showed no significant (N.S.) difference between therapies ( p ≥0.05, One Way ANOVA).
    Immunoblotting, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 1021 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Synaptic Systems immunoblotting
    BI-1-associated lysosomal activity leads to ER stress regulation. ( a ) Neo and BI-1 cells were cultured in serum-free medium with 10 ng/ml TGF- β 1 for 0, 12, 24, 36, or 48 h. ( b ) Neo and BI-1 cells were cultured in serum-free medium with 10 ng/ml TGF- β 1 in the presence or absence of 10 nM bafilomycin for 48 h. Neo and BI-1 cells were transfected with non-specific or V-ATPase specific siRNA and <t>immunoblotting</t> was performed with anti-V-ATPase antibody. ( c ) Separately, siRNA-transfected Neo and BI-1 cells were cultured in serum-free medium with 10 ng/ml TGF- β 1 for 48 h. Immunoblotting was performed with anti-GRP78, CHOP, IRE-1 α , sXBP-1, PERK, p-eIF2 α , eIF2 α , p-JNK, JNK1, ATF6 α (50KD), and β -actin antibodies. NS, non-specific siRNA; V-siRNA, V-ATPase siRNA
    Immunoblotting, supplied by Synaptic Systems, used in various techniques. Bioz Stars score: 92/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson immunoblotting
    Downregulation of SRC-2 in MCF-7 cells induces distinct changes in global gene expression profiles. (A). Quantification of SRC-2 mRNA expression in shRNA lentivirus-infected MCF-7 cells. mRNA levels of SRC-2 in a MCF-7 cell line infected with shRNA targeting SRC-2 (SRC-2 shRNA) were compared to the expression in a control shRNA MCF-7 cell line (Ctr shRNA), and in a MCF-7 cell line transduced with shRNA lentivirus targeting SRC-3 (SRC-3 shRNA). The mRNA expression of SRC-2 is relative to TBP mRNA. The results are representative of at least three independent experiments. (B). Western blotting analyses of SRC-2-depleted MCF-7 cells. MCF-7 cells infected with shRNA lentivirus targeting SRC-2 (SRC-2 shRNA) or a negative control shRNA empty vector (Ctr shRNA), were grown in phenol red-free DMEM supplemented with charcoaled stripped FBS (5%) and 17β-estradiol (10 nM) for two days. The Ctr shRNA cells were then treated with either Vehicle or 8-CPT-cAMP (150 µM), IBMX (50 µM) and forskolin (10 µM) (cAMP) for 24 hours. <t>Immunoblotting</t> was performed with anti-TIF2 antibody and anti-GAPDH antibody. The results shown are representative of at least three independent experiments. (C). Microarray analyses of five RNA samples isolated from five individual cell samples of shRNA control MCF-7 cells (Ctr shRNA), SRC-2 KD MCF-7 cells (SRC-2 shRNA) and control shRNA cells treated with cAMP elevating agents, as described in A. A Venn diagram shows the number of individual and overlapping sets of genes differentially expressed after SRC-2 KD (SRC-2 shRNA) and after treatment with cAMP elevating agents. To examine which genes were similarly differentially expressed between the two treated groups when compared to control, a SAM analysis with overlapping genes was performed. The fold change cut-off value ≥1.5, and q-value = 0, was used to determine differentially expressed genes.
    Immunoblotting, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 2316 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Bethyl immunoblotting
    Regulation of the cell cycle by B55α and B55β. ( A ) Metaphase-arrested Xenopus egg extracts were supplemented with recombinant B55α or control buffer for 10 min. These extracts were then released into interphase by the addition of Calcium at time 0. The extract samples were collected at the indicated time points and analyzed by <t>immunoblotting.</t> Phosphorylation (band-shift) of Cdc27 and Cdc25, and the global phosphorylation of Cdk substrates are markers of mitosis. ( B ) Interphase Xenopus egg extracts were supplemented with recombinant B55α or control buffer. The extract samples were collected at the indicated time points and analyzed by immunoblotting. Mitosis is indicated by Cdc27 phosphorylation. ( C ) MBP-B55α and B55β were purified and examined by Coomassie staining. ( D ) Interphase Xenopus egg extracts were supplemented with recombinant B55α, B55β, or control buffer. The extract samples were collected at the indicated time points and analyzed by immunoblotting. Mitosis is indicated by Cdc27 phosphorylation.
    Immunoblotting, supplied by Bethyl, used in various techniques. Bioz Stars score: 92/100, based on 456 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    MBL International immunoblotting
    Flubendazole activates JNK1 and Bcl-2 and relieves Beclin 1 from negative regulation. ( a ) HeLa cells treated with DMSO or flubendazole (5 μg ml −1 ) were lysed and subjected to <t>immunoblotting.</t> ( b ) Coimmunoprecipitation analysis of interaction between Beclin 1 and Bcl-2 (HEK293T lysates) in cells treated with DMSO or 5 μg ml −1 flubendazole for 2 h. ( c ) HeLa cells knocked down for ATAT1 and treated with DMSO or 5 μg ml −1 flubendazole (for 2 h) were lysed and subjected to immunoblotting. ( d ) High-content analysis of the abundance of LC3 puncta in HeLa cells knockdown for ATAT1 or control cells treated with flubendazole or DMSO for 45 min. Flub, flubendazole; statistics, mean±s.e.; Student's t- test. * P
    Immunoblotting, supplied by MBL International, used in various techniques. Bioz Stars score: 92/100, based on 73 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Nacalai immunoblotting
    MEP50/PRMT5 complex-mediated GLI1 methylation inhibits the interaction of GLI1 with its E3 ligase complex, ITCH/NUMB, resulting in GLI1 stabilisation. a Interaction of GLI1 and endogenous ITCH or NUMB from stably PRMT5-knockdown or MEP50-knockdown C3H10T1/2 cells. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. MG132 (50 μM) was applied for 4 h before harvesting. b Interaction of GLI1 mutants with endogenous ITCH or NUMB in C3H10T1/2 cells. The cells were transfected as indicated. At 48 h post-transfection, 50 μM MG132 was applied for 4 h, and then the cells were lysed and subjected to immunoprecipitation with an anti-HA antibody, followed by <t>immunoblotting</t> with antibodies against the indicated proteins. c In vivo ubiquitination of HA-GLI1-RK mutants. Cells were transfected and cultured for 24 h, followed by treatment with 50 µM MG132 for 4 h before harvesting. Ubiquitinated GLI1 was detected by immuoprecipitation with an anti-HA (3F10) antibody and immunoblotting with anti-FLAG (upper panel) or anti-HA (lower panel) antibodies. The asterisk denotes non-specific bands. d Schematic diagram of the mechanism of PRMT5/MEP50-mediated GLI1 stabilisation. When the HH signalling pathway inactivates, the ITCH/NUMB E3 ligase complex binds to and ubiquitinates GLI1 for proteasomal degradation. In turn, under HH signalling pathway activation, the MEP50/PRMT5 complex methylates GLI1 to dissociate the ITCH/NUMB complex from GLI1, resulting in GLI1 stabilisation. Unprocessed original scans of blots are shown in Supplementary Fig. 6
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    OriGene immunoblotting
    MEP50/PRMT5 complex-mediated GLI1 methylation inhibits the interaction of GLI1 with its E3 ligase complex, ITCH/NUMB, resulting in GLI1 stabilisation. a Interaction of GLI1 and endogenous ITCH or NUMB from stably PRMT5-knockdown or MEP50-knockdown C3H10T1/2 cells. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. MG132 (50 μM) was applied for 4 h before harvesting. b Interaction of GLI1 mutants with endogenous ITCH or NUMB in C3H10T1/2 cells. The cells were transfected as indicated. At 48 h post-transfection, 50 μM MG132 was applied for 4 h, and then the cells were lysed and subjected to immunoprecipitation with an anti-HA antibody, followed by <t>immunoblotting</t> with antibodies against the indicated proteins. c In vivo ubiquitination of HA-GLI1-RK mutants. Cells were transfected and cultured for 24 h, followed by treatment with 50 µM MG132 for 4 h before harvesting. Ubiquitinated GLI1 was detected by immuoprecipitation with an anti-HA (3F10) antibody and immunoblotting with anti-FLAG (upper panel) or anti-HA (lower panel) antibodies. The asterisk denotes non-specific bands. d Schematic diagram of the mechanism of PRMT5/MEP50-mediated GLI1 stabilisation. When the HH signalling pathway inactivates, the ITCH/NUMB E3 ligase complex binds to and ubiquitinates GLI1 for proteasomal degradation. In turn, under HH signalling pathway activation, the MEP50/PRMT5 complex methylates GLI1 to dissociate the ITCH/NUMB complex from GLI1, resulting in GLI1 stabilisation. Unprocessed original scans of blots are shown in Supplementary Fig. 6
    Immunoblotting, supplied by OriGene, used in various techniques. Bioz Stars score: 93/100, based on 201 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    prosci incorporated immunoblotting
    Expression of interleukin-34 (IL-34) in human rheumatoid arthritis (RA) synovium and synovial fluid (SF) . ( A ) IL-34 and macrophage colony-stimulating factor (M-CSF) mRNA levels in synovia from patients with RA ( n = 3) and osteoarthritis (OA) ( n = 3) were determined by reverse transcriptase PCR (RT-PCR). Synovium were homogenized and lysed, and total RNA was extracted as described in Materials and Methods. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels were detected as a control. ( B ) Representative immunohistochemical images of OA or RA synovia stained with antibodies against IL-34 or isotype controls. Images are shown at 200× (upper) and 400× (lower) magnification. Scale bars = 100 μm. ( C ) Synovial fluid (SF) from RA patients ( n = 7) (RA SF) was collected and the concentrations of M-CSF and IL-34 were measured using an enzyme-linked immunosorbent assay (ELISA) assay. ( D ) Expression of IL-34 protein in fibroblast-like synovial cells (FLS) from RA patients ( n = 6) was determined by <t>immunoblotting</t> against human IL-34. Whole-cell lysates of RA FLS cells were resolved by SDS-PAGE and followed by immunoblotting with anti-human IL-34 and anti-β-actin antibodies.
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    Carl Roth GmbH immunoblotting
    Effect of HDACi on tumor suppressor proteins p53, p21, and p27 and on DBS marker pH2AX Analysis of tumor suppressor protein and pH2AX expression was performed by <t>immunoblotting</t> after 48 h treatment with HDACi SAHA, LBH-589, and PXD101. One representative blot out of three is shown. β-actin was used as loading control. Δ represents fold change normalized to controls (mean±SD of n = 3).
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    Cytoskeleton Inc immunoblotting
    Pharmacological inhibition of actin glutathionylation prevents DNA release. (A) <t>Immunoblotting.</t> Actin glutathionylation in activated mouse and human neutrophils is dependent on NADPH oxidase activation. Human and mouse neutrophils were analyzed after short-term stimulation (total 35 min) with the indicated triggers. C5a activation was performed in a time-dependent manner. No glutathionylated actin at the expected size (42 kD) was detected in Nox2 −/− mouse neutrophils or in 50-µM-DPI–pretreated human neutrophils. The ratio of glutathionylated actin to total actin is shown as actin glutathionylation in the bar graph (right). Data are representative of three independent experiments. (B) Confocal microscopy. DNA release was analyzed after short-term stimulation (total 35 min) of control human neutrophils with the indicated triggers in the presence and absence of the inhibitors as designated: 50 µM Na-arsenite and 2 µM CdCl 2 . Right: Quantification of released dsDNA in supernatants of activated neutrophils. Data are means ± SEM. *, P
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    Enzo Biochem immunoblotting
    Fluorescence properties of ER-targeted roGFP-iL mutants in the ER ( A ) Fluorescence images of HeLa cells transfected with ER-targeted roGFP-iL and its mutants were acquired (green) after fixation with 4% PFA. The cells were also co-stained with anti-GFP (red) and anti-Hsp47 (blue, ER marker). ( B ) <t>Immunoblotting</t> of ER-targeted roGFP-iL and its mutants with anti-GFP in 1% NP-40-soluble (S) and -insoluble (P) cell fractions.
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    MIKROGEN immunoblotting
    Fluorescence properties of ER-targeted roGFP-iL mutants in the ER ( A ) Fluorescence images of HeLa cells transfected with ER-targeted roGFP-iL and its mutants were acquired (green) after fixation with 4% PFA. The cells were also co-stained with anti-GFP (red) and anti-Hsp47 (blue, ER marker). ( B ) <t>Immunoblotting</t> of ER-targeted roGFP-iL and its mutants with anti-GFP in 1% NP-40-soluble (S) and -insoluble (P) cell fractions.
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    Image Search Results


    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

    ( A , B ): Effect of Resveratrol and/or 5-FU on NF-κB activation and NF-κB-regulated gene end-products involved in apoptosis, metastasis induced by TNF-β in HCT116 and HCT116R in inflammatory microenvironment. Alginate cultures of HCT116 ( A ) and HCT116R ( B ) were treated for 10 days as described in detail in “ Section 2 ”. Immunoblotting with whole cell lysates was performed with antibodies against p65-NF-κB, CXCR4, MMP-9 and cleaved-caspase-3. Western blots shown are representative of three independent experiments. The housekeeping protein β-actin served as a positive loading control in all experiments. ( C , D ): Effect of resveratrol and/or 5-FU on TNF-β-induced epithelial-to-mesenchymal transition of CRC cells in tumor microenvironment cultures. Colorectal cancer cells in alginate culture were treated as described in Materials and Methods. After 10 days whole cell lysates of HCT116 ( C ) and HCT116R ( D ) were subjected to western blotting with antibodies against vimentin, E-cadherin and slug. Western blots shown are representative of three independent experiments. The housekeeping protein β-actin served as a positive loading control in all experiments.

    Journal: Nutrients

    Article Title: Resveratrol Chemosensitizes TNF-β-Induced Survival of 5-FU-Treated Colorectal Cancer Cells

    doi: 10.3390/nu10070888

    Figure Lengend Snippet: ( A , B ): Effect of Resveratrol and/or 5-FU on NF-κB activation and NF-κB-regulated gene end-products involved in apoptosis, metastasis induced by TNF-β in HCT116 and HCT116R in inflammatory microenvironment. Alginate cultures of HCT116 ( A ) and HCT116R ( B ) were treated for 10 days as described in detail in “ Section 2 ”. Immunoblotting with whole cell lysates was performed with antibodies against p65-NF-κB, CXCR4, MMP-9 and cleaved-caspase-3. Western blots shown are representative of three independent experiments. The housekeeping protein β-actin served as a positive loading control in all experiments. ( C , D ): Effect of resveratrol and/or 5-FU on TNF-β-induced epithelial-to-mesenchymal transition of CRC cells in tumor microenvironment cultures. Colorectal cancer cells in alginate culture were treated as described in Materials and Methods. After 10 days whole cell lysates of HCT116 ( C ) and HCT116R ( D ) were subjected to western blotting with antibodies against vimentin, E-cadherin and slug. Western blots shown are representative of three independent experiments. The housekeeping protein β-actin served as a positive loading control in all experiments.

    Article Snippet: Secondary alkaline phosphatase–linked sheep anti-mouse and sheep anti-rabbit antibodies for immunoblotting were from EMD Millipore (Schwalbach, Germany) and secondary antibodies for fluorescence labeling from Dianova (Hamburg, Germany).

    Techniques: Activation Assay, Western Blot

    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

    Mutational Analysis of the A-ST Interface (A) Mutation of ST residues predicted to interact with PP2A Aα. Top: Expression of ST in whole cell lysates (WCL). Middle: Isolation of PP2A Aα complexes and immunoblotting with anti-PP2A Aα antibodies. Bottom: Isolation of PP2A Aα complexes and immunoblotting for ST. (B) Mutation of PP2A Aα residues predicted to interact with ST. Top: Isolation of PP2A Aα complexes and immunoblotting with anti-PP2A Aα. Bottom: Isolation of PP2A Aα complexes and immunoblotting for ST.

    Journal: PLoS Biology

    Article Title: Structural Basis of PP2A Inhibition by Small t Antigen

    doi: 10.1371/journal.pbio.0050202

    Figure Lengend Snippet: Mutational Analysis of the A-ST Interface (A) Mutation of ST residues predicted to interact with PP2A Aα. Top: Expression of ST in whole cell lysates (WCL). Middle: Isolation of PP2A Aα complexes and immunoblotting with anti-PP2A Aα antibodies. Bottom: Isolation of PP2A Aα complexes and immunoblotting for ST. (B) Mutation of PP2A Aα residues predicted to interact with ST. Top: Isolation of PP2A Aα complexes and immunoblotting with anti-PP2A Aα. Bottom: Isolation of PP2A Aα complexes and immunoblotting for ST.

    Article Snippet: For immunoblotting, we used affinity-purified polyclonal antibodies against SV40 ST [ ] and monoclonal antibodies (clone 6F9) against Aα (Abcam, http://www.abcam.com ).

    Techniques: Mutagenesis, Expressing, Isolation

    Analysis of MDCK cells with reduced expression of GEF-H1. (A) MDCK cells transfected with a plasmid driving the expression of a control RNA duplex (control-RD c1) or RNA duplex targeting different regions of cGEF-H1 (GEF-RD-I c6 and GEF-RD-II c4) were harvested, and expression was analyzed by immunoblotting with mAb B4/7. ZO-1 and α-tubulin were immunoblotted as controls. The reduction of GEF-H1 expression in all clones used for the functional analysis was at least 50%. (B–D) MDCK cells were cultured on filters for 9 d and were then analyzed by immunofluorescence (B shows labelings with mAb B4/7 and a polyclonal anti–ZO-1 antibody; Bar, 8 μm), by measuring TER (C), and by determining paracellular permeability of 4 kD FITC-dextran (D). The values represent means ± SD of four clones for cells with reduced cGEF-H1 expression (GEF-RD) and two clones for control transfections (control-RD). The decrease in paracellular permeability of GEF-RD cells is significant (p

    Journal: The Journal of Cell Biology

    Article Title: Identification of a tight junction-associated guanine nucleotide exchange factor that activates Rho and regulates paracellular permeability

    doi: 10.1083/jcb.200211047

    Figure Lengend Snippet: Analysis of MDCK cells with reduced expression of GEF-H1. (A) MDCK cells transfected with a plasmid driving the expression of a control RNA duplex (control-RD c1) or RNA duplex targeting different regions of cGEF-H1 (GEF-RD-I c6 and GEF-RD-II c4) were harvested, and expression was analyzed by immunoblotting with mAb B4/7. ZO-1 and α-tubulin were immunoblotted as controls. The reduction of GEF-H1 expression in all clones used for the functional analysis was at least 50%. (B–D) MDCK cells were cultured on filters for 9 d and were then analyzed by immunofluorescence (B shows labelings with mAb B4/7 and a polyclonal anti–ZO-1 antibody; Bar, 8 μm), by measuring TER (C), and by determining paracellular permeability of 4 kD FITC-dextran (D). The values represent means ± SD of four clones for cells with reduced cGEF-H1 expression (GEF-RD) and two clones for control transfections (control-RD). The decrease in paracellular permeability of GEF-RD cells is significant (p

    Article Snippet: Immunoblotting was done as described previously using HRP-conjugated secondary antibodies and the ECL chemiluminescence detection system (Amersham Biosciences).

    Techniques: Expressing, Transfection, Plasmid Preparation, Clone Assay, Functional Assay, Cell Culture, Immunofluorescence, Permeability

    Functional analysis of TJs of cells overexpressing cGEF-H1. (A) Wild-type (wt) and cGEF-H1–overexpressing (3A4) cells, cultured for 1 wk on polycarbonate filters, were fixed and processed for double immunofluorescence using mAb B4/7 and anti–ZO-1 antibodies. Images obtained with constant microscope sensitivity settings are shown. Bar, 10 μm. (B and C) MDCK cells were cultured on filters for 1 wk and were then analyzed first by measuring TER (B) and, after a time of recovery, by determining paracellular permeability of 4 kD FITC-dextran (C) or 400 kD FITC-dextran (D). At the end, the cultures were analyzed for overexpression of cGEF-H1 by immunoblotting (examples are shown in Fig. 3 A). All values of transfected cells represent means ± SD of several independent clones: four clones were analyzed for cGEF-H1+I, three clones for cGEF-H1-I, and three control clones that were derived from a transfection of the empty vector. The increases in 4 kD FITC-dextran permeability of overexpressing cells are significant (cGEF-H1+I, p

    Journal: The Journal of Cell Biology

    Article Title: Identification of a tight junction-associated guanine nucleotide exchange factor that activates Rho and regulates paracellular permeability

    doi: 10.1083/jcb.200211047

    Figure Lengend Snippet: Functional analysis of TJs of cells overexpressing cGEF-H1. (A) Wild-type (wt) and cGEF-H1–overexpressing (3A4) cells, cultured for 1 wk on polycarbonate filters, were fixed and processed for double immunofluorescence using mAb B4/7 and anti–ZO-1 antibodies. Images obtained with constant microscope sensitivity settings are shown. Bar, 10 μm. (B and C) MDCK cells were cultured on filters for 1 wk and were then analyzed first by measuring TER (B) and, after a time of recovery, by determining paracellular permeability of 4 kD FITC-dextran (C) or 400 kD FITC-dextran (D). At the end, the cultures were analyzed for overexpression of cGEF-H1 by immunoblotting (examples are shown in Fig. 3 A). All values of transfected cells represent means ± SD of several independent clones: four clones were analyzed for cGEF-H1+I, three clones for cGEF-H1-I, and three control clones that were derived from a transfection of the empty vector. The increases in 4 kD FITC-dextran permeability of overexpressing cells are significant (cGEF-H1+I, p

    Article Snippet: Immunoblotting was done as described previously using HRP-conjugated secondary antibodies and the ECL chemiluminescence detection system (Amersham Biosciences).

    Techniques: Functional Assay, Cell Culture, Immunofluorescence, Microscopy, Permeability, Over Expression, Transfection, Clone Assay, Derivative Assay, Plasmid Preparation

    Activation of Rho by cGEF-H1. (A) Total cell extracts of wild-type (wt) or transfected MDCK cells were immunoblotted with mAb B4/7. 3A3 and 3A4, clones overexpressing cGEF-H1; control, clone derived from control transfection. (B) Wild-type and transfected MDCK cells expressing truncated cGEF-H1 (HA-GEF-T) or wild-type cGEF-H1 were extracted and tested for the presence of activated Rho or Rac1 by performing pull-down assays with recombinant fusion proteins lacking (GST) or containing a GTPase-binding domains of (GST-GBD). The pulled-down fractions were analyzed by immunoblotting for the presence of Rho or Rac1. Increasing amounts of total cell extracts were loaded on the gels to normalize the amount of GTPases in different cell extracts (1X Total ex and 2X Total ex). Immunoblots were quantified by densitometric scanning, and the values obtained for the activated GTPase were divided by those obtained in total cell extracts. All ratios were then normalized to wild-type cells and expressed as fold activation (mean ± SD of three experiments; both increases in active Rho are significant, p

    Journal: The Journal of Cell Biology

    Article Title: Identification of a tight junction-associated guanine nucleotide exchange factor that activates Rho and regulates paracellular permeability

    doi: 10.1083/jcb.200211047

    Figure Lengend Snippet: Activation of Rho by cGEF-H1. (A) Total cell extracts of wild-type (wt) or transfected MDCK cells were immunoblotted with mAb B4/7. 3A3 and 3A4, clones overexpressing cGEF-H1; control, clone derived from control transfection. (B) Wild-type and transfected MDCK cells expressing truncated cGEF-H1 (HA-GEF-T) or wild-type cGEF-H1 were extracted and tested for the presence of activated Rho or Rac1 by performing pull-down assays with recombinant fusion proteins lacking (GST) or containing a GTPase-binding domains of (GST-GBD). The pulled-down fractions were analyzed by immunoblotting for the presence of Rho or Rac1. Increasing amounts of total cell extracts were loaded on the gels to normalize the amount of GTPases in different cell extracts (1X Total ex and 2X Total ex). Immunoblots were quantified by densitometric scanning, and the values obtained for the activated GTPase were divided by those obtained in total cell extracts. All ratios were then normalized to wild-type cells and expressed as fold activation (mean ± SD of three experiments; both increases in active Rho are significant, p

    Article Snippet: Immunoblotting was done as described previously using HRP-conjugated secondary antibodies and the ECL chemiluminescence detection system (Amersham Biosciences).

    Techniques: Activation Assay, Transfection, Clone Assay, Derivative Assay, Expressing, Recombinant, Binding Assay, Western Blot

    STX1, STX4 and Syb are required for the fusion of secretory carriers with the plasma membrane. A) Clone 3 cells were mock transfected (TransFast only) or transfected with dsRNA targeting the indicated genes. After 96 hours, the cells were incubated with AP21998 at 25°C for 80 minutes and their mean fluorescence determined using flow cytometry. The amount of cargo remaining in the cells was calculated and plotted (Error bars show experimental range for six repeats). B) Clone 3 cells were mock transfected (TransFast only) or transfected with dsRNA targeting the indicated genes. After 96 hours, the cells were incubated with AP21998 at 25°C for 80 minutes and the media and cells harvested for immunoblotting. Solid arrowhead indicates unprocessed cargo and unfilled arrowhead furin processed cargo. The amount of processed GH in the media was quantified using densitometry and plotted in C (Error bars show experimental range for two repeats). *The apparent increase in STX1 and STX4 levels by immunoblotting when STX5 and Syb are depleted is not because of a change in total STX1 levels but is due to a change in its extractability from cells. No difference in STX1 or STX4 levels were observed when cells are directly prepared in Laemmli sample buffer ( S2 Fig ). D) Clone 3 cells were either mock transfected or transfected with dsRNA targeting STX1 and STX4. After 72 hours the cells were seeded onto coverslips. The next day, the cells were incubated with AP21998 at 25°C for 80 minutes. The cells were then fixed and stained for the Golgi marker GM130. Scale Bar 10μm.

    Journal: PLoS Genetics

    Article Title: VAMP3/Syb and YKT6 are required for the fusion of constitutive secretory carriers with the plasma membrane

    doi: 10.1371/journal.pgen.1006698

    Figure Lengend Snippet: STX1, STX4 and Syb are required for the fusion of secretory carriers with the plasma membrane. A) Clone 3 cells were mock transfected (TransFast only) or transfected with dsRNA targeting the indicated genes. After 96 hours, the cells were incubated with AP21998 at 25°C for 80 minutes and their mean fluorescence determined using flow cytometry. The amount of cargo remaining in the cells was calculated and plotted (Error bars show experimental range for six repeats). B) Clone 3 cells were mock transfected (TransFast only) or transfected with dsRNA targeting the indicated genes. After 96 hours, the cells were incubated with AP21998 at 25°C for 80 minutes and the media and cells harvested for immunoblotting. Solid arrowhead indicates unprocessed cargo and unfilled arrowhead furin processed cargo. The amount of processed GH in the media was quantified using densitometry and plotted in C (Error bars show experimental range for two repeats). *The apparent increase in STX1 and STX4 levels by immunoblotting when STX5 and Syb are depleted is not because of a change in total STX1 levels but is due to a change in its extractability from cells. No difference in STX1 or STX4 levels were observed when cells are directly prepared in Laemmli sample buffer ( S2 Fig ). D) Clone 3 cells were either mock transfected or transfected with dsRNA targeting STX1 and STX4. After 72 hours the cells were seeded onto coverslips. The next day, the cells were incubated with AP21998 at 25°C for 80 minutes. The cells were then fixed and stained for the Golgi marker GM130. Scale Bar 10μm.

    Article Snippet: Secondary antibodies for immunoblotting were purchased from Jackson ImmunoResearch Laboratories.

    Techniques: Transfection, Incubation, Fluorescence, Flow Cytometry, Cytometry, Staining, Marker

    YKT6 functions on multiple intracellular pathways. A) Clone 3 cells were mock transfected (TransFast only) or transfected with dsRNA targeting the indicated genes. After 96 hours, the cells were incubated with AP21998 at 25°C for 80 minutes and their mean fluorescence determined using flow cytometry. The amount of cargo remaining in the cells was calculated and plotted (Error bars show experimental range for six repeats). B) Clone 3 cells were mock transfected (TransFast only) or transfected with dsRNA targeting the indicated genes. After 96 hours, the cells were incubated with AP21998 at 25°C for 80 minutes and the media and cells harvested for immunoblotting. Solid arrowhead indicates unprocessed cargo and unfilled arrowhead furin processed cargo. The amount of processed GH in the media was quantified using densitometry and plotted in C (Error bars show experimental range for two repeats). D) Clone 3 cells were either mock transfected or transfected with dsRNA targeting YKT6 and Sec22b. After 72 hours the cells were seeded onto coverslips. The next day, the cells were incubated with AP21998 at 25°C for 80 minutes. The cells were then fixed and stained for the Golgi marker GM130. Scale Bar 10μm.

    Journal: PLoS Genetics

    Article Title: VAMP3/Syb and YKT6 are required for the fusion of constitutive secretory carriers with the plasma membrane

    doi: 10.1371/journal.pgen.1006698

    Figure Lengend Snippet: YKT6 functions on multiple intracellular pathways. A) Clone 3 cells were mock transfected (TransFast only) or transfected with dsRNA targeting the indicated genes. After 96 hours, the cells were incubated with AP21998 at 25°C for 80 minutes and their mean fluorescence determined using flow cytometry. The amount of cargo remaining in the cells was calculated and plotted (Error bars show experimental range for six repeats). B) Clone 3 cells were mock transfected (TransFast only) or transfected with dsRNA targeting the indicated genes. After 96 hours, the cells were incubated with AP21998 at 25°C for 80 minutes and the media and cells harvested for immunoblotting. Solid arrowhead indicates unprocessed cargo and unfilled arrowhead furin processed cargo. The amount of processed GH in the media was quantified using densitometry and plotted in C (Error bars show experimental range for two repeats). D) Clone 3 cells were either mock transfected or transfected with dsRNA targeting YKT6 and Sec22b. After 72 hours the cells were seeded onto coverslips. The next day, the cells were incubated with AP21998 at 25°C for 80 minutes. The cells were then fixed and stained for the Golgi marker GM130. Scale Bar 10μm.

    Article Snippet: Secondary antibodies for immunoblotting were purchased from Jackson ImmunoResearch Laboratories.

    Techniques: Transfection, Incubation, Fluorescence, Flow Cytometry, Cytometry, Staining, Marker

    Syb and YKT6 have a role in the fusion of secretory carriers with the plasma membrane. A) Clone 3 cells were mock transfected (TransFast only) or transfected with dsRNA targeting the indicated genes. After 96 hours, the cells were incubated with AP21998 at 25°C for 80 minutes and their mean fluorescence determined using flow cytometry. The amount of cargo remaining in the cells was calculated and plotted (Error bars show experimental range for six repeats). B) Clone 3 cells were mock transfected (TransFast only) or transfected with dsRNA targeting the indicated genes. After 96 hours, the cells were incubated with AP21998 at 25°C for 80 minutes and the media and cells harvested for immunoblotting. Solid arrowhead indicates unprocessed cargo and unfilled arrowhead furin processed cargo. The amount of processed GH in the media was quantified using densitometry and plotted in C (Error bars show experimental range for two repeats). D) Clone 3 cells were either mock transfected or transfected with dsRNA targeting YKT6 and Syb. After 72 hours the cells were seeded onto coverslips. The next day, the cells were incubated with AP21998 at 25°C for 80 minutes. The cells were then fixed and stained for the Golgi marker GM130. Scale Bar 10μm. E) To determine which SNAREs interact with STX1 a native immunoprecipitation was performed from Drosophila cells stably expressing HA tagged STX1. The isolated protein complexes were separated by SDS-PAGE and the major bands identified using mass-spectrometry.

    Journal: PLoS Genetics

    Article Title: VAMP3/Syb and YKT6 are required for the fusion of constitutive secretory carriers with the plasma membrane

    doi: 10.1371/journal.pgen.1006698

    Figure Lengend Snippet: Syb and YKT6 have a role in the fusion of secretory carriers with the plasma membrane. A) Clone 3 cells were mock transfected (TransFast only) or transfected with dsRNA targeting the indicated genes. After 96 hours, the cells were incubated with AP21998 at 25°C for 80 minutes and their mean fluorescence determined using flow cytometry. The amount of cargo remaining in the cells was calculated and plotted (Error bars show experimental range for six repeats). B) Clone 3 cells were mock transfected (TransFast only) or transfected with dsRNA targeting the indicated genes. After 96 hours, the cells were incubated with AP21998 at 25°C for 80 minutes and the media and cells harvested for immunoblotting. Solid arrowhead indicates unprocessed cargo and unfilled arrowhead furin processed cargo. The amount of processed GH in the media was quantified using densitometry and plotted in C (Error bars show experimental range for two repeats). D) Clone 3 cells were either mock transfected or transfected with dsRNA targeting YKT6 and Syb. After 72 hours the cells were seeded onto coverslips. The next day, the cells were incubated with AP21998 at 25°C for 80 minutes. The cells were then fixed and stained for the Golgi marker GM130. Scale Bar 10μm. E) To determine which SNAREs interact with STX1 a native immunoprecipitation was performed from Drosophila cells stably expressing HA tagged STX1. The isolated protein complexes were separated by SDS-PAGE and the major bands identified using mass-spectrometry.

    Article Snippet: Secondary antibodies for immunoblotting were purchased from Jackson ImmunoResearch Laboratories.

    Techniques: Transfection, Incubation, Fluorescence, Flow Cytometry, Cytometry, Staining, Marker, Immunoprecipitation, Stable Transfection, Expressing, Isolation, SDS Page, Mass Spectrometry

    Development of a novel assay for measuring secretion in Drosophila cells. A) Schematic of the reporter construct used to measure secretion (DD, dimerisation domain; FCS, furin cleave site; hGH, human growth hormone and numbers indicate amino acids). B) Transport kinetics of the reporter construct were determined by incubating C3 cells with AP21998 at 25°C for the indicated times and the mean fluorescence of the cells measured using flow cytometry. The amount of cargo remaining in the cells after the addition of AP21998 was calculated as a ratio between the control sample (no AP21998) and the experimental samples (+AP21998). C) Clone 3 cells were mock transfected (TransFast only) or transfected with dsRNA targeting the indicated genes. After 96 hours, the cells were incubated with AP21998 at 25°C for 80 minutes and their mean fluorescence determined using flow cytometry. The red histogram indicates the fluorescent intensity of the control sample, no AP21998 and the blue histogram shows the fluorescent intensity of the cells incubated with AP21998. D) The amount of cargo remaining in the cells was calculated as in B and plotted (Error bars show experimental range for three repeats). E) Clone 3 cells were mock transfected (TransFast only) or transfected with dsRNA targeting the indicated genes. After 96 hours, the cells were incubated with AP21998 at 25°C for 80 minutes and the media and cells harvested for immunoblotting. Solid arrowhead indicates unprocessed cargo and unfilled arrowhead furin processed cargo. F) Clone 3 cells were either mock transfected or transfected with dsRNA targeting STX5 and ROP. After 72 hours the cells were seeded onto coverslips. The next day, the cells were incubated with AP21998 at 25°C for 80 minutes. The cells were then fixed and stained for the Golgi marker GM130. Scale Bar 10μm.

    Journal: PLoS Genetics

    Article Title: VAMP3/Syb and YKT6 are required for the fusion of constitutive secretory carriers with the plasma membrane

    doi: 10.1371/journal.pgen.1006698

    Figure Lengend Snippet: Development of a novel assay for measuring secretion in Drosophila cells. A) Schematic of the reporter construct used to measure secretion (DD, dimerisation domain; FCS, furin cleave site; hGH, human growth hormone and numbers indicate amino acids). B) Transport kinetics of the reporter construct were determined by incubating C3 cells with AP21998 at 25°C for the indicated times and the mean fluorescence of the cells measured using flow cytometry. The amount of cargo remaining in the cells after the addition of AP21998 was calculated as a ratio between the control sample (no AP21998) and the experimental samples (+AP21998). C) Clone 3 cells were mock transfected (TransFast only) or transfected with dsRNA targeting the indicated genes. After 96 hours, the cells were incubated with AP21998 at 25°C for 80 minutes and their mean fluorescence determined using flow cytometry. The red histogram indicates the fluorescent intensity of the control sample, no AP21998 and the blue histogram shows the fluorescent intensity of the cells incubated with AP21998. D) The amount of cargo remaining in the cells was calculated as in B and plotted (Error bars show experimental range for three repeats). E) Clone 3 cells were mock transfected (TransFast only) or transfected with dsRNA targeting the indicated genes. After 96 hours, the cells were incubated with AP21998 at 25°C for 80 minutes and the media and cells harvested for immunoblotting. Solid arrowhead indicates unprocessed cargo and unfilled arrowhead furin processed cargo. F) Clone 3 cells were either mock transfected or transfected with dsRNA targeting STX5 and ROP. After 72 hours the cells were seeded onto coverslips. The next day, the cells were incubated with AP21998 at 25°C for 80 minutes. The cells were then fixed and stained for the Golgi marker GM130. Scale Bar 10μm.

    Article Snippet: Secondary antibodies for immunoblotting were purchased from Jackson ImmunoResearch Laboratories.

    Techniques: Construct, Fluorescence, Flow Cytometry, Cytometry, Transfection, Incubation, Staining, Marker

    SNAP24 and SNAP29 mediate the fusion of secretory carriers with the plasma membrane. A) Clone 3 cells were mock transfected (TransFast only) or transfected with dsRNA targeting the indicated genes. After 96 hours, the cells were incubated with DD solubiliser at 25°C for 80 minutes and their mean fluorescence determined using flow cytometry. The amount of cargo remaining in the cells was calculated and plotted (Error bars show experimental range for three repeats). B) Clone 3 cells were mock transfected (TransFast only) or transfected with dsRNA targeting the indicated genes. After 96 hours, the cells were incubated with DD solubiliser at 25°C for 80 minutes and the media and cells harvested for immunoblotting. Solid arrowhead indicates unprocessed cargo and unfilled arrowhead furin processed cargo. The amount of processed GH in the media was quantified using densitometry and plotted in C (Error bars show experimental range for two repeats). D) Clone 3 cells were either mock transfected or transfected with dsRNA targeting the indicated genes. After 72 hours the cells were seeded onto coverslips. The next day, the cells were incubated with AP21998 at 25°C for 80 minutes. The cells were then fixed and stained for the Golgi marker GM130. Scale Bar 10μm.

    Journal: PLoS Genetics

    Article Title: VAMP3/Syb and YKT6 are required for the fusion of constitutive secretory carriers with the plasma membrane

    doi: 10.1371/journal.pgen.1006698

    Figure Lengend Snippet: SNAP24 and SNAP29 mediate the fusion of secretory carriers with the plasma membrane. A) Clone 3 cells were mock transfected (TransFast only) or transfected with dsRNA targeting the indicated genes. After 96 hours, the cells were incubated with DD solubiliser at 25°C for 80 minutes and their mean fluorescence determined using flow cytometry. The amount of cargo remaining in the cells was calculated and plotted (Error bars show experimental range for three repeats). B) Clone 3 cells were mock transfected (TransFast only) or transfected with dsRNA targeting the indicated genes. After 96 hours, the cells were incubated with DD solubiliser at 25°C for 80 minutes and the media and cells harvested for immunoblotting. Solid arrowhead indicates unprocessed cargo and unfilled arrowhead furin processed cargo. The amount of processed GH in the media was quantified using densitometry and plotted in C (Error bars show experimental range for two repeats). D) Clone 3 cells were either mock transfected or transfected with dsRNA targeting the indicated genes. After 72 hours the cells were seeded onto coverslips. The next day, the cells were incubated with AP21998 at 25°C for 80 minutes. The cells were then fixed and stained for the Golgi marker GM130. Scale Bar 10μm.

    Article Snippet: Secondary antibodies for immunoblotting were purchased from Jackson ImmunoResearch Laboratories.

    Techniques: Transfection, Incubation, Fluorescence, Flow Cytometry, Cytometry, Staining, Marker

    Verapamil restores lysosomal degradation of autophagosomes in liver of obese mice 4 month-old C57BL/6 male mice kept on HFD for two months were subjected to daily administration of PBS (Con, n = 4) or verapamil (Ver, 25 mg/Kg body weight, i.p., n = 3) for 10 days. LFD-kept mice ( n = 5) of same age were used as a negative control. ( a , b ) Levels of LC3-II from 1% Triton X-100 insoluble fraction of livers were analyzed by immunoblotting ( a ) and quantified ( b ). ( c , d ) Frozen sections of indicated livers were subjected to LC3/LAMP1 immunostaining and DAPI counterstaining ( c ). Co-localization between LC3 and LAMP1 was quantified ( d ). Boxed areas in fluorescence images are magnified in right-most panels ( c ). Scale bars, 5 μm ( c ). All data are shown as mean ± s.e.m. *** P

    Journal: Nature communications

    Article Title: Pharmacological correction of obesity-induced autophagy arrest using calcium channel blockers

    doi: 10.1038/ncomms5834

    Figure Lengend Snippet: Verapamil restores lysosomal degradation of autophagosomes in liver of obese mice 4 month-old C57BL/6 male mice kept on HFD for two months were subjected to daily administration of PBS (Con, n = 4) or verapamil (Ver, 25 mg/Kg body weight, i.p., n = 3) for 10 days. LFD-kept mice ( n = 5) of same age were used as a negative control. ( a , b ) Levels of LC3-II from 1% Triton X-100 insoluble fraction of livers were analyzed by immunoblotting ( a ) and quantified ( b ). ( c , d ) Frozen sections of indicated livers were subjected to LC3/LAMP1 immunostaining and DAPI counterstaining ( c ). Co-localization between LC3 and LAMP1 was quantified ( d ). Boxed areas in fluorescence images are magnified in right-most panels ( c ). Scale bars, 5 μm ( c ). All data are shown as mean ± s.e.m. *** P

    Article Snippet: Immunoblotting Unless otherwise noted, cells and tissues were lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl pH 7.4; 150 mM NaCl; 1% deoxycholate Na; 1% NP-40; 0.1% SDS and complete protease inhibitor cocktail (Roche)).

    Techniques: Mouse Assay, Negative Control, Immunostaining, Fluorescence

    Calcium channel blockers suppress saturated fatty acid-induced protein inclusion formation HepG2 cells were treated with BSA (Con or (−)), PA (500 μM), PA + verapamil (Ver, 50 μM) or PA + nicardipine (Nic, 100 μM) for 9 hr. ( a – d ) After each treatment, cells were loaded with a calcium indicator X-Rhod-1-AM ( a , b ) or Fura-2-AM ( c , d ). Calcium levels were visualized by laser confocal microscopy ( a ) or by dual fluorescent microscopy ( c , 340/380 nm ratio image) and quantified ( b , d ; n = 8 and 30, respectively). ( e – h ) Cells with indicated treatments were subjected to solubility fractionation. 1% Triton X-100-insoluble fractions were dissolved in 2% SDS, analyzed by immunoblotting ( e , f ) and quantified ( g , h ) ( n = 3). ( i – l ) Cells with indicated treatments were subjected to immunostaining with indicated antibodies ( i , k ). DNA was stained with DAPI (blue). Amount of aggregated proteins was quantified ( j ) ( n = 10). Co-localization between LAMP1 and LC3 was quantified ( l ) ( n = 4). Boxed areas in fluorescence images are magnified in right-most panels ( i , k ). Scale bars, 10 μm ( a ), 20 μm ( c ), 5 μm ( i , k ). All data are shown as mean ± s.e.m. * P

    Journal: Nature communications

    Article Title: Pharmacological correction of obesity-induced autophagy arrest using calcium channel blockers

    doi: 10.1038/ncomms5834

    Figure Lengend Snippet: Calcium channel blockers suppress saturated fatty acid-induced protein inclusion formation HepG2 cells were treated with BSA (Con or (−)), PA (500 μM), PA + verapamil (Ver, 50 μM) or PA + nicardipine (Nic, 100 μM) for 9 hr. ( a – d ) After each treatment, cells were loaded with a calcium indicator X-Rhod-1-AM ( a , b ) or Fura-2-AM ( c , d ). Calcium levels were visualized by laser confocal microscopy ( a ) or by dual fluorescent microscopy ( c , 340/380 nm ratio image) and quantified ( b , d ; n = 8 and 30, respectively). ( e – h ) Cells with indicated treatments were subjected to solubility fractionation. 1% Triton X-100-insoluble fractions were dissolved in 2% SDS, analyzed by immunoblotting ( e , f ) and quantified ( g , h ) ( n = 3). ( i – l ) Cells with indicated treatments were subjected to immunostaining with indicated antibodies ( i , k ). DNA was stained with DAPI (blue). Amount of aggregated proteins was quantified ( j ) ( n = 10). Co-localization between LAMP1 and LC3 was quantified ( l ) ( n = 4). Boxed areas in fluorescence images are magnified in right-most panels ( i , k ). Scale bars, 10 μm ( a ), 20 μm ( c ), 5 μm ( i , k ). All data are shown as mean ± s.e.m. * P

    Article Snippet: Immunoblotting Unless otherwise noted, cells and tissues were lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl pH 7.4; 150 mM NaCl; 1% deoxycholate Na; 1% NP-40; 0.1% SDS and complete protease inhibitor cocktail (Roche)).

    Techniques: Confocal Microscopy, Microscopy, Solubility, Fractionation, Immunostaining, Staining, Fluorescence

    Verapamil relieves hepatosteatosis of obese mice 4 month-old C57BL/6 male mice kept on HFD for two months were subjected to daily administration of PBS (Con, n = 4) or verapamil (Ver, 25 mg kg −1 body weight, i.p., n = 3) for 10 days. LFD-kept mice ( n = 5) of same age were used as a negative control. ( a ) Body weight was daily monitored during injection period. ( b ) Daily food consumption was measured during injection period. ( c ) Livers were harvested from indicated mice and photographed. ( d , e ) Total liver mass ( d ) and total epididymal white adipose tissue (eWAT) mass ( e ) were measured from indicated mice. ( f – h ) Relative mRNA expression was analyzed from eWAT of indicated mice through quantitative RT-PCR. ( i , j ) Liver sections were analyzed by hematoxylin and eosin (H E, upper panels) and Oil Red O (ORO, lower panels) staining ( i ). ORO densities were quantified ( j ). ( k , l ) Calcium-induced CaMKII autophosphorylation in livers was analyzed by immunoblotting ( k ) and quantified ( l ). ( m ) Primary hepatocytes from 2 month-old C57BL/6 mice kept on LFD were treated with BSA (Con), PA (500 μM), PA + verapamil (Ver, 50 μM) or PA + nicardipine (Nic, 100 μM) for 12 hr. After each treatment, cells were loaded with a calcium indicator Fura-2-AM. Calcium levels were visualized by dual fluorescent microscopy at 340/380 nm, and ratio of fluorescence intensities of Fura-2-AM at 340 nm over 380 nm was quantified ( n = 17). Scale bars, 1 cm ( c ), 200 μm ( i ). All data are shown as mean ± s.e.m. * P

    Journal: Nature communications

    Article Title: Pharmacological correction of obesity-induced autophagy arrest using calcium channel blockers

    doi: 10.1038/ncomms5834

    Figure Lengend Snippet: Verapamil relieves hepatosteatosis of obese mice 4 month-old C57BL/6 male mice kept on HFD for two months were subjected to daily administration of PBS (Con, n = 4) or verapamil (Ver, 25 mg kg −1 body weight, i.p., n = 3) for 10 days. LFD-kept mice ( n = 5) of same age were used as a negative control. ( a ) Body weight was daily monitored during injection period. ( b ) Daily food consumption was measured during injection period. ( c ) Livers were harvested from indicated mice and photographed. ( d , e ) Total liver mass ( d ) and total epididymal white adipose tissue (eWAT) mass ( e ) were measured from indicated mice. ( f – h ) Relative mRNA expression was analyzed from eWAT of indicated mice through quantitative RT-PCR. ( i , j ) Liver sections were analyzed by hematoxylin and eosin (H E, upper panels) and Oil Red O (ORO, lower panels) staining ( i ). ORO densities were quantified ( j ). ( k , l ) Calcium-induced CaMKII autophosphorylation in livers was analyzed by immunoblotting ( k ) and quantified ( l ). ( m ) Primary hepatocytes from 2 month-old C57BL/6 mice kept on LFD were treated with BSA (Con), PA (500 μM), PA + verapamil (Ver, 50 μM) or PA + nicardipine (Nic, 100 μM) for 12 hr. After each treatment, cells were loaded with a calcium indicator Fura-2-AM. Calcium levels were visualized by dual fluorescent microscopy at 340/380 nm, and ratio of fluorescence intensities of Fura-2-AM at 340 nm over 380 nm was quantified ( n = 17). Scale bars, 1 cm ( c ), 200 μm ( i ). All data are shown as mean ± s.e.m. * P

    Article Snippet: Immunoblotting Unless otherwise noted, cells and tissues were lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl pH 7.4; 150 mM NaCl; 1% deoxycholate Na; 1% NP-40; 0.1% SDS and complete protease inhibitor cocktail (Roche)).

    Techniques: Mouse Assay, Negative Control, Injection, Expressing, Quantitative RT-PCR, Staining, Microscopy, Fluorescence

    Verapamil suppresses obesity-induced accumulation of protein aggregates 4 month-old C57BL/6 male mice kept on HFD for two months were subjected to daily administration of PBS (Con, n = 4) or verapamil (Ver, 25 mg kg −1 body weight, i.p., n = 3) for 10 days. LFD-kept mice ( n = 5) of same age were used as a negative control. ( a – d ) Livers were subjected to solubility fractionation. 1% Triton X-100 soluble ( a ) and insoluble ( b ) fractions were analyzed by immunoblotting ( a , b ) and quantified ( c , d ). ( e , f ) Paraffin sections of indicated livers were subjected to p62 immunostaining and hematoxylin counterstaining ( e ). p62-positive areas were quantified ( f ). Scale bar, 200 μm ( e ). All data are shown as mean ± s.e.m. * P

    Journal: Nature communications

    Article Title: Pharmacological correction of obesity-induced autophagy arrest using calcium channel blockers

    doi: 10.1038/ncomms5834

    Figure Lengend Snippet: Verapamil suppresses obesity-induced accumulation of protein aggregates 4 month-old C57BL/6 male mice kept on HFD for two months were subjected to daily administration of PBS (Con, n = 4) or verapamil (Ver, 25 mg kg −1 body weight, i.p., n = 3) for 10 days. LFD-kept mice ( n = 5) of same age were used as a negative control. ( a – d ) Livers were subjected to solubility fractionation. 1% Triton X-100 soluble ( a ) and insoluble ( b ) fractions were analyzed by immunoblotting ( a , b ) and quantified ( c , d ). ( e , f ) Paraffin sections of indicated livers were subjected to p62 immunostaining and hematoxylin counterstaining ( e ). p62-positive areas were quantified ( f ). Scale bar, 200 μm ( e ). All data are shown as mean ± s.e.m. * P

    Article Snippet: Immunoblotting Unless otherwise noted, cells and tissues were lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl pH 7.4; 150 mM NaCl; 1% deoxycholate Na; 1% NP-40; 0.1% SDS and complete protease inhibitor cocktail (Roche)).

    Techniques: Mouse Assay, Negative Control, Solubility, Fractionation, Immunostaining

    Saturated fatty acids induce protein inclusions and arrest autophagy ( a – f ) HepG2 cells were treated with BSA (Con), 500 μM palmitic acid (PA) or 100 nM rapamycin (Rap) for 9 hr and subjected to following analyses. ( a , c – f ) Cells were stained with ubiquitin (Ub), p62, LC3 and LAMP1 antibodies and DAPI (blue). Boxed areas are magnified in right-most panels ( c , d ). Co-localization between LAMP1 and LC3 staining ( e ) was quantified ( f ) ( n = 3). ( b ) Cells were subjected to serial protein extraction (solubility fractionation) with indicated concentration of Triton X-100 (TX100) or sodium dodecyl sulfate (SDS) and analyzed by immunoblotting with indicated antibodies. ( g , h ) HepG2 cells stably transduced with mCherry (mCh)-GFP-LC3-expressing retroviruses were treated with Con, Rap or PA for 9 hr and examined under a live confocal microscope ( g ). Yellow dots represent autophagosomes while red dots indicate autolysosomes in which GFP signal was faded out. Number of autolysosomes was quantified ( h ) ( n = 7). Scale bars, 5 μm. All data are shown as mean ± s.e.m. *** P

    Journal: Nature communications

    Article Title: Pharmacological correction of obesity-induced autophagy arrest using calcium channel blockers

    doi: 10.1038/ncomms5834

    Figure Lengend Snippet: Saturated fatty acids induce protein inclusions and arrest autophagy ( a – f ) HepG2 cells were treated with BSA (Con), 500 μM palmitic acid (PA) or 100 nM rapamycin (Rap) for 9 hr and subjected to following analyses. ( a , c – f ) Cells were stained with ubiquitin (Ub), p62, LC3 and LAMP1 antibodies and DAPI (blue). Boxed areas are magnified in right-most panels ( c , d ). Co-localization between LAMP1 and LC3 staining ( e ) was quantified ( f ) ( n = 3). ( b ) Cells were subjected to serial protein extraction (solubility fractionation) with indicated concentration of Triton X-100 (TX100) or sodium dodecyl sulfate (SDS) and analyzed by immunoblotting with indicated antibodies. ( g , h ) HepG2 cells stably transduced with mCherry (mCh)-GFP-LC3-expressing retroviruses were treated with Con, Rap or PA for 9 hr and examined under a live confocal microscope ( g ). Yellow dots represent autophagosomes while red dots indicate autolysosomes in which GFP signal was faded out. Number of autolysosomes was quantified ( h ) ( n = 7). Scale bars, 5 μm. All data are shown as mean ± s.e.m. *** P

    Article Snippet: Immunoblotting Unless otherwise noted, cells and tissues were lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl pH 7.4; 150 mM NaCl; 1% deoxycholate Na; 1% NP-40; 0.1% SDS and complete protease inhibitor cocktail (Roche)).

    Techniques: Staining, Protein Extraction, Solubility, Fractionation, Concentration Assay, Stable Transfection, Transduction, Expressing, Microscopy

    Interaction of EphA4 with GHR. (A) Schemes of wild-type (WT) and a cytoplasmic deletion mutant of EphA4. The numbers represent the amino acid numbers counting from the one encoded by the initiation codon. TM, transmembrane domain; JM, juxtamembrane domain. Dotted blue rectangles indicate the deleted amino-acid regions. (B) Binding between the extracellular domains of EphA4 and GHR. EphA4(WT)-3Flag or EphA4(Δcyto)-3Flag was transiently co-expressed with GHR(WT)-6myc in HEK293T cells. These proteins were subjected to co-immunoprecipitation (IP), fractionation with SDS-PAGE, and detection by immunoblotting (IB) using the indicated antibodies. Arrowheads indicate the molecules shown in the figure. The other bands are regarded as non-specific bands, compared with the result without EphA4 expression (see also S1 Fig ). (C) Binding between the cytoplasmic domains of EphA4 and GHR. GHR(WT)-6myc and the chimeric protein in which the cytoplasmic domain of EphA4 was fused to the extracellular domain of ephrin-B2 (B2-A4-3Flag) were co-expressed in HEK293T cells and their interaction was examined by IB following IP and SDS-PAGE fractionation. The bands at approximately 100 kDa for GHR-6myc transfectants probably correspond to the GHR molecules modified with carbohydrates. (D) Control experiment for (C) showing no interaction between GHR and ephrin-B2. Ephrin-B2-3Flag was transiently co-expressed with GHR(WT)-6myc in HEK293T cells and their interaction was examined as described in (C). Some of the molecules that have extracellular domains (EphA4, ephrin-B2, and B2-A4) show broad bands, with the upper bands represented by the carbohydrate-modified proteins. All the IP and IB studies in this figure were repeated 3 times to confirm reproducibility, and representative results are shown.

    Journal: PLoS ONE

    Article Title: Molecular interactions of EphA4, growth hormone receptor, Janus kinase 2, and signal transducer and activator of transcription 5B

    doi: 10.1371/journal.pone.0180785

    Figure Lengend Snippet: Interaction of EphA4 with GHR. (A) Schemes of wild-type (WT) and a cytoplasmic deletion mutant of EphA4. The numbers represent the amino acid numbers counting from the one encoded by the initiation codon. TM, transmembrane domain; JM, juxtamembrane domain. Dotted blue rectangles indicate the deleted amino-acid regions. (B) Binding between the extracellular domains of EphA4 and GHR. EphA4(WT)-3Flag or EphA4(Δcyto)-3Flag was transiently co-expressed with GHR(WT)-6myc in HEK293T cells. These proteins were subjected to co-immunoprecipitation (IP), fractionation with SDS-PAGE, and detection by immunoblotting (IB) using the indicated antibodies. Arrowheads indicate the molecules shown in the figure. The other bands are regarded as non-specific bands, compared with the result without EphA4 expression (see also S1 Fig ). (C) Binding between the cytoplasmic domains of EphA4 and GHR. GHR(WT)-6myc and the chimeric protein in which the cytoplasmic domain of EphA4 was fused to the extracellular domain of ephrin-B2 (B2-A4-3Flag) were co-expressed in HEK293T cells and their interaction was examined by IB following IP and SDS-PAGE fractionation. The bands at approximately 100 kDa for GHR-6myc transfectants probably correspond to the GHR molecules modified with carbohydrates. (D) Control experiment for (C) showing no interaction between GHR and ephrin-B2. Ephrin-B2-3Flag was transiently co-expressed with GHR(WT)-6myc in HEK293T cells and their interaction was examined as described in (C). Some of the molecules that have extracellular domains (EphA4, ephrin-B2, and B2-A4) show broad bands, with the upper bands represented by the carbohydrate-modified proteins. All the IP and IB studies in this figure were repeated 3 times to confirm reproducibility, and representative results are shown.

    Article Snippet: Immunoblotting following immunoprecipitation revealed that JAK2 bound to EphA4(WT), EphA4(ΔJM), EphA4(KD), and EphA4(ΔJM,KD) and barely bound to EphA4(2M) and EphA4(3M) ( left panel).

    Techniques: Mutagenesis, Binding Assay, Immunoprecipitation, Fractionation, SDS Page, Expressing, Modification

    ADAM9 reduction therapy decreased tumor volume by inhibiting cancer cell proliferation. (a) Schematic of ADAM9 knockdown therapy experiment. Mice were injected subcutaneously with PC3 cells in both flanks, and tumor size was measured twice a week until the size reached approximately 200 mm 3 . Mice then received injections of either PBS into both tumor sites (n = 5) or shGFP lentivirus into the left side tumor and shADAM9 into the right side (n = 10). Viruses were injected and tumors measured weekly for 6 consecutive weeks. (b) After shADAM9 therapy, tumor volumes did not increase compared to shGFP therapy or PBS controls (** p ≤0.01, Student’s t test). (c) Immunoblotting assay of ADAM9 expression confirms decreased ADAM9 expression after shADAM9 therapy. EF-1α was used as a loading control (* p ≤0.05; ** p ≤0.01, Student’s t test). (d) Statistical analysis of ki67 staining. shADAM9 therapy significantly decreased cell proliferation activities in PC3 tumors. PBS and shGFP therapy showed no difference in the proliferation index (** p ≤0.001, One Way ANOVA). (e) Statistical analysis of TUNEL staining showed no significant (N.S.) difference between therapies ( p ≥0.05, One Way ANOVA).

    Journal: PLoS ONE

    Article Title: In Vivo Targeting of ADAM9 Gene Expression Using Lentivirus-Delivered shRNA Suppresses Prostate Cancer Growth by Regulating REG4 Dependent Cell Cycle Progression

    doi: 10.1371/journal.pone.0053795

    Figure Lengend Snippet: ADAM9 reduction therapy decreased tumor volume by inhibiting cancer cell proliferation. (a) Schematic of ADAM9 knockdown therapy experiment. Mice were injected subcutaneously with PC3 cells in both flanks, and tumor size was measured twice a week until the size reached approximately 200 mm 3 . Mice then received injections of either PBS into both tumor sites (n = 5) or shGFP lentivirus into the left side tumor and shADAM9 into the right side (n = 10). Viruses were injected and tumors measured weekly for 6 consecutive weeks. (b) After shADAM9 therapy, tumor volumes did not increase compared to shGFP therapy or PBS controls (** p ≤0.01, Student’s t test). (c) Immunoblotting assay of ADAM9 expression confirms decreased ADAM9 expression after shADAM9 therapy. EF-1α was used as a loading control (* p ≤0.05; ** p ≤0.01, Student’s t test). (d) Statistical analysis of ki67 staining. shADAM9 therapy significantly decreased cell proliferation activities in PC3 tumors. PBS and shGFP therapy showed no difference in the proliferation index (** p ≤0.001, One Way ANOVA). (e) Statistical analysis of TUNEL staining showed no significant (N.S.) difference between therapies ( p ≥0.05, One Way ANOVA).

    Article Snippet: Overexpressed REG4 was confirmed by immunoblotting (R & D System).

    Techniques: Mouse Assay, Injection, Expressing, Staining, TUNEL Assay

    BI-1-associated lysosomal activity leads to ER stress regulation. ( a ) Neo and BI-1 cells were cultured in serum-free medium with 10 ng/ml TGF- β 1 for 0, 12, 24, 36, or 48 h. ( b ) Neo and BI-1 cells were cultured in serum-free medium with 10 ng/ml TGF- β 1 in the presence or absence of 10 nM bafilomycin for 48 h. Neo and BI-1 cells were transfected with non-specific or V-ATPase specific siRNA and immunoblotting was performed with anti-V-ATPase antibody. ( c ) Separately, siRNA-transfected Neo and BI-1 cells were cultured in serum-free medium with 10 ng/ml TGF- β 1 for 48 h. Immunoblotting was performed with anti-GRP78, CHOP, IRE-1 α , sXBP-1, PERK, p-eIF2 α , eIF2 α , p-JNK, JNK1, ATF6 α (50KD), and β -actin antibodies. NS, non-specific siRNA; V-siRNA, V-ATPase siRNA

    Journal: Cell Death & Disease

    Article Title: BAX inhibitor-1-associated V-ATPase glycosylation enhances collagen degradation in pulmonary fibrosis

    doi: 10.1038/cddis.2014.86

    Figure Lengend Snippet: BI-1-associated lysosomal activity leads to ER stress regulation. ( a ) Neo and BI-1 cells were cultured in serum-free medium with 10 ng/ml TGF- β 1 for 0, 12, 24, 36, or 48 h. ( b ) Neo and BI-1 cells were cultured in serum-free medium with 10 ng/ml TGF- β 1 in the presence or absence of 10 nM bafilomycin for 48 h. Neo and BI-1 cells were transfected with non-specific or V-ATPase specific siRNA and immunoblotting was performed with anti-V-ATPase antibody. ( c ) Separately, siRNA-transfected Neo and BI-1 cells were cultured in serum-free medium with 10 ng/ml TGF- β 1 for 48 h. Immunoblotting was performed with anti-GRP78, CHOP, IRE-1 α , sXBP-1, PERK, p-eIF2 α , eIF2 α , p-JNK, JNK1, ATF6 α (50KD), and β -actin antibodies. NS, non-specific siRNA; V-siRNA, V-ATPase siRNA

    Article Snippet: Anti-rabbit polyclonal V-ATPase antibody for immunoblotting was purchased from Synaptic Systems (Goettingen, Germany).

    Techniques: Activity Assay, Cell Culture, Transfection

    Lysosomal V-ATPase is highly expressed in BI-1 cells. ( a ) Neo and BI-1 cells were cultured in serum-free medium with or without 5 ng/ml TGF- β 1 for 0, 12, 24, 36, or 48 h. Immunoblotting was performed with antibodies against the V-ATPase V0a1 subunit or β -actin. Ratio of glycosylated to unglycosylated V-ATPase was quantified as shown in the right panel. * P

    Journal: Cell Death & Disease

    Article Title: BAX inhibitor-1-associated V-ATPase glycosylation enhances collagen degradation in pulmonary fibrosis

    doi: 10.1038/cddis.2014.86

    Figure Lengend Snippet: Lysosomal V-ATPase is highly expressed in BI-1 cells. ( a ) Neo and BI-1 cells were cultured in serum-free medium with or without 5 ng/ml TGF- β 1 for 0, 12, 24, 36, or 48 h. Immunoblotting was performed with antibodies against the V-ATPase V0a1 subunit or β -actin. Ratio of glycosylated to unglycosylated V-ATPase was quantified as shown in the right panel. * P

    Article Snippet: Anti-rabbit polyclonal V-ATPase antibody for immunoblotting was purchased from Synaptic Systems (Goettingen, Germany).

    Techniques: Cell Culture

    BI-1 enhances N-glycosylation activity in the ER during TGF- β 1 treatment. ( a ) Neo and BI-1 cells were cultured with 5 ng/ml TGF- β 1 for 0, 24, or 48 h. O-glynase, Endo-H, or PNGase-F was added to the extracts followed by 30-min incubation. Immunoblotting was then performed with anti-V-ATPase antibody. ( b ) V-ATPase activity in the lysosome fractions isolated from 5 ng/ml TGF- β 1-treated Neo or BI-1 cells in the presence or absence of PNGase-F was measured and quantified. * P

    Journal: Cell Death & Disease

    Article Title: BAX inhibitor-1-associated V-ATPase glycosylation enhances collagen degradation in pulmonary fibrosis

    doi: 10.1038/cddis.2014.86

    Figure Lengend Snippet: BI-1 enhances N-glycosylation activity in the ER during TGF- β 1 treatment. ( a ) Neo and BI-1 cells were cultured with 5 ng/ml TGF- β 1 for 0, 24, or 48 h. O-glynase, Endo-H, or PNGase-F was added to the extracts followed by 30-min incubation. Immunoblotting was then performed with anti-V-ATPase antibody. ( b ) V-ATPase activity in the lysosome fractions isolated from 5 ng/ml TGF- β 1-treated Neo or BI-1 cells in the presence or absence of PNGase-F was measured and quantified. * P

    Article Snippet: Anti-rabbit polyclonal V-ATPase antibody for immunoblotting was purchased from Synaptic Systems (Goettingen, Germany).

    Techniques: Activity Assay, Cell Culture, Incubation, Isolation

    Downregulation of SRC-2 in MCF-7 cells induces distinct changes in global gene expression profiles. (A). Quantification of SRC-2 mRNA expression in shRNA lentivirus-infected MCF-7 cells. mRNA levels of SRC-2 in a MCF-7 cell line infected with shRNA targeting SRC-2 (SRC-2 shRNA) were compared to the expression in a control shRNA MCF-7 cell line (Ctr shRNA), and in a MCF-7 cell line transduced with shRNA lentivirus targeting SRC-3 (SRC-3 shRNA). The mRNA expression of SRC-2 is relative to TBP mRNA. The results are representative of at least three independent experiments. (B). Western blotting analyses of SRC-2-depleted MCF-7 cells. MCF-7 cells infected with shRNA lentivirus targeting SRC-2 (SRC-2 shRNA) or a negative control shRNA empty vector (Ctr shRNA), were grown in phenol red-free DMEM supplemented with charcoaled stripped FBS (5%) and 17β-estradiol (10 nM) for two days. The Ctr shRNA cells were then treated with either Vehicle or 8-CPT-cAMP (150 µM), IBMX (50 µM) and forskolin (10 µM) (cAMP) for 24 hours. Immunoblotting was performed with anti-TIF2 antibody and anti-GAPDH antibody. The results shown are representative of at least three independent experiments. (C). Microarray analyses of five RNA samples isolated from five individual cell samples of shRNA control MCF-7 cells (Ctr shRNA), SRC-2 KD MCF-7 cells (SRC-2 shRNA) and control shRNA cells treated with cAMP elevating agents, as described in A. A Venn diagram shows the number of individual and overlapping sets of genes differentially expressed after SRC-2 KD (SRC-2 shRNA) and after treatment with cAMP elevating agents. To examine which genes were similarly differentially expressed between the two treated groups when compared to control, a SAM analysis with overlapping genes was performed. The fold change cut-off value ≥1.5, and q-value = 0, was used to determine differentially expressed genes.

    Journal: PLoS ONE

    Article Title: Downregulation of Steroid Receptor Coactivator-2 Modulates Estrogen-Responsive Genes and Stimulates Proliferation of MCF-7 Breast Cancer Cells

    doi: 10.1371/journal.pone.0070096

    Figure Lengend Snippet: Downregulation of SRC-2 in MCF-7 cells induces distinct changes in global gene expression profiles. (A). Quantification of SRC-2 mRNA expression in shRNA lentivirus-infected MCF-7 cells. mRNA levels of SRC-2 in a MCF-7 cell line infected with shRNA targeting SRC-2 (SRC-2 shRNA) were compared to the expression in a control shRNA MCF-7 cell line (Ctr shRNA), and in a MCF-7 cell line transduced with shRNA lentivirus targeting SRC-3 (SRC-3 shRNA). The mRNA expression of SRC-2 is relative to TBP mRNA. The results are representative of at least three independent experiments. (B). Western blotting analyses of SRC-2-depleted MCF-7 cells. MCF-7 cells infected with shRNA lentivirus targeting SRC-2 (SRC-2 shRNA) or a negative control shRNA empty vector (Ctr shRNA), were grown in phenol red-free DMEM supplemented with charcoaled stripped FBS (5%) and 17β-estradiol (10 nM) for two days. The Ctr shRNA cells were then treated with either Vehicle or 8-CPT-cAMP (150 µM), IBMX (50 µM) and forskolin (10 µM) (cAMP) for 24 hours. Immunoblotting was performed with anti-TIF2 antibody and anti-GAPDH antibody. The results shown are representative of at least three independent experiments. (C). Microarray analyses of five RNA samples isolated from five individual cell samples of shRNA control MCF-7 cells (Ctr shRNA), SRC-2 KD MCF-7 cells (SRC-2 shRNA) and control shRNA cells treated with cAMP elevating agents, as described in A. A Venn diagram shows the number of individual and overlapping sets of genes differentially expressed after SRC-2 KD (SRC-2 shRNA) and after treatment with cAMP elevating agents. To examine which genes were similarly differentially expressed between the two treated groups when compared to control, a SAM analysis with overlapping genes was performed. The fold change cut-off value ≥1.5, and q-value = 0, was used to determine differentially expressed genes.

    Article Snippet: Primary antibodies used in the immunoblotting experiments were mouse monoclonal anti-TIF2 (BD Biosciences, San Jose, CA) and mouse monoclonal anti-GAPDH (Chemicon International, Temecula, CA).

    Techniques: Expressing, shRNA, Infection, Transduction, Western Blot, Negative Control, Plasmid Preparation, Cycling Probe Technology, Microarray, Isolation

    Regulation of the cell cycle by B55α and B55β. ( A ) Metaphase-arrested Xenopus egg extracts were supplemented with recombinant B55α or control buffer for 10 min. These extracts were then released into interphase by the addition of Calcium at time 0. The extract samples were collected at the indicated time points and analyzed by immunoblotting. Phosphorylation (band-shift) of Cdc27 and Cdc25, and the global phosphorylation of Cdk substrates are markers of mitosis. ( B ) Interphase Xenopus egg extracts were supplemented with recombinant B55α or control buffer. The extract samples were collected at the indicated time points and analyzed by immunoblotting. Mitosis is indicated by Cdc27 phosphorylation. ( C ) MBP-B55α and B55β were purified and examined by Coomassie staining. ( D ) Interphase Xenopus egg extracts were supplemented with recombinant B55α, B55β, or control buffer. The extract samples were collected at the indicated time points and analyzed by immunoblotting. Mitosis is indicated by Cdc27 phosphorylation.

    Journal: Scientific Reports

    Article Title: Protein interactomes of protein phosphatase 2A B55 regulatory subunits reveal B55-mediated regulation of replication protein A under replication stress

    doi: 10.1038/s41598-018-21040-6

    Figure Lengend Snippet: Regulation of the cell cycle by B55α and B55β. ( A ) Metaphase-arrested Xenopus egg extracts were supplemented with recombinant B55α or control buffer for 10 min. These extracts were then released into interphase by the addition of Calcium at time 0. The extract samples were collected at the indicated time points and analyzed by immunoblotting. Phosphorylation (band-shift) of Cdc27 and Cdc25, and the global phosphorylation of Cdk substrates are markers of mitosis. ( B ) Interphase Xenopus egg extracts were supplemented with recombinant B55α or control buffer. The extract samples were collected at the indicated time points and analyzed by immunoblotting. Mitosis is indicated by Cdc27 phosphorylation. ( C ) MBP-B55α and B55β were purified and examined by Coomassie staining. ( D ) Interphase Xenopus egg extracts were supplemented with recombinant B55α, B55β, or control buffer. The extract samples were collected at the indicated time points and analyzed by immunoblotting. Mitosis is indicated by Cdc27 phosphorylation.

    Article Snippet: Antibodies used in immunoblotting include: Chk1 phospho-S317, Mcm2, Ube3a, Incenp, Smc2 antibodies from Bethyl Laboratories (Montgomery, TX); B55α antibody from Abcam (Cambridge, MA); β-actin, phospho-Cdk substrate and Plk1 antibodies from Cell Signaling Technology (Beverly, MA); Mastl antibody from Millipore (Billerica, MA), and phospho-RPA S4/8 and S33 antibodies as previously characterized .

    Techniques: Recombinant, Electrophoretic Mobility Shift Assay, Purification, Staining

    Validation of B55α and B55β-associated proteins. B55α or B55β pull-down was performed in Xenopus egg extracts. A control pull-down was performed using the same volume of amylose beads that were not conjugated with proteins. The extract input (approximately 20%), control (ctr) pull-down and B55α or B55β pull-down samples were analyzed by immunoblotting using PP2A, Mcm2, Ube3a, Mastl, Plk1, Incenp, Smc2, and MBP antibodies.

    Journal: Scientific Reports

    Article Title: Protein interactomes of protein phosphatase 2A B55 regulatory subunits reveal B55-mediated regulation of replication protein A under replication stress

    doi: 10.1038/s41598-018-21040-6

    Figure Lengend Snippet: Validation of B55α and B55β-associated proteins. B55α or B55β pull-down was performed in Xenopus egg extracts. A control pull-down was performed using the same volume of amylose beads that were not conjugated with proteins. The extract input (approximately 20%), control (ctr) pull-down and B55α or B55β pull-down samples were analyzed by immunoblotting using PP2A, Mcm2, Ube3a, Mastl, Plk1, Incenp, Smc2, and MBP antibodies.

    Article Snippet: Antibodies used in immunoblotting include: Chk1 phospho-S317, Mcm2, Ube3a, Incenp, Smc2 antibodies from Bethyl Laboratories (Montgomery, TX); B55α antibody from Abcam (Cambridge, MA); β-actin, phospho-Cdk substrate and Plk1 antibodies from Cell Signaling Technology (Beverly, MA); Mastl antibody from Millipore (Billerica, MA), and phospho-RPA S4/8 and S33 antibodies as previously characterized .

    Techniques:

    RPA and B55α association. ( A ) RPA2 IP was performed in HeLa cell lysates as described in Material and Methods. The input at 10%, control IP (with blank beads), and RPA2 IP were analyzed by immunoblotting for B55α and RPA2. ( B ) B55α IP was performed in HeLa cell lysates. The input at 10%, control IP (with blank beads), and B55α IP were analyzed by immunoblotting for B55α and RPA2. ( C ) HA-B55α was expressed in HeLa cells, which were treated with or without HU (1 mM, 24 h). HA IP was performed in the cell lysates. The input at 10%, control IP (with blank beads), and B55α IP were analyzed by immunoblotting for B55α and RPA2. ( D ) RPA2 IP was performed in the lysates of HeLa cells that were pre-treated with HU (1 mM, 24 h) or mock-treated. The input at 10%, control IP (with blank beads), and B55α IP were analyzed by immunoblotting for B55α and RPA2. ( E ) The level of B55α in RPA2 IP was examined by immunoblotting and quantified using ImageJ. The mean values and standard deviations were calculated from three independent experiments. Statistical significance was analyzed using an unpaired 2-tailed Student’s t-test. A p-value

    Journal: Scientific Reports

    Article Title: Protein interactomes of protein phosphatase 2A B55 regulatory subunits reveal B55-mediated regulation of replication protein A under replication stress

    doi: 10.1038/s41598-018-21040-6

    Figure Lengend Snippet: RPA and B55α association. ( A ) RPA2 IP was performed in HeLa cell lysates as described in Material and Methods. The input at 10%, control IP (with blank beads), and RPA2 IP were analyzed by immunoblotting for B55α and RPA2. ( B ) B55α IP was performed in HeLa cell lysates. The input at 10%, control IP (with blank beads), and B55α IP were analyzed by immunoblotting for B55α and RPA2. ( C ) HA-B55α was expressed in HeLa cells, which were treated with or without HU (1 mM, 24 h). HA IP was performed in the cell lysates. The input at 10%, control IP (with blank beads), and B55α IP were analyzed by immunoblotting for B55α and RPA2. ( D ) RPA2 IP was performed in the lysates of HeLa cells that were pre-treated with HU (1 mM, 24 h) or mock-treated. The input at 10%, control IP (with blank beads), and B55α IP were analyzed by immunoblotting for B55α and RPA2. ( E ) The level of B55α in RPA2 IP was examined by immunoblotting and quantified using ImageJ. The mean values and standard deviations were calculated from three independent experiments. Statistical significance was analyzed using an unpaired 2-tailed Student’s t-test. A p-value

    Article Snippet: Antibodies used in immunoblotting include: Chk1 phospho-S317, Mcm2, Ube3a, Incenp, Smc2 antibodies from Bethyl Laboratories (Montgomery, TX); B55α antibody from Abcam (Cambridge, MA); β-actin, phospho-Cdk substrate and Plk1 antibodies from Cell Signaling Technology (Beverly, MA); Mastl antibody from Millipore (Billerica, MA), and phospho-RPA S4/8 and S33 antibodies as previously characterized .

    Techniques: Recombinase Polymerase Amplification

    B55α-mediates RPA2 dephosphorylation. ( A ) HeLa cells with or without expression of HA-B55α, were treated with HU (1 mM, 24 h) as indicated. The cell lysates were analyzed by immunoblotting for RPA2, B55α, phospho-RPA2 Ser-4/Ser-8, Ser-33, phospho-Chk1 Ser-317, and β-actin. ( B ) The level of RPA2 S4/8 phosphorylation was examined by immunoblotting, as in panel A, and quantified using ImageJ. The mean values and standard deviations were calculated from three independent experiments. Statistical significance was analyzed using an unpaired 2-tailed Student’s t-test. A p-value

    Journal: Scientific Reports

    Article Title: Protein interactomes of protein phosphatase 2A B55 regulatory subunits reveal B55-mediated regulation of replication protein A under replication stress

    doi: 10.1038/s41598-018-21040-6

    Figure Lengend Snippet: B55α-mediates RPA2 dephosphorylation. ( A ) HeLa cells with or without expression of HA-B55α, were treated with HU (1 mM, 24 h) as indicated. The cell lysates were analyzed by immunoblotting for RPA2, B55α, phospho-RPA2 Ser-4/Ser-8, Ser-33, phospho-Chk1 Ser-317, and β-actin. ( B ) The level of RPA2 S4/8 phosphorylation was examined by immunoblotting, as in panel A, and quantified using ImageJ. The mean values and standard deviations were calculated from three independent experiments. Statistical significance was analyzed using an unpaired 2-tailed Student’s t-test. A p-value

    Article Snippet: Antibodies used in immunoblotting include: Chk1 phospho-S317, Mcm2, Ube3a, Incenp, Smc2 antibodies from Bethyl Laboratories (Montgomery, TX); B55α antibody from Abcam (Cambridge, MA); β-actin, phospho-Cdk substrate and Plk1 antibodies from Cell Signaling Technology (Beverly, MA); Mastl antibody from Millipore (Billerica, MA), and phospho-RPA S4/8 and S33 antibodies as previously characterized .

    Techniques: De-Phosphorylation Assay, Expressing

    Flubendazole activates JNK1 and Bcl-2 and relieves Beclin 1 from negative regulation. ( a ) HeLa cells treated with DMSO or flubendazole (5 μg ml −1 ) were lysed and subjected to immunoblotting. ( b ) Coimmunoprecipitation analysis of interaction between Beclin 1 and Bcl-2 (HEK293T lysates) in cells treated with DMSO or 5 μg ml −1 flubendazole for 2 h. ( c ) HeLa cells knocked down for ATAT1 and treated with DMSO or 5 μg ml −1 flubendazole (for 2 h) were lysed and subjected to immunoblotting. ( d ) High-content analysis of the abundance of LC3 puncta in HeLa cells knockdown for ATAT1 or control cells treated with flubendazole or DMSO for 45 min. Flub, flubendazole; statistics, mean±s.e.; Student's t- test. * P

    Journal: Nature Communications

    Article Title: Pharmaceutical screen identifies novel target processes for activation of autophagy with a broad translational potential

    doi: 10.1038/ncomms9620

    Figure Lengend Snippet: Flubendazole activates JNK1 and Bcl-2 and relieves Beclin 1 from negative regulation. ( a ) HeLa cells treated with DMSO or flubendazole (5 μg ml −1 ) were lysed and subjected to immunoblotting. ( b ) Coimmunoprecipitation analysis of interaction between Beclin 1 and Bcl-2 (HEK293T lysates) in cells treated with DMSO or 5 μg ml −1 flubendazole for 2 h. ( c ) HeLa cells knocked down for ATAT1 and treated with DMSO or 5 μg ml −1 flubendazole (for 2 h) were lysed and subjected to immunoblotting. ( d ) High-content analysis of the abundance of LC3 puncta in HeLa cells knockdown for ATAT1 or control cells treated with flubendazole or DMSO for 45 min. Flub, flubendazole; statistics, mean±s.e.; Student's t- test. * P

    Article Snippet: Anti-LC3B (PM036) used for immunoblotting was from MBL International (Woburn, MA).

    Techniques: High Content Screening

    MEP50/PRMT5 complex-mediated GLI1 methylation inhibits the interaction of GLI1 with its E3 ligase complex, ITCH/NUMB, resulting in GLI1 stabilisation. a Interaction of GLI1 and endogenous ITCH or NUMB from stably PRMT5-knockdown or MEP50-knockdown C3H10T1/2 cells. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. MG132 (50 μM) was applied for 4 h before harvesting. b Interaction of GLI1 mutants with endogenous ITCH or NUMB in C3H10T1/2 cells. The cells were transfected as indicated. At 48 h post-transfection, 50 μM MG132 was applied for 4 h, and then the cells were lysed and subjected to immunoprecipitation with an anti-HA antibody, followed by immunoblotting with antibodies against the indicated proteins. c In vivo ubiquitination of HA-GLI1-RK mutants. Cells were transfected and cultured for 24 h, followed by treatment with 50 µM MG132 for 4 h before harvesting. Ubiquitinated GLI1 was detected by immuoprecipitation with an anti-HA (3F10) antibody and immunoblotting with anti-FLAG (upper panel) or anti-HA (lower panel) antibodies. The asterisk denotes non-specific bands. d Schematic diagram of the mechanism of PRMT5/MEP50-mediated GLI1 stabilisation. When the HH signalling pathway inactivates, the ITCH/NUMB E3 ligase complex binds to and ubiquitinates GLI1 for proteasomal degradation. In turn, under HH signalling pathway activation, the MEP50/PRMT5 complex methylates GLI1 to dissociate the ITCH/NUMB complex from GLI1, resulting in GLI1 stabilisation. Unprocessed original scans of blots are shown in Supplementary Fig. 6

    Journal: Communications Biology

    Article Title: MEP50/PRMT5-mediated methylation activates GLI1 in Hedgehog signalling through inhibition of ubiquitination by the ITCH/NUMB complex

    doi: 10.1038/s42003-018-0275-4

    Figure Lengend Snippet: MEP50/PRMT5 complex-mediated GLI1 methylation inhibits the interaction of GLI1 with its E3 ligase complex, ITCH/NUMB, resulting in GLI1 stabilisation. a Interaction of GLI1 and endogenous ITCH or NUMB from stably PRMT5-knockdown or MEP50-knockdown C3H10T1/2 cells. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. MG132 (50 μM) was applied for 4 h before harvesting. b Interaction of GLI1 mutants with endogenous ITCH or NUMB in C3H10T1/2 cells. The cells were transfected as indicated. At 48 h post-transfection, 50 μM MG132 was applied for 4 h, and then the cells were lysed and subjected to immunoprecipitation with an anti-HA antibody, followed by immunoblotting with antibodies against the indicated proteins. c In vivo ubiquitination of HA-GLI1-RK mutants. Cells were transfected and cultured for 24 h, followed by treatment with 50 µM MG132 for 4 h before harvesting. Ubiquitinated GLI1 was detected by immuoprecipitation with an anti-HA (3F10) antibody and immunoblotting with anti-FLAG (upper panel) or anti-HA (lower panel) antibodies. The asterisk denotes non-specific bands. d Schematic diagram of the mechanism of PRMT5/MEP50-mediated GLI1 stabilisation. When the HH signalling pathway inactivates, the ITCH/NUMB E3 ligase complex binds to and ubiquitinates GLI1 for proteasomal degradation. In turn, under HH signalling pathway activation, the MEP50/PRMT5 complex methylates GLI1 to dissociate the ITCH/NUMB complex from GLI1, resulting in GLI1 stabilisation. Unprocessed original scans of blots are shown in Supplementary Fig. 6

    Article Snippet: Immunoprecipitation and immunoblotting Transfected or infected cells were lysed in TNE lysis buffer (1% Nonidet P-40, 10 mM Tris–HCl [pH 8.0], 150 mM NaCl, 1 mM EDTA, 1 mM NaF, 1 mM orthovanadate, 0.1 mM DTT, and a protease inhibitor cocktail [Nacalai Tesque]).

    Techniques: Methylation, Stable Transfection, Recombinant, Transfection, Immunoprecipitation, In Vivo, Cell Culture, Activation Assay

    GLI1 interacts with the MEP50/PRMT5 complex. a FLAG-GLI1 interacted with endogenous MEP50 and interaction of FLAG-GLI1 and MEP50 was increased by HH signalling pathway activation. C3H10T1/2 cells were transfected with FLAG-GLI1 or the empty vector for 24 h and then treated with 300 nM SAG for an additional 24 h. Interaction of FLAG-GLI1 and MEP50 was detected by immunoprecipitation with anti-FLAG antibody followed by immunoblot analysis using anti-FLAG and anti-MEP50 antibodies. b Schematic structures of MEP50 deletion mutants. c Mapping of the GLI1-binding region in MEP50 by immunoprecipitation analysis. HEK293T cells were transfected with Myc-MEP50 deletion mutants and FLAG-GLI1 plasmids for 24 h. Interaction of FLAG-GLI1 and Myc-MEP50 deletion mutants was detected by immunoprecipitation with anti-FLAG antibody followed by immunoblot analysis using anti-FLAG and anti-Myc antibodies. d Schematic of GLI1 deletion mutants. e GST pull-down assays to map the MEP50-binding region in GLI1. GST-GLI1 deletion mutants coupled to glutathione sepharose were incubated with immunoprecipitated Myc-MEP50 from HEK293T cells. Immunoblotting was performed with an anti-Myc antibody. In a and e , data represent one of three independent experiments with similar results. In c , data represent one of two independent experiments with similar results. Unprocessed original scans of blots are shown in Supplementary Fig. 6

    Journal: Communications Biology

    Article Title: MEP50/PRMT5-mediated methylation activates GLI1 in Hedgehog signalling through inhibition of ubiquitination by the ITCH/NUMB complex

    doi: 10.1038/s42003-018-0275-4

    Figure Lengend Snippet: GLI1 interacts with the MEP50/PRMT5 complex. a FLAG-GLI1 interacted with endogenous MEP50 and interaction of FLAG-GLI1 and MEP50 was increased by HH signalling pathway activation. C3H10T1/2 cells were transfected with FLAG-GLI1 or the empty vector for 24 h and then treated with 300 nM SAG for an additional 24 h. Interaction of FLAG-GLI1 and MEP50 was detected by immunoprecipitation with anti-FLAG antibody followed by immunoblot analysis using anti-FLAG and anti-MEP50 antibodies. b Schematic structures of MEP50 deletion mutants. c Mapping of the GLI1-binding region in MEP50 by immunoprecipitation analysis. HEK293T cells were transfected with Myc-MEP50 deletion mutants and FLAG-GLI1 plasmids for 24 h. Interaction of FLAG-GLI1 and Myc-MEP50 deletion mutants was detected by immunoprecipitation with anti-FLAG antibody followed by immunoblot analysis using anti-FLAG and anti-Myc antibodies. d Schematic of GLI1 deletion mutants. e GST pull-down assays to map the MEP50-binding region in GLI1. GST-GLI1 deletion mutants coupled to glutathione sepharose were incubated with immunoprecipitated Myc-MEP50 from HEK293T cells. Immunoblotting was performed with an anti-Myc antibody. In a and e , data represent one of three independent experiments with similar results. In c , data represent one of two independent experiments with similar results. Unprocessed original scans of blots are shown in Supplementary Fig. 6

    Article Snippet: Immunoprecipitation and immunoblotting Transfected or infected cells were lysed in TNE lysis buffer (1% Nonidet P-40, 10 mM Tris–HCl [pH 8.0], 150 mM NaCl, 1 mM EDTA, 1 mM NaF, 1 mM orthovanadate, 0.1 mM DTT, and a protease inhibitor cocktail [Nacalai Tesque]).

    Techniques: Activation Assay, Transfection, Plasmid Preparation, Immunoprecipitation, Binding Assay, Incubation

    MEP50/PRMT5 complex supports GLI1 activation through GLI1 stabilisation downstream of the HH signalling pathway. a–c Endogenous GLI1/MEP50/PRMT5 complex in C3H10T1/2 cells. Cells were treated with SAG for 36 h, and complex was detected by immunoprecipitation (IP) with anti-PRMT5 (D5P2T) ( a ), anti-MEP50 (ERP10708 [B]) ( b ), or anti-GLI1 (V812) ( c ) antibodies, followed by immunoblot (IB) with antibodies against indicated proteins. d Dissociation of PRMT5 and GLI1 in stable MEP50 knockdown C3H10T1/2 cells by siMEP50-m2. Cells were treated with SAG for 48 h and treated with 50 μM MG132 for 4 h. GLI1/PRMT5 complex was detected by immunoprecipitation with anti-PRMT5 (D5P2T) or anti-GLI1 (V812) antibodies, followed by immunoblot with indicated antibodies. e Immunoblot of endogenous GLI1 in C3H10T1/2 cells expressing MEP50 siRNAs (siMEP50-m1 or siMEP50-m2). f Immunoblot of endogenous GLI1 in C3H10T1/2 cells expressing two independent PRMT5 siRNAs. g Immunoblot of nuclear and cytoplasmic GLI1 and MEP50 in stable MEP50-knockdown (siMEP50-m2) or control siGFP-expressing cells treated with 300 nM SAG. Cells were treated with SAG for 24 h and separated into cytosol and nucleus fractions. h Immunoblot analysis of endogenous nuclear and cytoplasmic GLI1 and PRMT5 in stable PRMT5-knockdown (siPRMT5-m2) C3H10T1/2 cells. Cells were treated with 300 nM SAG for 24 h and then separated as in h . i In vivo ubiquitination of GLI1 in C3H10T1/2 cells with or without expression of siMEP50. FLAG-ubiquitin was transfected into C3H10T1/2 cells. After 48 h of transfection, then cells were treated with 50 µM MG132 for 4 h. Endogenous ubiquitinated GLI1 was immunoprecipitated with an anti-GLI1 (C-1) antibody, followed by immunoblotting with indicated antibodies. j In vivo ubiquitination of GLI1 in C3H10T1/2 cells with exogenous expression of PRMT5 or MEP50. FLAG-ubiquitin and HA-PRMT5, HA-PRMT5 G367A/R368A (inactive form of PRMT5), or Myc-MEP50 were transfected into C3H10T1/2 cells. After 48 h, the cells were treated with 50 µM MG132 for 4 h. Endogenous ubiquitinated GLI1 was detected as described in i . In a , i and j , data represent one of two independent experiments with similar results. Unprocessed original scans of blots are shown in Supplementary Fig. 6

    Journal: Communications Biology

    Article Title: MEP50/PRMT5-mediated methylation activates GLI1 in Hedgehog signalling through inhibition of ubiquitination by the ITCH/NUMB complex

    doi: 10.1038/s42003-018-0275-4

    Figure Lengend Snippet: MEP50/PRMT5 complex supports GLI1 activation through GLI1 stabilisation downstream of the HH signalling pathway. a–c Endogenous GLI1/MEP50/PRMT5 complex in C3H10T1/2 cells. Cells were treated with SAG for 36 h, and complex was detected by immunoprecipitation (IP) with anti-PRMT5 (D5P2T) ( a ), anti-MEP50 (ERP10708 [B]) ( b ), or anti-GLI1 (V812) ( c ) antibodies, followed by immunoblot (IB) with antibodies against indicated proteins. d Dissociation of PRMT5 and GLI1 in stable MEP50 knockdown C3H10T1/2 cells by siMEP50-m2. Cells were treated with SAG for 48 h and treated with 50 μM MG132 for 4 h. GLI1/PRMT5 complex was detected by immunoprecipitation with anti-PRMT5 (D5P2T) or anti-GLI1 (V812) antibodies, followed by immunoblot with indicated antibodies. e Immunoblot of endogenous GLI1 in C3H10T1/2 cells expressing MEP50 siRNAs (siMEP50-m1 or siMEP50-m2). f Immunoblot of endogenous GLI1 in C3H10T1/2 cells expressing two independent PRMT5 siRNAs. g Immunoblot of nuclear and cytoplasmic GLI1 and MEP50 in stable MEP50-knockdown (siMEP50-m2) or control siGFP-expressing cells treated with 300 nM SAG. Cells were treated with SAG for 24 h and separated into cytosol and nucleus fractions. h Immunoblot analysis of endogenous nuclear and cytoplasmic GLI1 and PRMT5 in stable PRMT5-knockdown (siPRMT5-m2) C3H10T1/2 cells. Cells were treated with 300 nM SAG for 24 h and then separated as in h . i In vivo ubiquitination of GLI1 in C3H10T1/2 cells with or without expression of siMEP50. FLAG-ubiquitin was transfected into C3H10T1/2 cells. After 48 h of transfection, then cells were treated with 50 µM MG132 for 4 h. Endogenous ubiquitinated GLI1 was immunoprecipitated with an anti-GLI1 (C-1) antibody, followed by immunoblotting with indicated antibodies. j In vivo ubiquitination of GLI1 in C3H10T1/2 cells with exogenous expression of PRMT5 or MEP50. FLAG-ubiquitin and HA-PRMT5, HA-PRMT5 G367A/R368A (inactive form of PRMT5), or Myc-MEP50 were transfected into C3H10T1/2 cells. After 48 h, the cells were treated with 50 µM MG132 for 4 h. Endogenous ubiquitinated GLI1 was detected as described in i . In a , i and j , data represent one of two independent experiments with similar results. Unprocessed original scans of blots are shown in Supplementary Fig. 6

    Article Snippet: Immunoprecipitation and immunoblotting Transfected or infected cells were lysed in TNE lysis buffer (1% Nonidet P-40, 10 mM Tris–HCl [pH 8.0], 150 mM NaCl, 1 mM EDTA, 1 mM NaF, 1 mM orthovanadate, 0.1 mM DTT, and a protease inhibitor cocktail [Nacalai Tesque]).

    Techniques: Activation Assay, Immunoprecipitation, Expressing, In Vivo, Transfection

    Expression of interleukin-34 (IL-34) in human rheumatoid arthritis (RA) synovium and synovial fluid (SF) . ( A ) IL-34 and macrophage colony-stimulating factor (M-CSF) mRNA levels in synovia from patients with RA ( n = 3) and osteoarthritis (OA) ( n = 3) were determined by reverse transcriptase PCR (RT-PCR). Synovium were homogenized and lysed, and total RNA was extracted as described in Materials and Methods. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels were detected as a control. ( B ) Representative immunohistochemical images of OA or RA synovia stained with antibodies against IL-34 or isotype controls. Images are shown at 200× (upper) and 400× (lower) magnification. Scale bars = 100 μm. ( C ) Synovial fluid (SF) from RA patients ( n = 7) (RA SF) was collected and the concentrations of M-CSF and IL-34 were measured using an enzyme-linked immunosorbent assay (ELISA) assay. ( D ) Expression of IL-34 protein in fibroblast-like synovial cells (FLS) from RA patients ( n = 6) was determined by immunoblotting against human IL-34. Whole-cell lysates of RA FLS cells were resolved by SDS-PAGE and followed by immunoblotting with anti-human IL-34 and anti-β-actin antibodies.

    Journal: Arthritis Research & Therapy

    Article Title: Interleukin-34 produced by human fibroblast-like synovial cells in rheumatoid arthritis supports osteoclastogenesis

    doi: 10.1186/ar3693

    Figure Lengend Snippet: Expression of interleukin-34 (IL-34) in human rheumatoid arthritis (RA) synovium and synovial fluid (SF) . ( A ) IL-34 and macrophage colony-stimulating factor (M-CSF) mRNA levels in synovia from patients with RA ( n = 3) and osteoarthritis (OA) ( n = 3) were determined by reverse transcriptase PCR (RT-PCR). Synovium were homogenized and lysed, and total RNA was extracted as described in Materials and Methods. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels were detected as a control. ( B ) Representative immunohistochemical images of OA or RA synovia stained with antibodies against IL-34 or isotype controls. Images are shown at 200× (upper) and 400× (lower) magnification. Scale bars = 100 μm. ( C ) Synovial fluid (SF) from RA patients ( n = 7) (RA SF) was collected and the concentrations of M-CSF and IL-34 were measured using an enzyme-linked immunosorbent assay (ELISA) assay. ( D ) Expression of IL-34 protein in fibroblast-like synovial cells (FLS) from RA patients ( n = 6) was determined by immunoblotting against human IL-34. Whole-cell lysates of RA FLS cells were resolved by SDS-PAGE and followed by immunoblotting with anti-human IL-34 and anti-β-actin antibodies.

    Article Snippet: Rabbit anti-human IL-34 for immunoblotting was from ProSci Incorporated (Poway, CA, USA).

    Techniques: Expressing, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Immunohistochemistry, Staining, Enzyme-linked Immunosorbent Assay, SDS Page

    Effect of HDACi on tumor suppressor proteins p53, p21, and p27 and on DBS marker pH2AX Analysis of tumor suppressor protein and pH2AX expression was performed by immunoblotting after 48 h treatment with HDACi SAHA, LBH-589, and PXD101. One representative blot out of three is shown. β-actin was used as loading control. Δ represents fold change normalized to controls (mean±SD of n = 3).

    Journal: Oncotarget

    Article Title: Histone deacetylase inhibitors vorinostat and panobinostat induce G1 cell cycle arrest and apoptosis in multidrug resistant sarcoma cell lines

    doi: 10.18632/oncotarget.20460

    Figure Lengend Snippet: Effect of HDACi on tumor suppressor proteins p53, p21, and p27 and on DBS marker pH2AX Analysis of tumor suppressor protein and pH2AX expression was performed by immunoblotting after 48 h treatment with HDACi SAHA, LBH-589, and PXD101. One representative blot out of three is shown. β-actin was used as loading control. Δ represents fold change normalized to controls (mean±SD of n = 3).

    Article Snippet: Western blot analysis For immunoblotting, whole cell protein extracts were prepared with lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 50 mM NaF, 1 mM EDTA, 10-% NP-40, 1% Triton-X and protease inhibitors), subjected to SDS-PAGE (10 or 12%) and blotted onto PVDF membranes (Roth, Karlsruhe, Germany).

    Techniques: Marker, Expressing

    Expression of Class I HDACs in SW-982 and SW-1353 cells Protein expression of Class I HDAC members HDAC1, -2, -3, and -8 in SW-982 and SW-1353 cells was evaluated by immunoblotting under control conditions and in the presence of the IC 50 and IC 75 concentrations of the HDACi SAHA, LBH-589, and PXD101 for 48 h. β-actin was used as loading control. Δ, fold change normalized to controls (mean±SD of n = 3).

    Journal: Oncotarget

    Article Title: Histone deacetylase inhibitors vorinostat and panobinostat induce G1 cell cycle arrest and apoptosis in multidrug resistant sarcoma cell lines

    doi: 10.18632/oncotarget.20460

    Figure Lengend Snippet: Expression of Class I HDACs in SW-982 and SW-1353 cells Protein expression of Class I HDAC members HDAC1, -2, -3, and -8 in SW-982 and SW-1353 cells was evaluated by immunoblotting under control conditions and in the presence of the IC 50 and IC 75 concentrations of the HDACi SAHA, LBH-589, and PXD101 for 48 h. β-actin was used as loading control. Δ, fold change normalized to controls (mean±SD of n = 3).

    Article Snippet: Western blot analysis For immunoblotting, whole cell protein extracts were prepared with lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 50 mM NaF, 1 mM EDTA, 10-% NP-40, 1% Triton-X and protease inhibitors), subjected to SDS-PAGE (10 or 12%) and blotted onto PVDF membranes (Roth, Karlsruhe, Germany).

    Techniques: Expressing

    Pharmacological inhibition of actin glutathionylation prevents DNA release. (A) Immunoblotting. Actin glutathionylation in activated mouse and human neutrophils is dependent on NADPH oxidase activation. Human and mouse neutrophils were analyzed after short-term stimulation (total 35 min) with the indicated triggers. C5a activation was performed in a time-dependent manner. No glutathionylated actin at the expected size (42 kD) was detected in Nox2 −/− mouse neutrophils or in 50-µM-DPI–pretreated human neutrophils. The ratio of glutathionylated actin to total actin is shown as actin glutathionylation in the bar graph (right). Data are representative of three independent experiments. (B) Confocal microscopy. DNA release was analyzed after short-term stimulation (total 35 min) of control human neutrophils with the indicated triggers in the presence and absence of the inhibitors as designated: 50 µM Na-arsenite and 2 µM CdCl 2 . Right: Quantification of released dsDNA in supernatants of activated neutrophils. Data are means ± SEM. *, P

    Journal: The Journal of Cell Biology

    Article Title: ROS and glutathionylation balance cytoskeletal dynamics in neutrophil extracellular trap formation

    doi: 10.1083/jcb.201611168

    Figure Lengend Snippet: Pharmacological inhibition of actin glutathionylation prevents DNA release. (A) Immunoblotting. Actin glutathionylation in activated mouse and human neutrophils is dependent on NADPH oxidase activation. Human and mouse neutrophils were analyzed after short-term stimulation (total 35 min) with the indicated triggers. C5a activation was performed in a time-dependent manner. No glutathionylated actin at the expected size (42 kD) was detected in Nox2 −/− mouse neutrophils or in 50-µM-DPI–pretreated human neutrophils. The ratio of glutathionylated actin to total actin is shown as actin glutathionylation in the bar graph (right). Data are representative of three independent experiments. (B) Confocal microscopy. DNA release was analyzed after short-term stimulation (total 35 min) of control human neutrophils with the indicated triggers in the presence and absence of the inhibitors as designated: 50 µM Na-arsenite and 2 µM CdCl 2 . Right: Quantification of released dsDNA in supernatants of activated neutrophils. Data are means ± SEM. *, P

    Article Snippet: Rabbit polyclonal anti–pan-actin and sheep anti-tubulin for immunoblotting were purchased from Cytoskeleton, Inc.

    Techniques: Inhibition, Activation Assay, Confocal Microscopy

    Cys374 is required for actin glutathionylation, actin polymerization, and NET formation. (A) Glutathionylated proteins in activated human neutrophil lysates were immunoprecipitated with anti–GSH antibody (time-dependent after C5a stimulation) and immunoblotted by using anti–pan-actin antibody. A total cell lysates (Input) was loaded as control. Data are representative of five independent experiments. (B) Immunoblotting. Actin glutathionylation in activated Hoxb8 mouse neutrophils overexpressing EGFP-β-actin-Cys 374 (WT), point-mutated Cys 374 to Ala 374 , or Cys 374 to Glu 374 was detected by anti–GSH antibody. The overexpression of WT and mutant forms of EGFP-β-actin was confirmed by immunoblotting with anti–GFP antibody. The ratio of glutathionylated EGFP-β-actin to total EGFP-β-actin was calculated as actin-glutathionylation in the bar graph (right panel). (C) Confocal microscopy. F-actin distribution and morphological changes were analyzed after short-term stimulation (total 35 min) of WT and overexpressed EGFP-β-actin-Cys 374 (WT), point-mutated Cys 374 to Ala 374 , and Cys 374 to Glu 374 mouse neutrophils with the indicated triggers. Right: Quantification of F-actin polymerization. (D) Confocal microscopy. DNA release was analyzed after short-term stimulation (total 35 min) of WT and overexpressed EGFP-β-actin-Cys 374 (WT), point-mutated Cys 374 to Ala 374 , and Cys 374 to Glu 374 mouse neutrophils with the indicated triggers. Right: Quantification of released dsDNA in supernatants of activated neutrophils. Data are means ± SEM. ***, P

    Journal: The Journal of Cell Biology

    Article Title: ROS and glutathionylation balance cytoskeletal dynamics in neutrophil extracellular trap formation

    doi: 10.1083/jcb.201611168

    Figure Lengend Snippet: Cys374 is required for actin glutathionylation, actin polymerization, and NET formation. (A) Glutathionylated proteins in activated human neutrophil lysates were immunoprecipitated with anti–GSH antibody (time-dependent after C5a stimulation) and immunoblotted by using anti–pan-actin antibody. A total cell lysates (Input) was loaded as control. Data are representative of five independent experiments. (B) Immunoblotting. Actin glutathionylation in activated Hoxb8 mouse neutrophils overexpressing EGFP-β-actin-Cys 374 (WT), point-mutated Cys 374 to Ala 374 , or Cys 374 to Glu 374 was detected by anti–GSH antibody. The overexpression of WT and mutant forms of EGFP-β-actin was confirmed by immunoblotting with anti–GFP antibody. The ratio of glutathionylated EGFP-β-actin to total EGFP-β-actin was calculated as actin-glutathionylation in the bar graph (right panel). (C) Confocal microscopy. F-actin distribution and morphological changes were analyzed after short-term stimulation (total 35 min) of WT and overexpressed EGFP-β-actin-Cys 374 (WT), point-mutated Cys 374 to Ala 374 , and Cys 374 to Glu 374 mouse neutrophils with the indicated triggers. Right: Quantification of F-actin polymerization. (D) Confocal microscopy. DNA release was analyzed after short-term stimulation (total 35 min) of WT and overexpressed EGFP-β-actin-Cys 374 (WT), point-mutated Cys 374 to Ala 374 , and Cys 374 to Glu 374 mouse neutrophils with the indicated triggers. Right: Quantification of released dsDNA in supernatants of activated neutrophils. Data are means ± SEM. ***, P

    Article Snippet: Rabbit polyclonal anti–pan-actin and sheep anti-tubulin for immunoblotting were purchased from Cytoskeleton, Inc.

    Techniques: Immunoprecipitation, Over Expression, Mutagenesis, Confocal Microscopy

    Fluorescence properties of ER-targeted roGFP-iL mutants in the ER ( A ) Fluorescence images of HeLa cells transfected with ER-targeted roGFP-iL and its mutants were acquired (green) after fixation with 4% PFA. The cells were also co-stained with anti-GFP (red) and anti-Hsp47 (blue, ER marker). ( B ) Immunoblotting of ER-targeted roGFP-iL and its mutants with anti-GFP in 1% NP-40-soluble (S) and -insoluble (P) cell fractions.

    Journal: Bioscience Reports

    Article Title: Development of a stable ERroGFP variant suitable for monitoring redox dynamics in the ER

    doi: 10.1042/BSR20160027

    Figure Lengend Snippet: Fluorescence properties of ER-targeted roGFP-iL mutants in the ER ( A ) Fluorescence images of HeLa cells transfected with ER-targeted roGFP-iL and its mutants were acquired (green) after fixation with 4% PFA. The cells were also co-stained with anti-GFP (red) and anti-Hsp47 (blue, ER marker). ( B ) Immunoblotting of ER-targeted roGFP-iL and its mutants with anti-GFP in 1% NP-40-soluble (S) and -insoluble (P) cell fractions.

    Article Snippet: Antibodies Anti-GFP JL-8 mouse monoclonal (Clontech) and anti-β-actin (Sigma–Aldrich) antibodies were used for immunoblotting and anti-Hsp47 SPA-470 mouse monoclonal (Enzo Life Sciences) and anti-GFP rabbit polyclonal (Life Technology) antibodies for immunostaining.

    Techniques: Fluorescence, Transfection, Staining, Marker