c-myc Search Results


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  • 99
    Thermo Fisher anti myc
    H-rev107 was associated with <t>H-RAS</t> and inhibited its palmitoylation. (A) HtTA cells plated in a 10-cm dish were transfected for 24 h with 0.1 μg of H-RAS along with <t>H-rev107-myc</t> or the control vector. Cell lysates were prepared as described in Methods. The interaction between H-rev107 and RAS was analyzed by immunoprecipitation followed by Western blot analysis. (B) HtTA cells plated in a 10-cm dish were transfected for 24 h with 0.1 μg of H-RAS along with 3 μg of H-rev107-myc or the control vector. Cell lysates were prepared, and acyl-biotin exchange analysis of H-RAS was performed as described in Methods. Aliquots containing 5 μg of protein including acylated RAS were biotinylated and then processed with streptavidin agarose resin followed by Western blot analysis. The input consisted of 300 ng of protein from the acyl-biotin exchange that was loaded. HC: heavy chain; NH 2 OH: hydroxylamine.
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    Millipore anti myc
    Immunoblot Analysis of SAGL1:eYFP and <t>MYC:CER3</t> in Transgenic Arabidopsis Plants, and Cuticle Permeability of Wild Type and sagl1 - 2 under Relatively Low and High Humidity Conditions. (A) Assay of SAGL1:eYFP protein levels under different humidity conditions. Seven-d–old wild-type (Col-0) and transgenic Arabidopsis seedlings overexpressing SAGL1 : eYFP grown in Petri dishes were transferred to soil. The seedlings were covered with an opaque plastic lid for 1 d ( > 90% RH), uncovered (50% to 60% RH) for the indicated time points, and subjected to immunoblot analysis. SAGL1:eYFP protein levels were determined in total protein extracts by immunoblot analysis using <t>anti-GFP</t> antibodies at the indicated time points. Equal protein loading was assured using Ponceau-S staining. (B) SAGL1:eYFP protein stability assay. Four-d–old wild-type (Col-0) and transgenic Arabidopsis seedlings overexpressing SAGL1 : eYFP were treated with the MG132 (100 μM) or DMSO before treatment with 100 μM of CHX for the indicated amounts of time. SAGL1:eYFP protein levels were determined in total protein extracts by immunoblot analysis using anti-GFP antibodies at the indicated time points. Equal protein loading was assured using Ponceau-S staining. (C) Seven-d–old transgenic wild-type (C24) and sagl1 - 1 seedlings overexpressing MYC : CER3 grown in Petri dishes were exposed to 50% to 60% RH for 0 and 12 h and subjected to immunoblot analysis. MYC:CER3 protein levels were determined in total protein extracts by immunoblot analysis using anti-MYC antibodies. Equal protein loading was assured using Ponceau-S staining. (D) Arabidopsis wild-type (Col-0) and sagl1 - 2 plants were transferred to soil after germination on half-strength MS medium and uncovered or covered with a lid to maintain high humidity ( > 90% RH). Four-week–old Arabidopsis wild-type and sagl1 - 2 leaves were immersed in TB O solution (0.05% with 0.01% TWEEN 20) for 10 min, washed with distilled water, and photographed.
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    94
    Santa Cruz Biotechnology anti myc
    TMEM192 siRNAs alleviated TIG1A-induced expression of autophagy-related proteins. HtTA cells plated in 6-well dishes were transfected with 0.5 μg of <t>TIG1B-myc</t> (A) or <t>TMEM192-Flag</t> (B) expression vector along with 30 nM TIG1, TMEM192, or NC siRNA for 48 h. Alternatively, cells were transfected with 0.5 μg of TIG1A-myc (C–E) expression vector along with the indicated siRNA (30 nM) for 48 h and were then cultured in serum-free medium for 6 h. Cell lysates were prepared, and the expression of TIG1, TMEM192, Beclin-1, or LC3B was determined using anti-MYC, anti-FLAG, anti-Beclin-1, or anti-LC3B antibodies, respectively. Experimental results are summarized as the mean percentage (± SD) of the ratio of LC3B-II to LC3B-I and the level of Beclin-1 with each sample normalized to the level of actin protein in two independent experiments (D, E). HtTA cells plated in 6-well dishes were transfected with 0.5 μg of TIG1A-myc expression vectors along with the indicated siRNA for 48 h and were then cultured in serum-free medium for 6 h. Cell lysates were prepared, and the level of Beclin-1 was detected using an enzyme immunoassay. Representative results of three independent experiments are shown. HtTA cells plated in triplicate in 24-well plates were transfected with 75 ng of TIG1A-myc expression vector, 150 ng pGFP-LC3 expression vector and with the indicated 30 nM siRNA for 48 h and were then cultured in serum-free medium for 6 h. Representative images with GFP-LC3 puncta formation (G). Bar chart indicating the percentage of cells with GFP-LC3 puncta formation from three independent experiments (H). Scale bar, 10 μm. *Indicates p value
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    94
    Cell Signaling Technology Inc c myc
    Expression of BCL-2 protein family members in A1 +/+ Eµ <t>-Myc</t> and A1 −/− Eµ -Myc lymphomas and premalignant cells. Western blots were performed on a pro/pre-B cell lymphomas from lymph nodes of sick A1 +/+ Eµ -Myc (lanes 1–4) and A1 − / − Eµ -Myc mice (lanes 5–8) and pre-malignant pro/pre-B cells (MACS-sorted CD19 + bone marrow (BM) samples) from healthy A1 +/+ Eµ -Myc and A1 − / − Eµ -Myc mice euthanased at 28–30 days of age (lanes 9–12); b B cell lymphomas from lymph nodes of A1 +/+ Eµ -Myc (lanes 1–4) and A1 −/− Eµ -Myc mice (lanes 5–8). The same premalignant samples and control lysates as in ( a ) were run to enable comparison between ( a ) and ( b ). Lane labels indicate the individual mouse number. WEHI-231 cells served as a positive control for A1 protein, tumour 1/8094 as a positive control for p53 and <t>p19ARF</t> proteins and β-ACTIN as a loading control. Independent blots are separated by a horizontal black line and molecular weight markers (kD) are indicated. Additional lymphomas were analysed (data not shown), in total: 8 pro/pre-B and 8 B A1 +/+ Eµ -Myc ; 7 pro/pre-B and 6 B A1 −/− Eµ -Myc
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    Santa Cruz Biotechnology c myc
    Increases in <t>c-Myc</t> expression in the mouse neuroinflammation model. A , ChIP assays showing increased recruitment of c-Myc onto the <t>Gnpat</t> promoter in the hippocampal tissues from mice injected with LPS (250 μg/kg/d, i.p., for 7 d), aged mice (16 months), and mice subjected to CRS compared with the saline-injected, adult (2 months), and sham groups, respectively. B , c-Myc expression was increased in the cortex of LPS, aged, and CRS mice. C , Immunohistochemical data for Iba-1, GFAP, c-Myc, and Gnpat expression in the cortex of LPS-injected mice and the saline group. Scale bar, 50 μm. D , Quantification of data in C showing increase in amoeboid Iba-1 + microglial cells and c-Myc and Gnpat expressions in LPS-injected mice ( n = 5). E , Protein expression of Gnpat, c-Myc, and β-actin (loading control) in the cortical tissue of three representative mice from the LPS and saline groups. Right panel shows the relative changes in the protein expression. F , Immunohistochemical data showing coexpression of c-Myc with the NeuN, Iba-1, and GFAP in the LPS and saline groups. Scale bar, 50 μm. G , Quantification of data in F showing increases in c-Myc protein in glial cells but not in neurons. H , Quantification data of Gnpat costained with the Iba-1, GFAP, and NeuN. The image data for this graph are available upon request. All of the image quantification data were derived from 5 mice in each group. More than 7 slices of each mouse brain were examined. ** p
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    99
    Cell Signaling Technology Inc anti myc
    Cooperative effects of OTX015 with a panel of drugs in ALCL in vitro models A. The combination of OTX015-CEP28122 effectively down-regulated <t>MYC</t> protein levels by Western blot in TS-Supm2 cells <t>(β-actin</t> was used as a loading control), resulting in a pronounced G1 cell cycle arrest with limited effects on cell viability according to FACScan. The effect was maximal after 48 h. B. OTX015 exposure (500 nM) for 30 minutes to 24 h in Karpas299 cells resulted in robust down-regulation of GLI1 mRNA, a known target of GANT61, following an initial rapid upregulation according to qRT-PCR. Exposure of in Karpas299 cells to increasing OTX015 concentrations for 24 h showed that the mRNA down-regulation was dose-dependent. C. The effects on cell viability of the GLI inhibitor GANT61 and OTX015 were measured as single agents and in combination according to the qRT-PCR. Concomitant exposure of OTX015 (500 nM) and GANT61 (2.5 or 5 μM) increased the percentage of dead cells after 24 and 48 h exposure. D. Cells treated with combined ibrutinib (10 μM) and OTX015 (500 nM) had reduced levels of P-STAT3 and P-ITK protein compared to treatment with either single agent, as per Western blot. β-actin was used as a loading control. E. Metabolic readout of ALCL cell lines treated with OTX015 (500 nM) in combination with ibrutinib (1.2 or 5 μM) for 48 h, determined using an ATPlite assay. F. Cell viability was determined after 48 h in multiple cell lines exposed to 500 nM OTX015 in combination with ibrutinib (2, 5, 10 μM), according to the ATPlite assay.
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    c myc  (Abcam)
    99
    Abcam c myc
    IKBKE influences AR mRNA expression via the Hippo pathway. LNCaP cells were reverse transfected in full media (FM) with either N/S or three pooled siRNAs against IKBKE . After 72 h, protein expression was assessed by ( A ) immunoblotting and ( B ) <t>YAP</t> , <t>c-MYC</t> and IKBKE mRNA expression determined by qPCR ( n = 3). ( C ) After 72 h steroid depletion, LNCaP cells were pre-treated with vehicle (DMSO) or increasing concentrations of IKBKE antagonist, CAY10576, for 8 h followed by treatment with vehicle or 1 nM R1881 for a further 24 h. YAP and c-MYC protein levels were assessed by immunoblotting. ( D ) LNCaP cells were reverse transfected in FM with either N/S or pooled siRNAs against IKBKE . After 72 h, MG132 (20 μM) was applied and incubated for a further 16 h. YAP levels were determined by immunoblotting. ( E ) LNCaP cells were reverse transfected in FM with either N/S or pooled siRNAs against IKBKE . After 72 h, nuclear-cytoplasmic fractionation was performed with YAP and c-MYC localization determined by immunoblotting. PARP was used as a nuclear loading control and α-tubulin as a cytoplasmic loading control. Densitometry was performed on YAP expression and normalized to loading controls using ImageJ software. Data is expressed as a % of total cellular YAP. ( F ) LNCaP cells were treated with either vehicle or CAY10576 (5 μM) or CYT387 (5 μM) for 16 h prior to nuclear-cytoplasmic fractionation. Nuclear YAP and c-MYC levels were determined by immunoblotting. ( G ) LNCaP cells were reverse transfected in FM with either N/S or pooled siRNAs against IKBKE and grown for 48 h prior to ChIP analysis using antibodies specific to c-MYC and isotype controls (IgG). Recruitment to the c-MYC binding site within the AR gene was assessed by qPCR. Data are an average of 3 independent experiments ± SEM. One-way ANOVA paired Dunnetts multiple comparison test * P
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    99
    Millipore monoclonal anti c myc antibody
    Interaction of <t>Loqs</t> isoforms with Dicer1 and Dicer2. ( A ) The Loqs-containing complexes were immunoprecipitated from S2 cells using anti-Loqs antibodies and the proteins contained in the immunoprecipitates were visualized by silver staining. The protein bands corresponding to Loqs-PA, Loqs-PB, and Loqs-PC/Loqs-PD are indicated. Two proteins coimmunopurified with Loqs were identified as Dicer1 and Dicer2 by mass spectrometry analyses. (n.i.) Nonimmune IgG used as a negative control. ( B ) Western blot analyses using anti-Dicer1 and anti-Dicer2 antibodies confirmed that the immunoprecipitates obtained using anti-Loqs antibodies (panel A ) contain both Dicer proteins, along with all Loqs isoforms. ( C ) Loqs-PA and Loqs-PB interact with Dicer1, whereas Loqs-PD interacts with Dicer2. Each Loqs isoform was expressed in S2 cells, and the anti-myc immunoprecipitated complexes were probed with anti-Dicer1 and anti-Dicer2 antibodies. (h.c.) Heavy chain of <t>anti-c-myc</t> antibody. ( D ) Deletion mutant analysis of Loqs-PD to determine the domain necessary for interacting with Dicer2. Various deletion mutants of Loqs-PD (m1–m5) were expressed in S2 cells, and their interactions with endogenous Dicer2 were examined. The Loqs-PD-m4 mutant interacted with Dicer2 as efficiently as wt Loqs-PD. The dsRNA-binding domains (light gray boxes) and C-terminal region (dark gray boxes) found exclusively in Loqs-PD but not in other Loqs isoforms, respectively, are indicated ( upper diagram). The lower panel shows immunoblotting results of the protein–protein interaction assays. EGFP was used as a negative control.
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    92
    Santa Cruz Biotechnology mouse anti myc
    Expression of <t>p300</t> and <t>MYC</t> in a panel of human B-lymphoma cell lines. Extracts from the indicated human B-lymphoma cell lines were analyzed by Western blotting for p300, MYC, and β-tubulin (as a loading control). In a given panel, all lanes contain
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    99
    Millipore anti c myc antibody
    Expression of berghepain-1 in liver stage parasites. HepG2 cells infected with BP1-myc parasites, and merosomes collected from these cultures, were fixed at the indicated timepoints and probed with <t>anti-c-myc</t> antibodies (magenta) to localize berghepain-1, and co-localized with antibodies against A. the cytosolic marker heat-shock protein 70 (HSP70), B. the ER chaperone <t>BIP</t> (green) and C. the membrane maker merozoite surface protein 1 (MSP-1). BP1-myc expression was low 24 h after invasion, but seen in early schizonts after 36 h of development. Host and parasite DNA were stained with DAPI (blue). Scale bars: 10 μm.
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    Cell Signaling Technology Inc anti c myc
    BTB reduces ER α , c-Myc, and <t>Cyclin</t> D1 protein expression levels in MCF-7, Ishikawa, and SKOV-3 cells but has no effect on ER α gene expression at mRNA level. Western blot analyses of ER α , c-Myc, and Cyclin D1 levels in control and 2.5 uM BTB treated MCF-7, Ishikawa, and SKOV-3 cells in the absence or presence of 10 nM E2. 50 μ g of total protein from cells was applied onto a 10% sodium dodecyl sulfate-polyacrylamide gel and subjected to electrophoresis followed by Western blot using anti-ER α , <t>anti-c-Myc,</t> or anti-Cyclin D1 antibodies. Representative graphs were shown from consistent results collected from three independent experiments.
    Anti C Myc, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1192 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Abcam anti myc
    <t>OXSR1</t> and STK39 are required for pre-TCR induced proliferation and regulation of <t>MYC</t> (A-C) Mean±SEM number of thymocytes at each developmental stage in Stk39 +/+ and Stk39 T243A/T243A mice (A) or in Rag1 -/- radiation chimeras reconstituted with fetal liver cells from Oxsr1 +/+ and Oxsr1 T185A/T185A embryos (B) or Oxsr1 +/+ Stk39 +/+ /RCE, Oxsr1 fl/fl Stk39 +/+ /RCE and Oxsr1 fl/fl Stk39 T243A/T243A /RCE embryos (C). The chimeric mice in C were analyzed 7 d after treatment with tamoxifen. Thymocyte subsets were identified by flow cytometry as shown in Figure 1A, E . (D) Left: flow cytometric analysis of DNA content in DN4 thymocytes from Rag1 -/- radiation chimeras reconstituted with fetal liver cells of the indicated genotypes. Gates show percentage of cells containing more than the diploid amount of DNA (S, G2 and M phases of cell cycle). Right: mean±SEM percentage of cells in S/G2/M phases of the cell cycle based on gate shown on histograms. (E) Left: flow cytometric analysis of MYC levels in DN4 thymocytes from Rag1 -/- radiation chimeras reconstituted with fetal liver cells of the indicated genotypes. Right: mean±SEM levels of MYC in DN4 thymocytes. (F) Mean±SEM number of thymocytes at each developmental stage in Rag1 -/- radiation chimeras reconstituted with liver cells from Slc12a2 +/+ and Slc12a2 -/- embryos. (G) Mean±SEM percentage of cells in each thymocyte population in chimeras reconstituted with Slc12a2 -/- fetal liver normalized to Slc12a2 +/+ control chimeras (set to 100%). * 0.01
    Anti Myc, supplied by Abcam, used in various techniques. Bioz Stars score: 96/100, based on 1191 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti c myc
    Overexpression of <t>MXD1</t> repressed c-Myc and miR-19a/b levels and impeded the malignant phenotypes of gastric cancer cells. ( a ) Western blot of anti-HA and <t>anti-c-Myc</t> using HA-tagged MXD1 over-expressing cells and the vector control cells. ( b and c ) Effects of MXD1 in in vitro migration and invasion assays. ( b ) Representative pictures show effect of MXD1 and si-MXD1 on the migration and invasion of gastric cancer cells. ( c ) Quantification of migration and invasion assays of B (** P ≤0.01 compared with the vector control). Each experiment was repeated at least three times. Error bars correspond to the mean±S.D. ( d ) Effects of MXD1 on in vitro proliferation in gastric cancer (** P ≤0.01, * P ≤0.05, respectively, compared with the vector control). Each experiment was repeated at least three times. Error bars correspond to the mean±S.D. ( e ) Real-time PCR show the expression levels of miR-19a and miR-19b in MXD1 over-expressing cells (** P
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    Covance anti myc
    Accumulated <t>LC3-positive</t> structures did not colocalize with phagophore and lysosome markers in DOX-treated TetON-GFPSTX17ΔNTD and TetON-GFPSTX17FL HeLa cells. (A-C) TetON-GFPSTX17ΔNTD and TetON-GFPSTX17FL HeLa cells were cultured with DOX (1.5 μg/ml) for 2 d. Cells were cultured in regular (Gr) or starvation (St) medium for 1 h and analyzed by immunofluorescence microscopy using antibodies against RB1CC1 (A), WIPI2 (B), and LAMP1 (C). Colocalization analysis was performed as described in Materials and Methods. Each correlation plot is derived from 30–61 cells in 3 different fields of view. The mean Pearson's correlation coefficient values ± SEM are shown on the plots and the graphs. The intensities of GFP-STX17 variants are represented on the x-axis. Asterisks indicate significant differences between <t>MYC-tagged</t> STX17 variants at p
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    92
    Santa Cruz Biotechnology rabbit anti myc
    Rmi1 -depletion causes a G2/M arrest during RTG. (A) Three Rmi1 -depleted mother-daughter cell pairs from an auxin-treated RMI1 -AID culture (MJL3899) taken 4 hr after shift to rich medium containing auxin. From left to right, differential interference contrast image, detection of DNA (DAPI), <t>beta-tubulin,</t> and Pds1 <t>-Myc.</t> See Materials and Methods for details. The bottom mother-daughter pair was scored as having undergone the metaphase–anaphase transition, based on the absence of Pds1 . (B) Percent of total cells in a control culture with a bud and lacking a bipolar spindle (white) or containing a bipolar spindle and nuclear Pds1 (red) or with a bipolar spindle but lacking nuclear Pds1 (blue). (C) As in (B), but in the presence of auxin. Data are from two experiments, error bars denote range.
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    Image Search Results


    H-rev107 was associated with H-RAS and inhibited its palmitoylation. (A) HtTA cells plated in a 10-cm dish were transfected for 24 h with 0.1 μg of H-RAS along with H-rev107-myc or the control vector. Cell lysates were prepared as described in Methods. The interaction between H-rev107 and RAS was analyzed by immunoprecipitation followed by Western blot analysis. (B) HtTA cells plated in a 10-cm dish were transfected for 24 h with 0.1 μg of H-RAS along with 3 μg of H-rev107-myc or the control vector. Cell lysates were prepared, and acyl-biotin exchange analysis of H-RAS was performed as described in Methods. Aliquots containing 5 μg of protein including acylated RAS were biotinylated and then processed with streptavidin agarose resin followed by Western blot analysis. The input consisted of 300 ng of protein from the acyl-biotin exchange that was loaded. HC: heavy chain; NH 2 OH: hydroxylamine.

    Journal: Journal of Biomedical Science

    Article Title: Phospholipase A/Acyltransferase enzyme activity of H-rev107 inhibits the H-RAS signaling pathway

    doi: 10.1186/1423-0127-21-36

    Figure Lengend Snippet: H-rev107 was associated with H-RAS and inhibited its palmitoylation. (A) HtTA cells plated in a 10-cm dish were transfected for 24 h with 0.1 μg of H-RAS along with H-rev107-myc or the control vector. Cell lysates were prepared as described in Methods. The interaction between H-rev107 and RAS was analyzed by immunoprecipitation followed by Western blot analysis. (B) HtTA cells plated in a 10-cm dish were transfected for 24 h with 0.1 μg of H-RAS along with 3 μg of H-rev107-myc or the control vector. Cell lysates were prepared, and acyl-biotin exchange analysis of H-RAS was performed as described in Methods. Aliquots containing 5 μg of protein including acylated RAS were biotinylated and then processed with streptavidin agarose resin followed by Western blot analysis. The input consisted of 300 ng of protein from the acyl-biotin exchange that was loaded. HC: heavy chain; NH 2 OH: hydroxylamine.

    Article Snippet: Cell lysates containing 500 μg of protein were first incubated for 2 h at 4°C with 3.2 μg of anti-MYC (Invitrogen, Carlsbad, CA, USA) or anti-RAS (Upstate Biotechnology) monoclonal antibody and then incubated for 2 h at 4°C with 20 μL of protein G plus/protein A agarose (Calbiochem, Cambridge, MA, USA).

    Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot

    AACOCF3 and MAFP eliminated H-rev107-mediated H-RAS palmitoylation. HtTA cells plated in a 10-cm dish were transfected for 24 h with 0.1 μg of H-RAS along with H-rev107-myc or the control vector in the presence of 10 μM AACOCF3 (A, B) , 1 μM palmostatin B (A) , 5 μM MAFP (B) , 2 μM BEL (B) or DMSO vehicle. Cell lysates were prepared, and acyl-biotin exchange analysis of H-RAS was performed as described in Methods. Levels of H-RAS and H-rev107 in the cell lysates are shown in the bottom panel. NH 2 OH: hydroxylamine.

    Journal: Journal of Biomedical Science

    Article Title: Phospholipase A/Acyltransferase enzyme activity of H-rev107 inhibits the H-RAS signaling pathway

    doi: 10.1186/1423-0127-21-36

    Figure Lengend Snippet: AACOCF3 and MAFP eliminated H-rev107-mediated H-RAS palmitoylation. HtTA cells plated in a 10-cm dish were transfected for 24 h with 0.1 μg of H-RAS along with H-rev107-myc or the control vector in the presence of 10 μM AACOCF3 (A, B) , 1 μM palmostatin B (A) , 5 μM MAFP (B) , 2 μM BEL (B) or DMSO vehicle. Cell lysates were prepared, and acyl-biotin exchange analysis of H-RAS was performed as described in Methods. Levels of H-RAS and H-rev107 in the cell lysates are shown in the bottom panel. NH 2 OH: hydroxylamine.

    Article Snippet: Cell lysates containing 500 μg of protein were first incubated for 2 h at 4°C with 3.2 μg of anti-MYC (Invitrogen, Carlsbad, CA, USA) or anti-RAS (Upstate Biotechnology) monoclonal antibody and then incubated for 2 h at 4°C with 20 μL of protein G plus/protein A agarose (Calbiochem, Cambridge, MA, USA).

    Techniques: Transfection, Plasmid Preparation

    H-rev107 associates and co-localizes with POR. HtTA cells plated in a 10-cm dish were transfected with H-rev107-myc and POR-Flag expression vector for 24 h. The cell lysates were prepared, and the interaction between H-rev107 and POR was analyzed by immunoprecipitation followed by western blot analysis. Immunoprecipitates were resolved by SDS-PAGE and immunoblotted using the anti-Flag antibody or anti-myc antibody (A). Total cellular extracts from HtTA or Huh7 cells were subjected to western blot analysis for H-rev107, POR, and actin (B). Huh7 cell lysates were prepared, and the interaction between H-rev107 and POR was analyzed by immunoprecipitation using anti-H-rev107- or POR-specific antibodies, respectively, followed by western blot analysis (C). HtTA cells were cotransfected with GFP-POR and H-rev107 expression vectors for 18 h. The cells were fixed and then incubated with anti-myc antibody followed by Alexa fluor 633 anti-mouse IgG antibody. The localization of H-rev107 (red), POR (green), and nuclei (blue) were analyzed using a laser scanning confocal microscope. Yellow fluorescence (arrows) evident in the merged images indicates co-localization of the H-rev107 and POR (D). The GST-tagged fusion proteins of H-rev107 (E) or POR (F) were purified, and the interaction between H-107 and POR was analyzed by GST pull down followed by Western blot analysis. Scale bar: 10 μm.

    Journal: PLoS ONE

    Article Title: H-rev107 Regulates Cytochrome P450 Reductase Activity and Increases Lipid Accumulation

    doi: 10.1371/journal.pone.0138586

    Figure Lengend Snippet: H-rev107 associates and co-localizes with POR. HtTA cells plated in a 10-cm dish were transfected with H-rev107-myc and POR-Flag expression vector for 24 h. The cell lysates were prepared, and the interaction between H-rev107 and POR was analyzed by immunoprecipitation followed by western blot analysis. Immunoprecipitates were resolved by SDS-PAGE and immunoblotted using the anti-Flag antibody or anti-myc antibody (A). Total cellular extracts from HtTA or Huh7 cells were subjected to western blot analysis for H-rev107, POR, and actin (B). Huh7 cell lysates were prepared, and the interaction between H-rev107 and POR was analyzed by immunoprecipitation using anti-H-rev107- or POR-specific antibodies, respectively, followed by western blot analysis (C). HtTA cells were cotransfected with GFP-POR and H-rev107 expression vectors for 18 h. The cells were fixed and then incubated with anti-myc antibody followed by Alexa fluor 633 anti-mouse IgG antibody. The localization of H-rev107 (red), POR (green), and nuclei (blue) were analyzed using a laser scanning confocal microscope. Yellow fluorescence (arrows) evident in the merged images indicates co-localization of the H-rev107 and POR (D). The GST-tagged fusion proteins of H-rev107 (E) or POR (F) were purified, and the interaction between H-107 and POR was analyzed by GST pull down followed by Western blot analysis. Scale bar: 10 μm.

    Article Snippet: Cells were then transfected with 500 ng of H-rev107 along with 500 ng pGFP-POR expression vector for 18 h. The cells were washed and fixed with 4% paraformaldehyde and then incubated with anti-MYC antibody followed by Alexa fluor 633 anti-mouse IgG antibody (Invitrogen).

    Techniques: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, SDS Page, Incubation, Microscopy, Fluorescence, Purification

    Immunoblot Analysis of SAGL1:eYFP and MYC:CER3 in Transgenic Arabidopsis Plants, and Cuticle Permeability of Wild Type and sagl1 - 2 under Relatively Low and High Humidity Conditions. (A) Assay of SAGL1:eYFP protein levels under different humidity conditions. Seven-d–old wild-type (Col-0) and transgenic Arabidopsis seedlings overexpressing SAGL1 : eYFP grown in Petri dishes were transferred to soil. The seedlings were covered with an opaque plastic lid for 1 d ( > 90% RH), uncovered (50% to 60% RH) for the indicated time points, and subjected to immunoblot analysis. SAGL1:eYFP protein levels were determined in total protein extracts by immunoblot analysis using anti-GFP antibodies at the indicated time points. Equal protein loading was assured using Ponceau-S staining. (B) SAGL1:eYFP protein stability assay. Four-d–old wild-type (Col-0) and transgenic Arabidopsis seedlings overexpressing SAGL1 : eYFP were treated with the MG132 (100 μM) or DMSO before treatment with 100 μM of CHX for the indicated amounts of time. SAGL1:eYFP protein levels were determined in total protein extracts by immunoblot analysis using anti-GFP antibodies at the indicated time points. Equal protein loading was assured using Ponceau-S staining. (C) Seven-d–old transgenic wild-type (C24) and sagl1 - 1 seedlings overexpressing MYC : CER3 grown in Petri dishes were exposed to 50% to 60% RH for 0 and 12 h and subjected to immunoblot analysis. MYC:CER3 protein levels were determined in total protein extracts by immunoblot analysis using anti-MYC antibodies. Equal protein loading was assured using Ponceau-S staining. (D) Arabidopsis wild-type (Col-0) and sagl1 - 2 plants were transferred to soil after germination on half-strength MS medium and uncovered or covered with a lid to maintain high humidity ( > 90% RH). Four-week–old Arabidopsis wild-type and sagl1 - 2 leaves were immersed in TB O solution (0.05% with 0.01% TWEEN 20) for 10 min, washed with distilled water, and photographed.

    Journal: The Plant Cell

    Article Title: The F-Box Protein SAGL1 and ECERIFERUM3 Regulate Cuticular Wax Biosynthesis in Response to Changes in Humidity in Arabidopsis [OPEN]

    doi: 10.1105/tpc.19.00152

    Figure Lengend Snippet: Immunoblot Analysis of SAGL1:eYFP and MYC:CER3 in Transgenic Arabidopsis Plants, and Cuticle Permeability of Wild Type and sagl1 - 2 under Relatively Low and High Humidity Conditions. (A) Assay of SAGL1:eYFP protein levels under different humidity conditions. Seven-d–old wild-type (Col-0) and transgenic Arabidopsis seedlings overexpressing SAGL1 : eYFP grown in Petri dishes were transferred to soil. The seedlings were covered with an opaque plastic lid for 1 d ( > 90% RH), uncovered (50% to 60% RH) for the indicated time points, and subjected to immunoblot analysis. SAGL1:eYFP protein levels were determined in total protein extracts by immunoblot analysis using anti-GFP antibodies at the indicated time points. Equal protein loading was assured using Ponceau-S staining. (B) SAGL1:eYFP protein stability assay. Four-d–old wild-type (Col-0) and transgenic Arabidopsis seedlings overexpressing SAGL1 : eYFP were treated with the MG132 (100 μM) or DMSO before treatment with 100 μM of CHX for the indicated amounts of time. SAGL1:eYFP protein levels were determined in total protein extracts by immunoblot analysis using anti-GFP antibodies at the indicated time points. Equal protein loading was assured using Ponceau-S staining. (C) Seven-d–old transgenic wild-type (C24) and sagl1 - 1 seedlings overexpressing MYC : CER3 grown in Petri dishes were exposed to 50% to 60% RH for 0 and 12 h and subjected to immunoblot analysis. MYC:CER3 protein levels were determined in total protein extracts by immunoblot analysis using anti-MYC antibodies. Equal protein loading was assured using Ponceau-S staining. (D) Arabidopsis wild-type (Col-0) and sagl1 - 2 plants were transferred to soil after germination on half-strength MS medium and uncovered or covered with a lid to maintain high humidity ( > 90% RH). Four-week–old Arabidopsis wild-type and sagl1 - 2 leaves were immersed in TB O solution (0.05% with 0.01% TWEEN 20) for 10 min, washed with distilled water, and photographed.

    Article Snippet: Epitope-tagged proteins were immunologically hybridized using anti-MYC (1:2,000 dilution; Millipore) and anti-GFP (1:8,000 dilution; Clontech) antibodies.

    Techniques: Transgenic Assay, Permeability, Staining, Stability Assay, Mass Spectrometry

    Immunoblot Analysis and Co-IP Assay of CER3 Associated with SAGL1. (A) and (B) MYC : CER3 and SAGL1 : eYFP (A) or mutated SAGL1ΔF : eYFP with a deleted F-box domain region (B) under the control of the CaMV 35S promoter were transiently coexpressed in N . benthamiana leaves. MG132 (100 μM) was used to inhibit the degradation of proteins by the 26S proteasome system. Anti-MYC or anti-GFP antibodies were used for immunoblot analysis to detect MYC:CER3 or SAGL1/SAGL1ΔF:eYFP. Equal protein loading was assured using Ponceau-S staining. (C) Co-IP assays to detect MYC:CER3 and SAGL1:eYFP interactions. Total protein extracts were obtained from N . benthamiana leaves infiltrated with Agrobacterium suspensions harboring the MYC:CER3 or SAGL1:eYFP construct. Input: Total protein extracts were subjected to immunoblot analysis using anti-GFP or anti-MYC antibodies. IP: α-MYC. Total protein extracts were immunoprecipitated with anti-MYC agarose and subjected to immunoblot analysis using anti-GFP or anti-MYC antibodies. (D) Ubiquitination assay of MYC:CER3. SAGL1:eYFP and MYC:CER3 were transiently coexpressed in N . benthamiana leaves. MG132 (100 μM) was infiltrated into the leaves to inhibit 26S proteasome activity. Total protein extracts obtained from N . benthamiana leaves were immunoprecipitated using anti-MYC conjugated to agarose beads and subjected to immunoblot analysis using anti-MYC or anti-Ub antibodies. WB, protein gel blotting (immunoblot analysis). Ub(n), poly-ubiquitination. (E) MYC:CER3 protein and transcripts levels in 14-d–old wild-type (C24) and sagl1 - 1 seedlings overexpressing MYC : CER3 under the control of the CaMV 35S promoter. MYC:CER3 protein levels were determined in total protein extracts by immunoblot analysis using anti-MYC antibodies. Equal protein loading was assured using Ponceau-S staining. MYC : CER3 transcript levels were determined by RT-PCR. PP2A was used as an endogenous reference gene.

    Journal: The Plant Cell

    Article Title: The F-Box Protein SAGL1 and ECERIFERUM3 Regulate Cuticular Wax Biosynthesis in Response to Changes in Humidity in Arabidopsis [OPEN]

    doi: 10.1105/tpc.19.00152

    Figure Lengend Snippet: Immunoblot Analysis and Co-IP Assay of CER3 Associated with SAGL1. (A) and (B) MYC : CER3 and SAGL1 : eYFP (A) or mutated SAGL1ΔF : eYFP with a deleted F-box domain region (B) under the control of the CaMV 35S promoter were transiently coexpressed in N . benthamiana leaves. MG132 (100 μM) was used to inhibit the degradation of proteins by the 26S proteasome system. Anti-MYC or anti-GFP antibodies were used for immunoblot analysis to detect MYC:CER3 or SAGL1/SAGL1ΔF:eYFP. Equal protein loading was assured using Ponceau-S staining. (C) Co-IP assays to detect MYC:CER3 and SAGL1:eYFP interactions. Total protein extracts were obtained from N . benthamiana leaves infiltrated with Agrobacterium suspensions harboring the MYC:CER3 or SAGL1:eYFP construct. Input: Total protein extracts were subjected to immunoblot analysis using anti-GFP or anti-MYC antibodies. IP: α-MYC. Total protein extracts were immunoprecipitated with anti-MYC agarose and subjected to immunoblot analysis using anti-GFP or anti-MYC antibodies. (D) Ubiquitination assay of MYC:CER3. SAGL1:eYFP and MYC:CER3 were transiently coexpressed in N . benthamiana leaves. MG132 (100 μM) was infiltrated into the leaves to inhibit 26S proteasome activity. Total protein extracts obtained from N . benthamiana leaves were immunoprecipitated using anti-MYC conjugated to agarose beads and subjected to immunoblot analysis using anti-MYC or anti-Ub antibodies. WB, protein gel blotting (immunoblot analysis). Ub(n), poly-ubiquitination. (E) MYC:CER3 protein and transcripts levels in 14-d–old wild-type (C24) and sagl1 - 1 seedlings overexpressing MYC : CER3 under the control of the CaMV 35S promoter. MYC:CER3 protein levels were determined in total protein extracts by immunoblot analysis using anti-MYC antibodies. Equal protein loading was assured using Ponceau-S staining. MYC : CER3 transcript levels were determined by RT-PCR. PP2A was used as an endogenous reference gene.

    Article Snippet: Epitope-tagged proteins were immunologically hybridized using anti-MYC (1:2,000 dilution; Millipore) and anti-GFP (1:8,000 dilution; Clontech) antibodies.

    Techniques: Co-Immunoprecipitation Assay, Staining, Construct, Immunoprecipitation, Ubiquitin Assay, Activity Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction

    Expression of COL7 mRNA and protein. (A) COL7 mRNA expression in response to high R:FR for the indicated period (15, 30, 60, 120, and 240min). Seedlings were grown in white light for 3 d, moved to low F:FR for 4 d, and then transferred to high R:FR (white light, R:FR ratio of 1.2). (B) COL7 mRNA expression in response to low R:FR. Seedlings were incubated in white light for 7 d and then transferred to low R:FR. Error bars represent the standard deviations of three independent replicates. (C, D) Representative immunoblot showing the level of COL7 protein in the 35S::MYC-COL7 line in response to red light (C) or far-red (D) light. Seedlings of the 35S::MYC-COL7 line were grown on MS plates for 5 d in white light followed by 3 d in the dark and then transferred to far-red light or red light. Immunoblots were probed with the anti-MYC antibody (MYC-COL7), stripped, and then probed with the anti-CRY1 antibody (CRY1). (G, H) The curves indicate the relative abundance of MYC-COL7 protein in samples in response to a period of red light (G) or far-red (H) light treatment, which was calculated by the formula (MYC-COL7/CRY1 [t n ])/(MYC-COL7/CRY1 [t 0 ]). The relative abundance of MYC-COL7 protein in the dark was set to 1. (E, F, I, J) COL7 protein level in the 35S::MYC-COL7 line in response to high R:FR (E, I) or low F:FR (F, J). The 35S::MYC-COL7 line was treated as mentioned in (A) and (B). ‘H’ represents high R:FR; ‘L’ represents low F:FR; ‘R’ represents red light; ‘FR’ represents far-red light; and ‘D’ represents dark. Similar results were obtained from three independent biological replicates.

    Journal: Journal of Experimental Botany

    Article Title: CONSTANS-LIKE 7 regulates branching and shade avoidance response in Arabidopsis

    doi: 10.1093/jxb/ers376

    Figure Lengend Snippet: Expression of COL7 mRNA and protein. (A) COL7 mRNA expression in response to high R:FR for the indicated period (15, 30, 60, 120, and 240min). Seedlings were grown in white light for 3 d, moved to low F:FR for 4 d, and then transferred to high R:FR (white light, R:FR ratio of 1.2). (B) COL7 mRNA expression in response to low R:FR. Seedlings were incubated in white light for 7 d and then transferred to low R:FR. Error bars represent the standard deviations of three independent replicates. (C, D) Representative immunoblot showing the level of COL7 protein in the 35S::MYC-COL7 line in response to red light (C) or far-red (D) light. Seedlings of the 35S::MYC-COL7 line were grown on MS plates for 5 d in white light followed by 3 d in the dark and then transferred to far-red light or red light. Immunoblots were probed with the anti-MYC antibody (MYC-COL7), stripped, and then probed with the anti-CRY1 antibody (CRY1). (G, H) The curves indicate the relative abundance of MYC-COL7 protein in samples in response to a period of red light (G) or far-red (H) light treatment, which was calculated by the formula (MYC-COL7/CRY1 [t n ])/(MYC-COL7/CRY1 [t 0 ]). The relative abundance of MYC-COL7 protein in the dark was set to 1. (E, F, I, J) COL7 protein level in the 35S::MYC-COL7 line in response to high R:FR (E, I) or low F:FR (F, J). The 35S::MYC-COL7 line was treated as mentioned in (A) and (B). ‘H’ represents high R:FR; ‘L’ represents low F:FR; ‘R’ represents red light; ‘FR’ represents far-red light; and ‘D’ represents dark. Similar results were obtained from three independent biological replicates.

    Article Snippet: Samples were harvested, frozen in liquid nitrogen, and ground in 4× SDS protein extraction buffer for subsequent SDS–PAGE and immunoblot analysis probed with anti-MYC antibody (Millipore, Cat. #05-724).

    Techniques: Expressing, Incubation, Mass Spectrometry, Western Blot

    The PIAS1-SnoN sumoylation pathway antagonizes TGFβ-suppression of E-cadherin promoter activity. A) SUMO loss of function SnoN associates with PIAS1. Lysates of 293T cells expressing PIAS1, SnoN (WT) or SUMO loss of function SnoN (EdA) mutant, alone or together were subjected to SnoN immunoprecipitation (α-FLAG IP) followed by sequential PIAS1 (α-MYC) and SnoN (α-SnoN) immunoblotting. Expression of PIAS1, SnoN, and actin in lysates were confirmed by MYC, SnoN, and actin immunoblotting. B) The SUMO loss of function SnoN mutant inhibits PIAS1 to block TGFβ-reduction of E-cadherin transcription. NMuMG cells stably expressing SnoN (WT) or SnoN (EdA) or stably transfected with the vector control plasmid (−) were transiently transfected with the E-cadherin-p-luciferase reporter and the β-gal plasmid together with an empty expression vector (−) or one encoding the PIAS1 protein (+) were subjected to luciferase and β-galactosidase assays (see MATERIALS and METHODS ). Each column is the mean (± SEM, n = 6) of percent reduction of E-cadherin-p-luciferase activity by TGFβ. C) The SUMO ligase mutant PIAS1 interacts with SnoN. Lysates of 293T cells expressing SnoN, PIAS1 (WT) or SUMO E3 ligase mutant PIAS1 (CS), alone or together were subjected to PIAS1 immunoprecipitation (α-MYC IP) followed by SnoN (α-SnoN) and PIAS1 (α-MYC) immunoblotting. Total lysates were immunoblotted as described in 7A. D) SUMO E3 ligase mutant PIAS1 reverses the effect of SnoN to inhibit TGFβ-repression of E-cadherin promoter activity. NMuMG cells stably expressing PIAS1 (WT) or PIAS1 (CS), or stably transfected with the vector control plasmid (−) were transiently transfected with the E-cadherin-p-luciferase reporter and the β-gal plasmid together with an empty expression vector (−) or one encoding the SnoN protein (+) were subjected to luciferase and β-galactosidase assays. Data from 6 independent experiments are presented as outlined in Figure 7B. * indicates statistical significant difference (P

    Journal: PLoS ONE

    Article Title: Suppression of TGF?-Induced Epithelial-Mesenchymal Transition Like Phenotype by a PIAS1 Regulated Sumoylation Pathway in NMuMG Epithelial Cells

    doi: 10.1371/journal.pone.0013971

    Figure Lengend Snippet: The PIAS1-SnoN sumoylation pathway antagonizes TGFβ-suppression of E-cadherin promoter activity. A) SUMO loss of function SnoN associates with PIAS1. Lysates of 293T cells expressing PIAS1, SnoN (WT) or SUMO loss of function SnoN (EdA) mutant, alone or together were subjected to SnoN immunoprecipitation (α-FLAG IP) followed by sequential PIAS1 (α-MYC) and SnoN (α-SnoN) immunoblotting. Expression of PIAS1, SnoN, and actin in lysates were confirmed by MYC, SnoN, and actin immunoblotting. B) The SUMO loss of function SnoN mutant inhibits PIAS1 to block TGFβ-reduction of E-cadherin transcription. NMuMG cells stably expressing SnoN (WT) or SnoN (EdA) or stably transfected with the vector control plasmid (−) were transiently transfected with the E-cadherin-p-luciferase reporter and the β-gal plasmid together with an empty expression vector (−) or one encoding the PIAS1 protein (+) were subjected to luciferase and β-galactosidase assays (see MATERIALS and METHODS ). Each column is the mean (± SEM, n = 6) of percent reduction of E-cadherin-p-luciferase activity by TGFβ. C) The SUMO ligase mutant PIAS1 interacts with SnoN. Lysates of 293T cells expressing SnoN, PIAS1 (WT) or SUMO E3 ligase mutant PIAS1 (CS), alone or together were subjected to PIAS1 immunoprecipitation (α-MYC IP) followed by SnoN (α-SnoN) and PIAS1 (α-MYC) immunoblotting. Total lysates were immunoblotted as described in 7A. D) SUMO E3 ligase mutant PIAS1 reverses the effect of SnoN to inhibit TGFβ-repression of E-cadherin promoter activity. NMuMG cells stably expressing PIAS1 (WT) or PIAS1 (CS), or stably transfected with the vector control plasmid (−) were transiently transfected with the E-cadherin-p-luciferase reporter and the β-gal plasmid together with an empty expression vector (−) or one encoding the SnoN protein (+) were subjected to luciferase and β-galactosidase assays. Data from 6 independent experiments are presented as outlined in Figure 7B. * indicates statistical significant difference (P

    Article Snippet: The protein contents of total cell lysate and immunoprecipitation samples were resolved by SDS-PAGE followed by immunoblotting using rabbit anti-SnoN, rabbit-anti ubiquitin (FL-76; Santa Cruz), mouse anti-E-cadherin (BD Transduction Laboratories), rabbit anti-PIAS1 (Epitomics), mouse anti-Smad2/3 (BD, Transduction Laboratories), rabbit anti-phospho Smad2 (Calbiochem), mouse anti-FLAG, mouse anti-MYC, and rabbit anti-actin (Sigma) as primary antibodies and horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody (Amersham Biosciences) as the secondary antibodies .

    Techniques: Activity Assay, Expressing, Mutagenesis, Immunoprecipitation, Blocking Assay, Stable Transfection, Transfection, Plasmid Preparation, Luciferase

    TMEM192 siRNAs alleviated TIG1A-induced expression of autophagy-related proteins. HtTA cells plated in 6-well dishes were transfected with 0.5 μg of TIG1B-myc (A) or TMEM192-Flag (B) expression vector along with 30 nM TIG1, TMEM192, or NC siRNA for 48 h. Alternatively, cells were transfected with 0.5 μg of TIG1A-myc (C–E) expression vector along with the indicated siRNA (30 nM) for 48 h and were then cultured in serum-free medium for 6 h. Cell lysates were prepared, and the expression of TIG1, TMEM192, Beclin-1, or LC3B was determined using anti-MYC, anti-FLAG, anti-Beclin-1, or anti-LC3B antibodies, respectively. Experimental results are summarized as the mean percentage (± SD) of the ratio of LC3B-II to LC3B-I and the level of Beclin-1 with each sample normalized to the level of actin protein in two independent experiments (D, E). HtTA cells plated in 6-well dishes were transfected with 0.5 μg of TIG1A-myc expression vectors along with the indicated siRNA for 48 h and were then cultured in serum-free medium for 6 h. Cell lysates were prepared, and the level of Beclin-1 was detected using an enzyme immunoassay. Representative results of three independent experiments are shown. HtTA cells plated in triplicate in 24-well plates were transfected with 75 ng of TIG1A-myc expression vector, 150 ng pGFP-LC3 expression vector and with the indicated 30 nM siRNA for 48 h and were then cultured in serum-free medium for 6 h. Representative images with GFP-LC3 puncta formation (G). Bar chart indicating the percentage of cells with GFP-LC3 puncta formation from three independent experiments (H). Scale bar, 10 μm. *Indicates p value

    Journal: Molecules and Cells

    Article Title: Tazarotene-Induced Gene 1 Enhanced Cervical Cell Autophagy through Transmembrane Protein 192

    doi: 10.14348/molcells.2016.0161

    Figure Lengend Snippet: TMEM192 siRNAs alleviated TIG1A-induced expression of autophagy-related proteins. HtTA cells plated in 6-well dishes were transfected with 0.5 μg of TIG1B-myc (A) or TMEM192-Flag (B) expression vector along with 30 nM TIG1, TMEM192, or NC siRNA for 48 h. Alternatively, cells were transfected with 0.5 μg of TIG1A-myc (C–E) expression vector along with the indicated siRNA (30 nM) for 48 h and were then cultured in serum-free medium for 6 h. Cell lysates were prepared, and the expression of TIG1, TMEM192, Beclin-1, or LC3B was determined using anti-MYC, anti-FLAG, anti-Beclin-1, or anti-LC3B antibodies, respectively. Experimental results are summarized as the mean percentage (± SD) of the ratio of LC3B-II to LC3B-I and the level of Beclin-1 with each sample normalized to the level of actin protein in two independent experiments (D, E). HtTA cells plated in 6-well dishes were transfected with 0.5 μg of TIG1A-myc expression vectors along with the indicated siRNA for 48 h and were then cultured in serum-free medium for 6 h. Cell lysates were prepared, and the level of Beclin-1 was detected using an enzyme immunoassay. Representative results of three independent experiments are shown. HtTA cells plated in triplicate in 24-well plates were transfected with 75 ng of TIG1A-myc expression vector, 150 ng pGFP-LC3 expression vector and with the indicated 30 nM siRNA for 48 h and were then cultured in serum-free medium for 6 h. Representative images with GFP-LC3 puncta formation (G). Bar chart indicating the percentage of cells with GFP-LC3 puncta formation from three independent experiments (H). Scale bar, 10 μm. *Indicates p value

    Article Snippet: After the membranes were blocked, they were incubated for 12 h at 4°C with anti-MYC, anti-FLAG, anti-TIG1 (Santa Cruz Biotechnology), anti-TMEM192 (Santa Cruz Biotechnology), anti-LC3B (Cell Signaling Technology, USA), anti-SQSTM1/p62 (Cell Signaling Technology), anti-Beclin-1 (Cell Signaling Technology), or antiactin (Sigma) antibody and then incubated with horseradish peroxidase-conjugated goat anti-mouse antibody at room temperature for 1 h. An ECL kit (Amersham, UK) was used to detect the substrate reaction.

    Techniques: Expressing, Transfection, Plasmid Preparation, Cell Culture, Enzyme-linked Immunosorbent Assay

    TIG1A induced the expression of autophagy-related proteins. HtTA cells plated in a 6-cm dish were transfected with 1.5 μg of TMEM192-Flag expression vector along with 1.5 μg of TIG1A-myc expression vector for 24 h. Cells were refreshed in medium without serum and treated with 200 nM rapamycin for 6 h. Cell lysates were prepared, and the levels of LC-3B, Beclin-1, SQSTM1/p62, TIG1A, and actin were determined by immunoblotting (A). Experimental results are summarized as the mean percentage (± SD) of the ratio of LC3B-II to LC3B-I and the level of Beclin-1 with each sample normalized to the level of actin protein in two independent experiments (B, C). HtTA cells plated in 6-well dishes were transfected with 0.5 μg of the indicated vectors or the control vector for 24 h and were then cultured in serum-free medium with 200 nM rapamycin for 6 h. Cell lysates were prepared, and the level of Beclin-1 was detected using an enzyme immunoassay. Representative results of three independent experiments are shown (D). HtTA cells plated in triplicate in 24-well plates were transfected with 75 ng of TIG1A-myc expression vector, 75 ng of TMEM192-Flag expression vector and 150 ng of pGFP-LC3 expression vector for 24 h. Cells were refreshed in medium without serum and treated with 400 nM rapamycin for 6 h. Representative images with GFP-LC3 puncta formation (E). Bar chart indicating the percentage of cells with GFP-LC3 puncta formation from three independent experiments (F). Scale bar, 10 μm. *Indicates p value

    Journal: Molecules and Cells

    Article Title: Tazarotene-Induced Gene 1 Enhanced Cervical Cell Autophagy through Transmembrane Protein 192

    doi: 10.14348/molcells.2016.0161

    Figure Lengend Snippet: TIG1A induced the expression of autophagy-related proteins. HtTA cells plated in a 6-cm dish were transfected with 1.5 μg of TMEM192-Flag expression vector along with 1.5 μg of TIG1A-myc expression vector for 24 h. Cells were refreshed in medium without serum and treated with 200 nM rapamycin for 6 h. Cell lysates were prepared, and the levels of LC-3B, Beclin-1, SQSTM1/p62, TIG1A, and actin were determined by immunoblotting (A). Experimental results are summarized as the mean percentage (± SD) of the ratio of LC3B-II to LC3B-I and the level of Beclin-1 with each sample normalized to the level of actin protein in two independent experiments (B, C). HtTA cells plated in 6-well dishes were transfected with 0.5 μg of the indicated vectors or the control vector for 24 h and were then cultured in serum-free medium with 200 nM rapamycin for 6 h. Cell lysates were prepared, and the level of Beclin-1 was detected using an enzyme immunoassay. Representative results of three independent experiments are shown (D). HtTA cells plated in triplicate in 24-well plates were transfected with 75 ng of TIG1A-myc expression vector, 75 ng of TMEM192-Flag expression vector and 150 ng of pGFP-LC3 expression vector for 24 h. Cells were refreshed in medium without serum and treated with 400 nM rapamycin for 6 h. Representative images with GFP-LC3 puncta formation (E). Bar chart indicating the percentage of cells with GFP-LC3 puncta formation from three independent experiments (F). Scale bar, 10 μm. *Indicates p value

    Article Snippet: After the membranes were blocked, they were incubated for 12 h at 4°C with anti-MYC, anti-FLAG, anti-TIG1 (Santa Cruz Biotechnology), anti-TMEM192 (Santa Cruz Biotechnology), anti-LC3B (Cell Signaling Technology, USA), anti-SQSTM1/p62 (Cell Signaling Technology), anti-Beclin-1 (Cell Signaling Technology), or antiactin (Sigma) antibody and then incubated with horseradish peroxidase-conjugated goat anti-mouse antibody at room temperature for 1 h. An ECL kit (Amersham, UK) was used to detect the substrate reaction.

    Techniques: Expressing, Transfection, Plasmid Preparation, Cell Culture, Enzyme-linked Immunosorbent Assay

    TIG1 interacts and co-localizes with TMEM192. HtTA cells plated in a 10-cm dish were transfected with 3 μg of TMEM192-Flag expression vector along with the TIG1A-myc or TIG1B-myc expression vector for 24 h. Cell lysates were prepared, and the interaction between TIG1 and TMEM192 was analyzed by immunoprecipitation followed by Western blot analysis. Immunoprecipitates were resolved by SDS-PAGE and immunoblotted using the anti-MYC antibody (A) or anti- FLAG antibody (B). Total cellular extracts from HtTA cells were subjected to Western blot analysis for TIG1, TMEM192, and actin. HtTA cells were co-transfected with EGFP-TMEM192 along with the TIG1A-myc or TIG1B-myc expression vectors for 18 h. The cells were fixed and then incubated with anti-MYC and anti-LAMP1 (lysosomal marker) antibodies followed by Alexa Fluor 633 goat anti-mouse IgG and Alexa Fluor 405 goat anti-rabbit IgG antibodies. The cells were then analyzed with a laser scanning confocal microscope. Scale bar: 10 μm. The localization of TIG1 (red), TMEM192 (green), and lysosomes (blue) was analyzed using a laser scanning confocal microscope. Arrows indicate co-localization of TIG1 and TMEM192 (C). Scale bar, 10 μm.

    Journal: Molecules and Cells

    Article Title: Tazarotene-Induced Gene 1 Enhanced Cervical Cell Autophagy through Transmembrane Protein 192

    doi: 10.14348/molcells.2016.0161

    Figure Lengend Snippet: TIG1 interacts and co-localizes with TMEM192. HtTA cells plated in a 10-cm dish were transfected with 3 μg of TMEM192-Flag expression vector along with the TIG1A-myc or TIG1B-myc expression vector for 24 h. Cell lysates were prepared, and the interaction between TIG1 and TMEM192 was analyzed by immunoprecipitation followed by Western blot analysis. Immunoprecipitates were resolved by SDS-PAGE and immunoblotted using the anti-MYC antibody (A) or anti- FLAG antibody (B). Total cellular extracts from HtTA cells were subjected to Western blot analysis for TIG1, TMEM192, and actin. HtTA cells were co-transfected with EGFP-TMEM192 along with the TIG1A-myc or TIG1B-myc expression vectors for 18 h. The cells were fixed and then incubated with anti-MYC and anti-LAMP1 (lysosomal marker) antibodies followed by Alexa Fluor 633 goat anti-mouse IgG and Alexa Fluor 405 goat anti-rabbit IgG antibodies. The cells were then analyzed with a laser scanning confocal microscope. Scale bar: 10 μm. The localization of TIG1 (red), TMEM192 (green), and lysosomes (blue) was analyzed using a laser scanning confocal microscope. Arrows indicate co-localization of TIG1 and TMEM192 (C). Scale bar, 10 μm.

    Article Snippet: After the membranes were blocked, they were incubated for 12 h at 4°C with anti-MYC, anti-FLAG, anti-TIG1 (Santa Cruz Biotechnology), anti-TMEM192 (Santa Cruz Biotechnology), anti-LC3B (Cell Signaling Technology, USA), anti-SQSTM1/p62 (Cell Signaling Technology), anti-Beclin-1 (Cell Signaling Technology), or antiactin (Sigma) antibody and then incubated with horseradish peroxidase-conjugated goat anti-mouse antibody at room temperature for 1 h. An ECL kit (Amersham, UK) was used to detect the substrate reaction.

    Techniques: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, SDS Page, Incubation, Marker, Microscopy

    Effects of TIG1 on expression of TMEM192 in HtTA cells. HtTA cells plated in 6-well dishes were transfected with 0.5 μg of TIG1A-myc (A) or TMEM192-flag (B) expression vector for 24 h. Total RNA was extracted and relative levels of the indicated mRNAs were measured by real-time RT-PCR. HtTA cells plated in a 6-cm dish were transfected with 0.5 μg of TMEM192-Flag expression vector along with 0.5–1.5 μg of TIG1A-myc (C) or TIG1B-myc (D) expression vector for 12 h. Alternatively, cells were transfected with 0.5–1.5 μg of TMEM192-Flag expression vector along with 0.5 μg of TIG1A-myc (E) or TIG1B-myc (F) expression vector for 12 h. Cell lysates were prepared, and the levels of TIG1A, TIG1B, and actin were determined by immunoblotting.

    Journal: Molecules and Cells

    Article Title: Tazarotene-Induced Gene 1 Enhanced Cervical Cell Autophagy through Transmembrane Protein 192

    doi: 10.14348/molcells.2016.0161

    Figure Lengend Snippet: Effects of TIG1 on expression of TMEM192 in HtTA cells. HtTA cells plated in 6-well dishes were transfected with 0.5 μg of TIG1A-myc (A) or TMEM192-flag (B) expression vector for 24 h. Total RNA was extracted and relative levels of the indicated mRNAs were measured by real-time RT-PCR. HtTA cells plated in a 6-cm dish were transfected with 0.5 μg of TMEM192-Flag expression vector along with 0.5–1.5 μg of TIG1A-myc (C) or TIG1B-myc (D) expression vector for 12 h. Alternatively, cells were transfected with 0.5–1.5 μg of TMEM192-Flag expression vector along with 0.5 μg of TIG1A-myc (E) or TIG1B-myc (F) expression vector for 12 h. Cell lysates were prepared, and the levels of TIG1A, TIG1B, and actin were determined by immunoblotting.

    Article Snippet: After the membranes were blocked, they were incubated for 12 h at 4°C with anti-MYC, anti-FLAG, anti-TIG1 (Santa Cruz Biotechnology), anti-TMEM192 (Santa Cruz Biotechnology), anti-LC3B (Cell Signaling Technology, USA), anti-SQSTM1/p62 (Cell Signaling Technology), anti-Beclin-1 (Cell Signaling Technology), or antiactin (Sigma) antibody and then incubated with horseradish peroxidase-conjugated goat anti-mouse antibody at room temperature for 1 h. An ECL kit (Amersham, UK) was used to detect the substrate reaction.

    Techniques: Expressing, Transfection, Plasmid Preparation, Quantitative RT-PCR

    miR675 Triggers MSI and Abnormal Gene Expression in the Human Mesenchymal Stem Cells Infected with rLV and rLV-miR675, Respectively (A) Microsatellite instability (MSI) analysis through dot blot (slot blot) using various biotin-labeling MSI probes (biotin-MSIs). (B) (a) Chromosome conformation capture (3C)-chromatin immunoprecipitation (ChIP) with anti-P300 and anti-Pol II. The chromatin was cross-linked, digested with restriction enzymes, and ligated under conditions that favor intramolecular ligation. Immediately after ligation, the chromatin was immunoprecipitated using an antibody (anti-P300, anti-Pol II) against the protein of interest. Thereafter, the cross-links were reversed and the DNA was purified further. The PCR anlysis was applied for detecting CyclinD1 promoter-enhancer coupling product using CyclinD1 promoter and enhancer primers. The CyclinD1 promoter and enhancer was the INPUT. (b) The quantitative analysis of ChIP-3C. (C) (a) Western blotting with anti-Rad51, anti-CDK2, anti-CyclinE, anti-CDK4, anti-CyclinD1, anti-PCNA, anti-ppRB, anti-E2F1, anti-P18, anti-P21, anti-PKM2, anti-c-Myc, and anti-Chk1. β-actin was the internal control. (b) The gray scan analysis of positive bands of western blotting.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: miR675 Accelerates Malignant Transformation of Mesenchymal Stem Cells by Blocking DNA Mismatch Repair

    doi: 10.1016/j.omtn.2018.11.010

    Figure Lengend Snippet: miR675 Triggers MSI and Abnormal Gene Expression in the Human Mesenchymal Stem Cells Infected with rLV and rLV-miR675, Respectively (A) Microsatellite instability (MSI) analysis through dot blot (slot blot) using various biotin-labeling MSI probes (biotin-MSIs). (B) (a) Chromosome conformation capture (3C)-chromatin immunoprecipitation (ChIP) with anti-P300 and anti-Pol II. The chromatin was cross-linked, digested with restriction enzymes, and ligated under conditions that favor intramolecular ligation. Immediately after ligation, the chromatin was immunoprecipitated using an antibody (anti-P300, anti-Pol II) against the protein of interest. Thereafter, the cross-links were reversed and the DNA was purified further. The PCR anlysis was applied for detecting CyclinD1 promoter-enhancer coupling product using CyclinD1 promoter and enhancer primers. The CyclinD1 promoter and enhancer was the INPUT. (b) The quantitative analysis of ChIP-3C. (C) (a) Western blotting with anti-Rad51, anti-CDK2, anti-CyclinE, anti-CDK4, anti-CyclinD1, anti-PCNA, anti-ppRB, anti-E2F1, anti-P18, anti-P21, anti-PKM2, anti-c-Myc, and anti-Chk1. β-actin was the internal control. (b) The gray scan analysis of positive bands of western blotting.

    Article Snippet: Antibodies and Primers Standard western immunoblotting procedures were used with the following antibodies: anti-H3K36me1 (Abcam), anti-H3K36me2 (Abcam), anti-H3K36me3 (Abcam), anti-SETD2 (Santa Cruz Biotechnology), anti-hMSH6 (Santa Cruz Biotechnology), anti-histone 3.3, anti-histone 3, anti-SKP2 (Santa Cruz Biotechnology), anti-biotin (Santa Cruz Biotechnology), anti-Ub (Santa Cruz Biotechnology), anti-P300 (Abcam), anti-RNA Pol II (Abcam), anti-CyclinE (Santa Cruz Biotechnology), anti-CDK4 (Santa Cruz Biotechnology), anti-CyclinD1 (Abcam), anti-PCNA (Abcam), anti-ppRB (Abcam), anti-E2F1 (Abcam), anti-P18 (Abcam), anti-P21/WAF1/Cip1 (Santa Cruz Biotechnology), anti-PKM2, anti-c-Myc (Santa Cruz Biotechnology), anti-Chk1 (Abcam), anti-P62 (Abcam), anti-KDM4A (Abcam), and anti-β-actin (Abcam).

    Techniques: Expressing, Infection, Dot Blot, Labeling, Chromatin Immunoprecipitation, Ligation, Immunoprecipitation, Purification, Polymerase Chain Reaction, Western Blot

    WDR 11 interacts with GLI ‐binding sites in PTCH 1 promoter Schematic diagram showing human PTCH1 gene locus. The filled boxes indicate the positions of different isoforms of exon 1. Two functional GLI‐binding sites (GliBS‐A and GliBS‐B) are shown with the consensus binding sequences underlined. ChIP assay was performed in HEK293 transfected with 6xMyc‐tagged WDR11 or empty vector, using anti‐Myc antibody or IgG. The qPCR amplification results of the putative GLI‐binding sites shown in (A) are shown as % of input (a portion of the sonicated chromatin template before immunoprecipitation). Data are presented as mean±SEM from three independent experiments. Two‐way ANOVA followed by Bonferroni's multiple comparison indicated that Myc‐WDR11 protein complex was present at both GliBS‐A (*** P = 0.0002) and GliBS‐B (* P = 0.042) sites at significantly higher levels compared to the empty vector‐transfected or IgG‐immunoprecipitated. Co‐IP Western blot confirmed the presence of endogenous GLI3FL and GLI3R in the Myc‐WDR11 immune complex. Agarose gel images showing the PCR amplification products of each target sequence from the input and ChIP samples. Source data are available online for this figure.

    Journal: EMBO Reports

    Article Title: WDR11‐mediated Hedgehog signalling defects underlie a new ciliopathy related to Kallmann syndrome

    doi: 10.15252/embr.201744632

    Figure Lengend Snippet: WDR 11 interacts with GLI ‐binding sites in PTCH 1 promoter Schematic diagram showing human PTCH1 gene locus. The filled boxes indicate the positions of different isoforms of exon 1. Two functional GLI‐binding sites (GliBS‐A and GliBS‐B) are shown with the consensus binding sequences underlined. ChIP assay was performed in HEK293 transfected with 6xMyc‐tagged WDR11 or empty vector, using anti‐Myc antibody or IgG. The qPCR amplification results of the putative GLI‐binding sites shown in (A) are shown as % of input (a portion of the sonicated chromatin template before immunoprecipitation). Data are presented as mean±SEM from three independent experiments. Two‐way ANOVA followed by Bonferroni's multiple comparison indicated that Myc‐WDR11 protein complex was present at both GliBS‐A (*** P = 0.0002) and GliBS‐B (* P = 0.042) sites at significantly higher levels compared to the empty vector‐transfected or IgG‐immunoprecipitated. Co‐IP Western blot confirmed the presence of endogenous GLI3FL and GLI3R in the Myc‐WDR11 immune complex. Agarose gel images showing the PCR amplification products of each target sequence from the input and ChIP samples. Source data are available online for this figure.

    Article Snippet: The pre‐cleared lysate was incubated with anti‐Myc antibody or mouse IgG with protein A/G‐agarose beads (Santa Cruz Biotechnology) overnight at 4°C.

    Techniques: Binding Assay, Functional Assay, Chromatin Immunoprecipitation, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Amplification, Sonication, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Sequencing

    WDR 11 forms a tertiary complex with EMX 1 and GLI 3 Co‐immunoprecipitation (co‐IP) analyses of HEK293 cells co‐transfected with 6xMyc‐tagged WDR11 and HA‐tagged EMX1 expression constructs either alone or together with GLI3 full‐length (GLI3FL) expression construct, demonstrating the formation of WDR11‐EMX1‐GLI3FL tertiary complex (left panel). In a reciprocal co‐IP experiment, 6xMyc‐tagged GLI3R was co‐transfected with HA‐tagged EMX1 either alone or together with untagged WDR11 expression construct to demonstrate the formation of GLI3R‐EMX1‐WDR11 complex. The disease‐associated mutant WDR11 showed a reduced binding capacity to EMX1 within this complex (right panel). Co‐IP analyses of mouse testes tissue lysates showed that endogenous WDR11 could bind to endogenous GLI3FL (190 kDa), GLI3R (83 kDa) and EMX1/2 (35 kDa) proteins. Tissue lysates were precipitated with anti‐WDR11 antibody or non‐immune IgG, and probed with antibodies against GLI3, WDR11 or EMX1/2. 6xMyc‐GLI3R and HA‐EMX1 were co‐transfected in WT or Wdr11 −/− MEFs and precipitated with anti‐Myc antibody and probed with EMX1 and GLI3 antibodies. EMX1‐GLI3 complex did not form in Wdr11 −/− MEFs, indicating that EMX1‐GLI3 failed to bind in the absence of WDR11. The relative expression ratios of endogenous GLI3FL/R normalized to the loading control (β‐actin) were assessed by Western blot in HEK293 cells after WDR11 overexpression (left panel), in MEFs of WT and Wdr11 −/− embryos (middle panel) and in tissue lysates of WT and Wdr11 −/− kidney (right panel). Band intensities of GLI3FL and GLI3R were quantified by ImageJ. Source data are available online for this figure.

    Journal: EMBO Reports

    Article Title: WDR11‐mediated Hedgehog signalling defects underlie a new ciliopathy related to Kallmann syndrome

    doi: 10.15252/embr.201744632

    Figure Lengend Snippet: WDR 11 forms a tertiary complex with EMX 1 and GLI 3 Co‐immunoprecipitation (co‐IP) analyses of HEK293 cells co‐transfected with 6xMyc‐tagged WDR11 and HA‐tagged EMX1 expression constructs either alone or together with GLI3 full‐length (GLI3FL) expression construct, demonstrating the formation of WDR11‐EMX1‐GLI3FL tertiary complex (left panel). In a reciprocal co‐IP experiment, 6xMyc‐tagged GLI3R was co‐transfected with HA‐tagged EMX1 either alone or together with untagged WDR11 expression construct to demonstrate the formation of GLI3R‐EMX1‐WDR11 complex. The disease‐associated mutant WDR11 showed a reduced binding capacity to EMX1 within this complex (right panel). Co‐IP analyses of mouse testes tissue lysates showed that endogenous WDR11 could bind to endogenous GLI3FL (190 kDa), GLI3R (83 kDa) and EMX1/2 (35 kDa) proteins. Tissue lysates were precipitated with anti‐WDR11 antibody or non‐immune IgG, and probed with antibodies against GLI3, WDR11 or EMX1/2. 6xMyc‐GLI3R and HA‐EMX1 were co‐transfected in WT or Wdr11 −/− MEFs and precipitated with anti‐Myc antibody and probed with EMX1 and GLI3 antibodies. EMX1‐GLI3 complex did not form in Wdr11 −/− MEFs, indicating that EMX1‐GLI3 failed to bind in the absence of WDR11. The relative expression ratios of endogenous GLI3FL/R normalized to the loading control (β‐actin) were assessed by Western blot in HEK293 cells after WDR11 overexpression (left panel), in MEFs of WT and Wdr11 −/− embryos (middle panel) and in tissue lysates of WT and Wdr11 −/− kidney (right panel). Band intensities of GLI3FL and GLI3R were quantified by ImageJ. Source data are available online for this figure.

    Article Snippet: The pre‐cleared lysate was incubated with anti‐Myc antibody or mouse IgG with protein A/G‐agarose beads (Santa Cruz Biotechnology) overnight at 4°C.

    Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Expressing, Construct, Mutagenesis, Binding Assay, Western Blot, Over Expression

    Effects of HIF ‐1α and c‐Myc on human EP 4 receptor promoter activity in low cellular density‐cultured HCA ‐7 cells. Cells were cultured under low (Low; 2 × 10 5 cells/each well of 6‐well plate), middle (Mid; 6 × 10 5 cells/each well of 6‐well plate), and high (High; 2 × 10 6 cells/each well of 6‐well plate) cellular density conditions in HCA ‐7 cells. (A) The luciferase activities of HCA ‐7 cells cultured under low cellular density conditions and transfected either with WT (−1238/+1), del 4 (−160/+1) or mut‐A,B‐del 3 of EP 4 promoter‐containing reporter gene plasmids concomitantly with either HA ‐control vector plasmids, HA ‐tagged HIF ‐1α expression plasmids, or FLAG ‐tagged c‐Myc expression plasmids. (B) An immunoblot analysis with an antibody against c‐Myc (upper panel) or β ‐tubulin (lower panel), and a histogram representing the ratio of c‐Myc to β ‐tubulin as assessed with pooled densitometric data (mean ± SD ) from three or more than three independent experiments in HCA ‐7 cells cultured under low, middle, or high cellular density conditions. (C, D) The luciferase activities of HCA ‐7 cells cultured under low cellular density conditions transfected with WT (−1238/+1) of EP 4 promoter‐containing reporter gene plasmids concomitantly with either HA ‐control vector plasmids or HA ‐tagged HIF ‐1α expression plasmids alone, or HA ‐tagged HIF ‐1α expression plasmids plus various amounts of FLAG ‐tagged c‐Myc expression plasmids (C); or with either HA ‐control vector plasmids or FLAG ‐tagged c‐Myc expression plasmids alone, or FLAG ‐tagged c‐Myc expression plasmids plus various amounts of HA ‐tagged HIF ‐1α expression plasmids (D). (E) Ch IP assay with anti‐Sp‐1, anti‐ HIF ‐1α and anti‐c‐Myc antibodies performed to verify the binding of Sp‐1, HIF ‐1α and c‐Myc to HRE point mutated WT (mut‐ HRE (−1238/+1)) of the EP 4 promoter in HCA ‐7 cells cultured under low and high cellular density conditions. (F) HCA ‐7 cells were cultured under low (Low; 2 × 10 5 cells/each well of 6‐well plate) or high (High; 2 × 10 6 cells/each well of 6‐well plate) cellular density conditions, and the hypoxia probe solution was then added to the medium in order to assess hypoxic areas, as described in the Materials Methods. Picture panels (A‐F) were from a representative experiment that was repeated three times. Data are normalized to low cellular density‐cultured HCA ‐7 cells transfected with control vector plasmid‐transfected cells (A, C, D), or immunoblot ratio of low cellular density‐cultured cells (B) as 100%. Data are the mean ± SD of three or more than three independent experiments. * P

    Journal: Pharmacology Research & Perspectives

    Article Title: Cellular density‐dependent increases in HIF‐1α compete with c‐Myc to down‐regulate human EP4 receptor promoter activity through Sp‐1‐binding region, et al. Cellular density‐dependent increases in HIF‐1α compete with c‐Myc to down‐regulate human EP4 receptor promoter activity through Sp‐1‐binding region

    doi: 10.1002/prp2.441

    Figure Lengend Snippet: Effects of HIF ‐1α and c‐Myc on human EP 4 receptor promoter activity in low cellular density‐cultured HCA ‐7 cells. Cells were cultured under low (Low; 2 × 10 5 cells/each well of 6‐well plate), middle (Mid; 6 × 10 5 cells/each well of 6‐well plate), and high (High; 2 × 10 6 cells/each well of 6‐well plate) cellular density conditions in HCA ‐7 cells. (A) The luciferase activities of HCA ‐7 cells cultured under low cellular density conditions and transfected either with WT (−1238/+1), del 4 (−160/+1) or mut‐A,B‐del 3 of EP 4 promoter‐containing reporter gene plasmids concomitantly with either HA ‐control vector plasmids, HA ‐tagged HIF ‐1α expression plasmids, or FLAG ‐tagged c‐Myc expression plasmids. (B) An immunoblot analysis with an antibody against c‐Myc (upper panel) or β ‐tubulin (lower panel), and a histogram representing the ratio of c‐Myc to β ‐tubulin as assessed with pooled densitometric data (mean ± SD ) from three or more than three independent experiments in HCA ‐7 cells cultured under low, middle, or high cellular density conditions. (C, D) The luciferase activities of HCA ‐7 cells cultured under low cellular density conditions transfected with WT (−1238/+1) of EP 4 promoter‐containing reporter gene plasmids concomitantly with either HA ‐control vector plasmids or HA ‐tagged HIF ‐1α expression plasmids alone, or HA ‐tagged HIF ‐1α expression plasmids plus various amounts of FLAG ‐tagged c‐Myc expression plasmids (C); or with either HA ‐control vector plasmids or FLAG ‐tagged c‐Myc expression plasmids alone, or FLAG ‐tagged c‐Myc expression plasmids plus various amounts of HA ‐tagged HIF ‐1α expression plasmids (D). (E) Ch IP assay with anti‐Sp‐1, anti‐ HIF ‐1α and anti‐c‐Myc antibodies performed to verify the binding of Sp‐1, HIF ‐1α and c‐Myc to HRE point mutated WT (mut‐ HRE (−1238/+1)) of the EP 4 promoter in HCA ‐7 cells cultured under low and high cellular density conditions. (F) HCA ‐7 cells were cultured under low (Low; 2 × 10 5 cells/each well of 6‐well plate) or high (High; 2 × 10 6 cells/each well of 6‐well plate) cellular density conditions, and the hypoxia probe solution was then added to the medium in order to assess hypoxic areas, as described in the Materials Methods. Picture panels (A‐F) were from a representative experiment that was repeated three times. Data are normalized to low cellular density‐cultured HCA ‐7 cells transfected with control vector plasmid‐transfected cells (A, C, D), or immunoblot ratio of low cellular density‐cultured cells (B) as 100%. Data are the mean ± SD of three or more than three independent experiments. * P

    Article Snippet: Incubations were conducted at 4°C for 16 hours in 5% bovine serum albumin (BSA) (Sigma) containing a 1:1000 dilution of an anti‐human EP4 receptor antibody (101775; Cayman Chemical, Ann Arbor, MI); a 1:1000 dilution of an anti‐Sp1 antibody (sc‐420; Santa Cruz Biotechnology, Santa Cruz, CA); a 1:1000 dilution of an anti‐c‐Myc antibody (sc‐40; Santa Cruz Biotechnology); a 1:1000 dilution of an anti‐HIF‐1α antibody (H1alpha67); or a 1:5000 dilution of an anti‐β ‐tubulin‐antibody (014‐25041; Wako).

    Techniques: Activity Assay, Cell Culture, High Content Screening, Luciferase, Transfection, Plasmid Preparation, Expressing, Binding Assay

    Expression of BCL-2 protein family members in A1 +/+ Eµ -Myc and A1 −/− Eµ -Myc lymphomas and premalignant cells. Western blots were performed on a pro/pre-B cell lymphomas from lymph nodes of sick A1 +/+ Eµ -Myc (lanes 1–4) and A1 − / − Eµ -Myc mice (lanes 5–8) and pre-malignant pro/pre-B cells (MACS-sorted CD19 + bone marrow (BM) samples) from healthy A1 +/+ Eµ -Myc and A1 − / − Eµ -Myc mice euthanased at 28–30 days of age (lanes 9–12); b B cell lymphomas from lymph nodes of A1 +/+ Eµ -Myc (lanes 1–4) and A1 −/− Eµ -Myc mice (lanes 5–8). The same premalignant samples and control lysates as in ( a ) were run to enable comparison between ( a ) and ( b ). Lane labels indicate the individual mouse number. WEHI-231 cells served as a positive control for A1 protein, tumour 1/8094 as a positive control for p53 and p19ARF proteins and β-ACTIN as a loading control. Independent blots are separated by a horizontal black line and molecular weight markers (kD) are indicated. Additional lymphomas were analysed (data not shown), in total: 8 pro/pre-B and 8 B A1 +/+ Eµ -Myc ; 7 pro/pre-B and 6 B A1 −/− Eµ -Myc

    Journal: Cell Death and Differentiation

    Article Title: Anti-apoptotic A1 is not essential for lymphoma development in Eµ-Myc mice but helps sustain transplanted Eµ-Myc tumour cells

    doi: 10.1038/s41418-017-0045-8

    Figure Lengend Snippet: Expression of BCL-2 protein family members in A1 +/+ Eµ -Myc and A1 −/− Eµ -Myc lymphomas and premalignant cells. Western blots were performed on a pro/pre-B cell lymphomas from lymph nodes of sick A1 +/+ Eµ -Myc (lanes 1–4) and A1 − / − Eµ -Myc mice (lanes 5–8) and pre-malignant pro/pre-B cells (MACS-sorted CD19 + bone marrow (BM) samples) from healthy A1 +/+ Eµ -Myc and A1 − / − Eµ -Myc mice euthanased at 28–30 days of age (lanes 9–12); b B cell lymphomas from lymph nodes of A1 +/+ Eµ -Myc (lanes 1–4) and A1 −/− Eµ -Myc mice (lanes 5–8). The same premalignant samples and control lysates as in ( a ) were run to enable comparison between ( a ) and ( b ). Lane labels indicate the individual mouse number. WEHI-231 cells served as a positive control for A1 protein, tumour 1/8094 as a positive control for p53 and p19ARF proteins and β-ACTIN as a loading control. Independent blots are separated by a horizontal black line and molecular weight markers (kD) are indicated. Additional lymphomas were analysed (data not shown), in total: 8 pro/pre-B and 8 B A1 +/+ Eµ -Myc ; 7 pro/pre-B and 6 B A1 −/− Eµ -Myc

    Article Snippet: Membranes were subsequently probed with the following antibodies: A1 (clone 6D6, WEHI mAB lab), BCL-2 (clone 7, BD Biosciences), BCL-XL (clone 44, BD Biosciences), MCL-1 (clone 19C4–15, WEHI mAb lab), PUMA (polyclonal, Abcam), BIM (clone 3C5, WEHI mAb lab), p53 (FL-393, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p19ARF (p19ARF exon 2, Rockland, Gilbertsville, PA, USA), c-MYC (D84C12, Cell Signaling Techology, Danvers, MA, USA) and β-ACTIN (clone AC-74, Sigma-Aldrich).

    Techniques: Expressing, Western Blot, Mouse Assay, Magnetic Cell Separation, Positive Control, Molecular Weight

    MAGE-A4 protects RAD18 from ubiquitin-mediated proteolysis. ( a ) Replicate plates of H1299 cells were transfected with siRNA against MAGE-A4, USP7 or with non-targeting control siRNA. After 48 h, one plate of each replicate was treated with 10 μM MG132 for 16 h. Extracts from control and MG132-treated cells were analysed by immunoblotting with antibodies against the indicated proteins. ( b ) 293T cells were co-transfected with an HA–RAD18 expression vector in combination with a CMV-MAGE-A4 plasmid or an empty vector for control. After 48 h, RAD18 complexes were immunoprecipitated with anti-HA antibodies. The resulting immune complexes were incubated in a rabbit reticulocyte lysate (RRL) to reconstitute ubiquitin-coupled proteolysis in vitro . Relative levels of RAD18 and MAGE-A4 were determined by immunoblotting and quantified using densitometry. ( c ) H1299 cells were transiently co-transfected with WT or mutant HA–RAD18 expression plasmids in combination with a MAGE-A4 expression vector (or empty vector control). Forty eight hours later, cells were harvested for immunoblot analysis of RAD18 and MAGE-A4. The white arrowhead indicates the RAD18Δ340–395 mutant protein band that is insensitive to MAGE-A4. ( d ) Replicate cultures of H1299 cells were transfected with an expression vector encoding MYC–TRIM69 or with an empty vector plasmid for control. Sixteen hours later, the cells were transfected with siRNA against MAGE-A4 or with a scrambled control siRNA and incubated for an additional 48 h before immunoblot analysis.

    Journal: Nature Communications

    Article Title: A neomorphic cancer cell-specific role of MAGE-A4 in trans-lesion synthesis

    doi: 10.1038/ncomms12105

    Figure Lengend Snippet: MAGE-A4 protects RAD18 from ubiquitin-mediated proteolysis. ( a ) Replicate plates of H1299 cells were transfected with siRNA against MAGE-A4, USP7 or with non-targeting control siRNA. After 48 h, one plate of each replicate was treated with 10 μM MG132 for 16 h. Extracts from control and MG132-treated cells were analysed by immunoblotting with antibodies against the indicated proteins. ( b ) 293T cells were co-transfected with an HA–RAD18 expression vector in combination with a CMV-MAGE-A4 plasmid or an empty vector for control. After 48 h, RAD18 complexes were immunoprecipitated with anti-HA antibodies. The resulting immune complexes were incubated in a rabbit reticulocyte lysate (RRL) to reconstitute ubiquitin-coupled proteolysis in vitro . Relative levels of RAD18 and MAGE-A4 were determined by immunoblotting and quantified using densitometry. ( c ) H1299 cells were transiently co-transfected with WT or mutant HA–RAD18 expression plasmids in combination with a MAGE-A4 expression vector (or empty vector control). Forty eight hours later, cells were harvested for immunoblot analysis of RAD18 and MAGE-A4. The white arrowhead indicates the RAD18Δ340–395 mutant protein band that is insensitive to MAGE-A4. ( d ) Replicate cultures of H1299 cells were transfected with an expression vector encoding MYC–TRIM69 or with an empty vector plasmid for control. Sixteen hours later, the cells were transfected with siRNA against MAGE-A4 or with a scrambled control siRNA and incubated for an additional 48 h before immunoblot analysis.

    Article Snippet: For immunoblotting, cell extracts or immunoprecipitates were separated by SDS-PAGE, transferred to nitrocellulose membranes, and incubated overnight with the following primary antibodies: PCNA (sc-56), Chk1 (sc-7898), β-actin (sc-130656), cyclin E (sc-198), GAPDH (sc-32233), MAGE-A4 (sc-292429), Pan-MAGE-A (sc71537) and GST (sc-53909) from Santa Cruz Biotech (Santa Cruz, CA); Polη (A301-231A), Polι (A301-304A), RAD6 (A300-281A), RAD18 (A301-340A) and USP7 (A300-033A) from Bethyl Laboratories (Montgomery, TX); p42 MAPK (9107) and MYC-Tag (2276) from Cell Signaling; γH2AX (05-636) from Millipore; and Cdc45 rat monoclonal antibody as previously described .

    Techniques: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Incubation, In Vitro, Mutagenesis

    NONO directly mediates STAT3 function in TNBC cells (A) Alignment of the STAT3 locus sequence. (B) RNA-IP was performed with anti-Myc ab or endogenous NONO ab in Myc-NONO-overexpressing MDA-MB-231 cells or MDA-MB-231 cells. After RNA-IP, the cells were analyzed by qRT-PCR with the indicated probes. (C) A dual-luciferase assay in HEK293T cells, which harbored a luciferase reporter vector containing the wild or mutant-type sequence of STAT3 locus. Luciferase activities were measured after transfecting the indicated constructs. (D) HEK293T cells were transfected with Flag-STAT3 or Myc-NONO alone or in combination. The cells were then lysed and co-immunoprecipitated with Myc ab, and western blotting was performed with Myc and Flag antibodies (left panel). MDA-MB-231 cell lysates were immunoprecipitated with IgG and NONO (center panel) and STAT3 (right panel) antibodies, and western blotting was performed with STAT3 and NONO antibodies. (E) Protein interaction amplitudes based on the correlation functions obtained in the cells co-expressing GFP and RFP (F) Computational docking model for human NONO (cyan) and STAT3 (olive) predicted using ClusPro 17 (see Materials and Methods). (G) Cell localization of NONO and STAT3 in MDA-MB-231 cells. The cells were immunostained with the indicated antibodies and visualized using microscopy. (H) Schematic of the CCND1 promoter region. ChIP assays were performed in MDA-MB-231 cells using a STAT3 or NONO antibody. Recruitment of NONO to the CCDN1 promoter via STAT3 was analyzed using primers specific to this promoter. IgG was used as an internal control. (I) Dual‐luciferase reporter gene assay to determine the STAT3 activity level following transfection of NONO, STAT3, and a STAT3-reporter into HEK293T cells. (J) The STAT3-reporter was transfected into shNONO (or shGFP) infected MDA-MB-231 cells and rescued by STAT3 re-introduction. The cells were then used to measure luciferase activity. (K and L) MDA-MB-231 cells were stably transfected with shNONO or shGFP. After transfection, the cells were treated with DMSO, cycloheximide (CHX; 50μg/ml), or actinomycin D (Act D; 1μM) and harvested at the indicated time points. Total proteins or RNAs were extracted from the indicated cells and analyzed by western blotting with the indicated antibodies or qRT-PCR, respectively. Western blot bands were quantified using the software ImageJ. STAT3 protein levels were normalized to those of β-actin. The relative STAT3 protein or RNA level was designated as the protein or RNA half-life. All results are shown as means plus standard deviations from three-independent replicates (* p

    Journal: Theranostics

    Article Title: RNA-binding protein NONO contributes to cancer cell growth and confers drug resistance as a theranostic target in TNBC

    doi: 10.7150/thno.45037

    Figure Lengend Snippet: NONO directly mediates STAT3 function in TNBC cells (A) Alignment of the STAT3 locus sequence. (B) RNA-IP was performed with anti-Myc ab or endogenous NONO ab in Myc-NONO-overexpressing MDA-MB-231 cells or MDA-MB-231 cells. After RNA-IP, the cells were analyzed by qRT-PCR with the indicated probes. (C) A dual-luciferase assay in HEK293T cells, which harbored a luciferase reporter vector containing the wild or mutant-type sequence of STAT3 locus. Luciferase activities were measured after transfecting the indicated constructs. (D) HEK293T cells were transfected with Flag-STAT3 or Myc-NONO alone or in combination. The cells were then lysed and co-immunoprecipitated with Myc ab, and western blotting was performed with Myc and Flag antibodies (left panel). MDA-MB-231 cell lysates were immunoprecipitated with IgG and NONO (center panel) and STAT3 (right panel) antibodies, and western blotting was performed with STAT3 and NONO antibodies. (E) Protein interaction amplitudes based on the correlation functions obtained in the cells co-expressing GFP and RFP (F) Computational docking model for human NONO (cyan) and STAT3 (olive) predicted using ClusPro 17 (see Materials and Methods). (G) Cell localization of NONO and STAT3 in MDA-MB-231 cells. The cells were immunostained with the indicated antibodies and visualized using microscopy. (H) Schematic of the CCND1 promoter region. ChIP assays were performed in MDA-MB-231 cells using a STAT3 or NONO antibody. Recruitment of NONO to the CCDN1 promoter via STAT3 was analyzed using primers specific to this promoter. IgG was used as an internal control. (I) Dual‐luciferase reporter gene assay to determine the STAT3 activity level following transfection of NONO, STAT3, and a STAT3-reporter into HEK293T cells. (J) The STAT3-reporter was transfected into shNONO (or shGFP) infected MDA-MB-231 cells and rescued by STAT3 re-introduction. The cells were then used to measure luciferase activity. (K and L) MDA-MB-231 cells were stably transfected with shNONO or shGFP. After transfection, the cells were treated with DMSO, cycloheximide (CHX; 50μg/ml), or actinomycin D (Act D; 1μM) and harvested at the indicated time points. Total proteins or RNAs were extracted from the indicated cells and analyzed by western blotting with the indicated antibodies or qRT-PCR, respectively. Western blot bands were quantified using the software ImageJ. STAT3 protein levels were normalized to those of β-actin. The relative STAT3 protein or RNA level was designated as the protein or RNA half-life. All results are shown as means plus standard deviations from three-independent replicates (* p

    Article Snippet: The following antibodies were used in this study: NONO (Millipore 05-950; Burlington, MA or Bethyl #A300-582A [587A]; Montgomery, TX, US), STAT3 (Cell Signaling #12640; Danvers, MA, US, or Abcam #ab119352, Cambridge, UK), phospho-STAT3 (Cell Signaling #9131), β-actin (Cell Signaling #4967), FLAG (Cell Signaling #2368), and MYC (Cell Signaling #2276).

    Techniques: Sequencing, Multiple Displacement Amplification, Quantitative RT-PCR, Luciferase, Plasmid Preparation, Mutagenesis, Construct, Transfection, Immunoprecipitation, Western Blot, Expressing, Microscopy, Chromatin Immunoprecipitation, Reporter Gene Assay, Activity Assay, Infection, Stable Transfection, Software

    Increases in c-Myc expression in the mouse neuroinflammation model. A , ChIP assays showing increased recruitment of c-Myc onto the Gnpat promoter in the hippocampal tissues from mice injected with LPS (250 μg/kg/d, i.p., for 7 d), aged mice (16 months), and mice subjected to CRS compared with the saline-injected, adult (2 months), and sham groups, respectively. B , c-Myc expression was increased in the cortex of LPS, aged, and CRS mice. C , Immunohistochemical data for Iba-1, GFAP, c-Myc, and Gnpat expression in the cortex of LPS-injected mice and the saline group. Scale bar, 50 μm. D , Quantification of data in C showing increase in amoeboid Iba-1 + microglial cells and c-Myc and Gnpat expressions in LPS-injected mice ( n = 5). E , Protein expression of Gnpat, c-Myc, and β-actin (loading control) in the cortical tissue of three representative mice from the LPS and saline groups. Right panel shows the relative changes in the protein expression. F , Immunohistochemical data showing coexpression of c-Myc with the NeuN, Iba-1, and GFAP in the LPS and saline groups. Scale bar, 50 μm. G , Quantification of data in F showing increases in c-Myc protein in glial cells but not in neurons. H , Quantification data of Gnpat costained with the Iba-1, GFAP, and NeuN. The image data for this graph are available upon request. All of the image quantification data were derived from 5 mice in each group. More than 7 slices of each mouse brain were examined. ** p

    Journal: The Journal of Neuroscience

    Article Title: Reduction of Ether-Type Glycerophospholipids, Plasmalogens, by NF-κB Signal Leading to Microglial Activation

    doi: 10.1523/JNEUROSCI.3941-15.2017

    Figure Lengend Snippet: Increases in c-Myc expression in the mouse neuroinflammation model. A , ChIP assays showing increased recruitment of c-Myc onto the Gnpat promoter in the hippocampal tissues from mice injected with LPS (250 μg/kg/d, i.p., for 7 d), aged mice (16 months), and mice subjected to CRS compared with the saline-injected, adult (2 months), and sham groups, respectively. B , c-Myc expression was increased in the cortex of LPS, aged, and CRS mice. C , Immunohistochemical data for Iba-1, GFAP, c-Myc, and Gnpat expression in the cortex of LPS-injected mice and the saline group. Scale bar, 50 μm. D , Quantification of data in C showing increase in amoeboid Iba-1 + microglial cells and c-Myc and Gnpat expressions in LPS-injected mice ( n = 5). E , Protein expression of Gnpat, c-Myc, and β-actin (loading control) in the cortical tissue of three representative mice from the LPS and saline groups. Right panel shows the relative changes in the protein expression. F , Immunohistochemical data showing coexpression of c-Myc with the NeuN, Iba-1, and GFAP in the LPS and saline groups. Scale bar, 50 μm. G , Quantification of data in F showing increases in c-Myc protein in glial cells but not in neurons. H , Quantification data of Gnpat costained with the Iba-1, GFAP, and NeuN. The image data for this graph are available upon request. All of the image quantification data were derived from 5 mice in each group. More than 7 slices of each mouse brain were examined. ** p

    Article Snippet: Membranes were then incubated at 4°C overnight with antibodies against GNPAT (Abcam catalog #ab75060 RRID:AB_2232364), c-MYC (Santa Cruz Biotechnology catalog #sc-764 RRID:AB_631276), Mycn (Calbiochem catalog #GTX16898 RRID:AB_422464), p65 (Cell Signaling Technology catalog #6956 also 6956S RRID:AB_10828935), Lamin A (BD Biosciences catalog #612162 RRID:AB_399533), Flag tag (MBL catalog #M185–3L RRID:AB_11123930), and β-Actin (Cell Signaling Technology catalog #sc-47778 RRID:AB_626632).

    Techniques: Expressing, Chromatin Immunoprecipitation, Mouse Assay, Injection, Immunohistochemistry, Derivative Assay

    Recruitment of NF-κB and Myc proteins onto the Gnpat promoter. A , Two possible Myc protein-binding sequences onto the mouse Gnpat promoter region are shown in red. B , C , ChIP assays show the basal recruitment of c-Myc ( B ) and Mycn ( C ) onto the Gnpat promoter (+43 to +171) in the indicated cultured cells. D , E , Immunoprecipitation (IP) data showing specific pull-down of c-Myc ( D ) and Mycn ( E ) by the antibodies. F , Promoter assays show the reduction of Gnpat promoter activity 48 h after NF-κB (p65 + p55)-expressing lentiviruses (1 × 10 5 transduction units/2000 cells/well of 96-well plate) infection. N2A cells were previously transfected with the luciferase promoter vectors (100 ng/2000 cells/well in 96-well plate). G , Luciferase promoter assays showing that overexpression of c-Myc and Mycn by lentiviral vectors (100 ng each) reduced the Gnpat promoter activity.

    Journal: The Journal of Neuroscience

    Article Title: Reduction of Ether-Type Glycerophospholipids, Plasmalogens, by NF-κB Signal Leading to Microglial Activation

    doi: 10.1523/JNEUROSCI.3941-15.2017

    Figure Lengend Snippet: Recruitment of NF-κB and Myc proteins onto the Gnpat promoter. A , Two possible Myc protein-binding sequences onto the mouse Gnpat promoter region are shown in red. B , C , ChIP assays show the basal recruitment of c-Myc ( B ) and Mycn ( C ) onto the Gnpat promoter (+43 to +171) in the indicated cultured cells. D , E , Immunoprecipitation (IP) data showing specific pull-down of c-Myc ( D ) and Mycn ( E ) by the antibodies. F , Promoter assays show the reduction of Gnpat promoter activity 48 h after NF-κB (p65 + p55)-expressing lentiviruses (1 × 10 5 transduction units/2000 cells/well of 96-well plate) infection. N2A cells were previously transfected with the luciferase promoter vectors (100 ng/2000 cells/well in 96-well plate). G , Luciferase promoter assays showing that overexpression of c-Myc and Mycn by lentiviral vectors (100 ng each) reduced the Gnpat promoter activity.

    Article Snippet: Membranes were then incubated at 4°C overnight with antibodies against GNPAT (Abcam catalog #ab75060 RRID:AB_2232364), c-MYC (Santa Cruz Biotechnology catalog #sc-764 RRID:AB_631276), Mycn (Calbiochem catalog #GTX16898 RRID:AB_422464), p65 (Cell Signaling Technology catalog #6956 also 6956S RRID:AB_10828935), Lamin A (BD Biosciences catalog #612162 RRID:AB_399533), Flag tag (MBL catalog #M185–3L RRID:AB_11123930), and β-Actin (Cell Signaling Technology catalog #sc-47778 RRID:AB_626632).

    Techniques: Protein Binding, Chromatin Immunoprecipitation, Cell Culture, Immunoprecipitation, Activity Assay, Expressing, Transduction, Infection, Transfection, Luciferase, Over Expression

    Suppression of Gnpat expression by NF-κB-induced c-Myc. A , Transfection of p65- and p50-expressing plasmids (1 μg each) in N2A cells (1 × 10 5 cells/well of 6-well plate) for 48 h increased c-Myc mRNA (left) and protein (right) expression. B , In the same experimental condition, p65 and p50 overexpression reduced Gnpat expression. C , MG6 cells (1 × 10 5 cells/well of 6 well platees) infected with c-Myc- and Mycn-expressing lentiviruses for 48 h showing the reduction of Gnpat expression compared with control lentivirus infection. D , Luciferase assays in N2A cells showing that the reduction of Gnpat promoter activity by NF-κB (p65 plus p50) (middle panel, third row) and c-Myc (right panel) was canceled by the deletion of Myc-binding sequences from the Gnpat promoter (first and second rows). The data represent six independent experiments. ** p

    Journal: The Journal of Neuroscience

    Article Title: Reduction of Ether-Type Glycerophospholipids, Plasmalogens, by NF-κB Signal Leading to Microglial Activation

    doi: 10.1523/JNEUROSCI.3941-15.2017

    Figure Lengend Snippet: Suppression of Gnpat expression by NF-κB-induced c-Myc. A , Transfection of p65- and p50-expressing plasmids (1 μg each) in N2A cells (1 × 10 5 cells/well of 6-well plate) for 48 h increased c-Myc mRNA (left) and protein (right) expression. B , In the same experimental condition, p65 and p50 overexpression reduced Gnpat expression. C , MG6 cells (1 × 10 5 cells/well of 6 well platees) infected with c-Myc- and Mycn-expressing lentiviruses for 48 h showing the reduction of Gnpat expression compared with control lentivirus infection. D , Luciferase assays in N2A cells showing that the reduction of Gnpat promoter activity by NF-κB (p65 plus p50) (middle panel, third row) and c-Myc (right panel) was canceled by the deletion of Myc-binding sequences from the Gnpat promoter (first and second rows). The data represent six independent experiments. ** p

    Article Snippet: Membranes were then incubated at 4°C overnight with antibodies against GNPAT (Abcam catalog #ab75060 RRID:AB_2232364), c-MYC (Santa Cruz Biotechnology catalog #sc-764 RRID:AB_631276), Mycn (Calbiochem catalog #GTX16898 RRID:AB_422464), p65 (Cell Signaling Technology catalog #6956 also 6956S RRID:AB_10828935), Lamin A (BD Biosciences catalog #612162 RRID:AB_399533), Flag tag (MBL catalog #M185–3L RRID:AB_11123930), and β-Actin (Cell Signaling Technology catalog #sc-47778 RRID:AB_626632).

    Techniques: Expressing, Transfection, Over Expression, Infection, Luciferase, Activity Assay, Binding Assay

    Overexpression of p65 and p50 reduces the expression of GNPAT in human cell lines. A , Western blot data showing an increase in c-MYC and decrease in GNPAT protein expression by the transient transfection of p65 and p50 expressing plasmids in SH-SY5Y cells for 48 h. B , Real-time PCR data performed after 48 h of overexpression showing the decrease in GNPAT expression by the overexpression of p65 and p50 in the cells. C , Position of c-MYC-binding sites and the primer locations for the ChIP assays onto the human GNPAT promoter. The number (+1) indicates the transcription start site located within the first exon. D , E , ChIP assays showing the recruitment of c-MYC proteins onto the binding sites of GNPAT promoter in the steady state ( D ) and after the overexpression of p65 and p50 proteins in the cells for 48 h ( E ). F , Schematic representing the cloned human GNPAT promoter regions in the luciferase vector. The short form of the promoter (−150 to +183) lacks the putative c-MYC-binding site ( C ). Promoter assays showed that the overexpression of p65 and p50 proteins could not reduce the promoter activity of the human GNPAT promoter lacking the c-MYC binding site ( n = 5).

    Journal: The Journal of Neuroscience

    Article Title: Reduction of Ether-Type Glycerophospholipids, Plasmalogens, by NF-κB Signal Leading to Microglial Activation

    doi: 10.1523/JNEUROSCI.3941-15.2017

    Figure Lengend Snippet: Overexpression of p65 and p50 reduces the expression of GNPAT in human cell lines. A , Western blot data showing an increase in c-MYC and decrease in GNPAT protein expression by the transient transfection of p65 and p50 expressing plasmids in SH-SY5Y cells for 48 h. B , Real-time PCR data performed after 48 h of overexpression showing the decrease in GNPAT expression by the overexpression of p65 and p50 in the cells. C , Position of c-MYC-binding sites and the primer locations for the ChIP assays onto the human GNPAT promoter. The number (+1) indicates the transcription start site located within the first exon. D , E , ChIP assays showing the recruitment of c-MYC proteins onto the binding sites of GNPAT promoter in the steady state ( D ) and after the overexpression of p65 and p50 proteins in the cells for 48 h ( E ). F , Schematic representing the cloned human GNPAT promoter regions in the luciferase vector. The short form of the promoter (−150 to +183) lacks the putative c-MYC-binding site ( C ). Promoter assays showed that the overexpression of p65 and p50 proteins could not reduce the promoter activity of the human GNPAT promoter lacking the c-MYC binding site ( n = 5).

    Article Snippet: Membranes were then incubated at 4°C overnight with antibodies against GNPAT (Abcam catalog #ab75060 RRID:AB_2232364), c-MYC (Santa Cruz Biotechnology catalog #sc-764 RRID:AB_631276), Mycn (Calbiochem catalog #GTX16898 RRID:AB_422464), p65 (Cell Signaling Technology catalog #6956 also 6956S RRID:AB_10828935), Lamin A (BD Biosciences catalog #612162 RRID:AB_399533), Flag tag (MBL catalog #M185–3L RRID:AB_11123930), and β-Actin (Cell Signaling Technology catalog #sc-47778 RRID:AB_626632).

    Techniques: Over Expression, Expressing, Western Blot, Transfection, Real-time Polymerase Chain Reaction, Binding Assay, Chromatin Immunoprecipitation, Clone Assay, Luciferase, Plasmid Preparation, Activity Assay

    ( See previous page ). MUL1 promotes ubiquitination of ULK1. ( A ) HEK 293T cells were transfected with MUL1-MYC, MUL1ΔR-MYC, or the empty MYC-vector together with GFP-ULK1 and HA-Ub. Ubiquitination assays were performed as described in Materials and Methods. The ubiquitination level of GFP-ULK1 was detected using an anti-HA antibody. ( B ) In vitro ubiquitination assays were performed as described in Materials and Methods. The ubiquitinated form of ULK1 was immunoblotted using an anti-ULK1 antibody. ( C ) MUL1 knockdown cells transfected with GFP-ULK1 were subjected to ubiquitination assays for analysis with an anti-Ub antibody. ( D ) Quantitative analysis of ubiquitinated GFP-ULK1 level as described in ( C ) (The intensity of bands was measured with Image J software. mean±SEM; from 3 independent experiments). ( E ) HEK 293T cells were transfected with the indicated plasmids for 24 h, and subsequently a ubiquitination assay was performed for analysis with an anti-Ub antibody. ( F ) HeLa cells transfected with GFP-ULK1 were treated with 5 μM of selenite for the indicated time, followed by ubiquitination assays for analysis with an anti-Ub antibody. ( G ) HeLa cells were treated with 5 μM of selenite for the indicated time, followed by ubiquitination assays for analysis with an anti-Ub antibody. ( H ) Scrambled RNA- or si MUL1 -transfected cells were treated with or without selenite for 12 h, followed by ubiquitination assays for analysis with an anti-Ub antibody. ( I ) NIH-3T3 cells were treated with 5 μM of selenite for the indicated time, followed by ubiquitination assays for analysis with an anti-Ub antibody. ( J ) NIH-3T3 cells were treated with selenite for the indicated time, with or without MG132, and subjected to western blotting analysis of the ULK1 protein level.

    Journal: Autophagy

    Article Title: Mitochondrial outer-membrane E3 ligase MUL1 ubiquitinates ULK1 and regulates selenite-induced mitophagy

    doi: 10.1080/15548627.2015.1017180

    Figure Lengend Snippet: ( See previous page ). MUL1 promotes ubiquitination of ULK1. ( A ) HEK 293T cells were transfected with MUL1-MYC, MUL1ΔR-MYC, or the empty MYC-vector together with GFP-ULK1 and HA-Ub. Ubiquitination assays were performed as described in Materials and Methods. The ubiquitination level of GFP-ULK1 was detected using an anti-HA antibody. ( B ) In vitro ubiquitination assays were performed as described in Materials and Methods. The ubiquitinated form of ULK1 was immunoblotted using an anti-ULK1 antibody. ( C ) MUL1 knockdown cells transfected with GFP-ULK1 were subjected to ubiquitination assays for analysis with an anti-Ub antibody. ( D ) Quantitative analysis of ubiquitinated GFP-ULK1 level as described in ( C ) (The intensity of bands was measured with Image J software. mean±SEM; from 3 independent experiments). ( E ) HEK 293T cells were transfected with the indicated plasmids for 24 h, and subsequently a ubiquitination assay was performed for analysis with an anti-Ub antibody. ( F ) HeLa cells transfected with GFP-ULK1 were treated with 5 μM of selenite for the indicated time, followed by ubiquitination assays for analysis with an anti-Ub antibody. ( G ) HeLa cells were treated with 5 μM of selenite for the indicated time, followed by ubiquitination assays for analysis with an anti-Ub antibody. ( H ) Scrambled RNA- or si MUL1 -transfected cells were treated with or without selenite for 12 h, followed by ubiquitination assays for analysis with an anti-Ub antibody. ( I ) NIH-3T3 cells were treated with 5 μM of selenite for the indicated time, followed by ubiquitination assays for analysis with an anti-Ub antibody. ( J ) NIH-3T3 cells were treated with selenite for the indicated time, with or without MG132, and subjected to western blotting analysis of the ULK1 protein level.

    Article Snippet: Santa Cruz Biotechnology: MYC (SC-40), GFP (SC-9996), HA (SC-7392), ULK1 (SC-33182).

    Techniques: Polyacrylamide Gel Electrophoresis, Transfection, Plasmid Preparation, In Vitro, Software, Ubiquitin Assay, Western Blot

    Overexpression of MUL1 and treatment with selenite promotes ULK1 degradation through the proteasome pathway. ( A ) HeLa cells were treated with CHX (10 μM, 12 h) and selenite (5 μM) for the indicated time, with or without MG132, and subjected to western blotting analysis of the ULK1. ( B ) Quantification of ULK1 protein levels in ( A ) (mean ±SEM, from 3 independent experiments). ( C ) HeLa cells were treated with the indicated agents (FCCP 5 μM; hypoxia with 1% O 2 ; selenite 5 μM), and then subjected to western blotting analysis of ULK1 (The intensity of indicated bands was measured with ImageJ software). ( D ) After transfection with plasmids as indicated, HeLa cells were treated with BAF (10 nM, 6 h) or MG132 (5 μM, 6 h) prior to harvesting, followed by western blotting analysis of the GFP-ULK1 level. ( E ) After transfection with MUL1-MYC as indicated, HeLa cells were treated with BAF (10 nM, 6 h) or MG132 (5 μM, 6 h), followed by western blotting analysis of the ULK1 protein level. ( F ) HeLa cells were transfected with MUL1-MYC or MUL1ΔR-MYC (MUL1 with absence of the RING finger domain) for 24 h, and subjected to western blotting analysis of the indicated protein levels (The intensity of indicated bands was measured with ImageJ software). ( G ) HeLa cells transfected with MUL1-MYC for 24 h, followed by treatment with CHX (10 μM) for the indicated time, with or without MG132, and subjected to western blotting analysis of the ULK1 protein level. ( H ) Quantification of ULK1 protein levels in ( G ). Mean ±SEM, from 3 independent experiments.

    Journal: Autophagy

    Article Title: Mitochondrial outer-membrane E3 ligase MUL1 ubiquitinates ULK1 and regulates selenite-induced mitophagy

    doi: 10.1080/15548627.2015.1017180

    Figure Lengend Snippet: Overexpression of MUL1 and treatment with selenite promotes ULK1 degradation through the proteasome pathway. ( A ) HeLa cells were treated with CHX (10 μM, 12 h) and selenite (5 μM) for the indicated time, with or without MG132, and subjected to western blotting analysis of the ULK1. ( B ) Quantification of ULK1 protein levels in ( A ) (mean ±SEM, from 3 independent experiments). ( C ) HeLa cells were treated with the indicated agents (FCCP 5 μM; hypoxia with 1% O 2 ; selenite 5 μM), and then subjected to western blotting analysis of ULK1 (The intensity of indicated bands was measured with ImageJ software). ( D ) After transfection with plasmids as indicated, HeLa cells were treated with BAF (10 nM, 6 h) or MG132 (5 μM, 6 h) prior to harvesting, followed by western blotting analysis of the GFP-ULK1 level. ( E ) After transfection with MUL1-MYC as indicated, HeLa cells were treated with BAF (10 nM, 6 h) or MG132 (5 μM, 6 h), followed by western blotting analysis of the ULK1 protein level. ( F ) HeLa cells were transfected with MUL1-MYC or MUL1ΔR-MYC (MUL1 with absence of the RING finger domain) for 24 h, and subjected to western blotting analysis of the indicated protein levels (The intensity of indicated bands was measured with ImageJ software). ( G ) HeLa cells transfected with MUL1-MYC for 24 h, followed by treatment with CHX (10 μM) for the indicated time, with or without MG132, and subjected to western blotting analysis of the ULK1 protein level. ( H ) Quantification of ULK1 protein levels in ( G ). Mean ±SEM, from 3 independent experiments.

    Article Snippet: Santa Cruz Biotechnology: MYC (SC-40), GFP (SC-9996), HA (SC-7392), ULK1 (SC-33182).

    Techniques: Over Expression, Western Blot, Software, Transfection

    ( See previous page ). MUL1 interacts with the ULK1 kinase in mammalian cells. ( A ) HeLa cells transfected with GFP-ULK1 (Green) were treated with 5 μM of selenite for the indicated time followed by staining with MitoTracker Red for mitochondria and DAPI (blue) for the nucleus. ( B ) HeLa cells were treated with 5 μM of selenite for the indicated times. Mitochondria were isolated and subsequently subjected to western blotting analysis of the indicated proteins. ( C ) Immunoprecipitation was performed with an ULK1 antibody. Coimmunoprecipitated endogenous MUL1 was detected through western blotting with an anti-MUL1 antibody. ( D ) ULK1-MYC was transfected into HEK 293T cells, and immunoprecipitation was performed with an anti-MYC antibody. Coimmunoprecipitated endogenous MUL1 was detected through western blotting with an anti-ULK1 antibody. ( E ) MUL1-MYC was transfected into HEK 293T cells, and immunoprecipitation was performed with an anti-MYC antibody. Coimmunoprecipitated endogenous ULK1 was detected through western blotting with an anti-ULK1 antibody. ( F ) HeLa cells transfected with MYC-vector or MUL1-MYC were treated with selenite for the indicated time and immunoprecipitation was performed with an anti-MYC antibody. Coimmunoprecipitated endogenous ULK1 level was detected through western blotting with an anti-ULK1 antibody. ( G ) Purified GST and GST-tagged MUL1 protein were used for the GST affinity isolation of endogenous ULK1, and blotted with an anti-ULK1 antibody. ( H ) GFP-MUL1 and ULK1-MYC were cotransfected with Flag-ATG13 or Flag vector, and immunoprecipitation was performed with an anti-Flag antibody. Coimmunoprecipitated ULK1 and MUL1 were detected through western blotting with anti-GFP and anti-MYC antibodies respectively. ( I ) Truncated forms of ULK1-MYC were constructed based on its functional domains. GFP-MUL1 was cotransfected with full-length or truncated forms of ULK1-MYC, and immunoprecipitation was performed with an anti-MYC antibody. Coimmunoprecipitated MUL1 was detected using an anti-GFP antibody. ( J ) Ulk1 −/− MEFs were transfected with exogenous wild-type ULK1 or truncation ULK1Δ CTD, followed by treatment with selenite for 12 h before western blotting analysis of the indicated proteins.

    Journal: Autophagy

    Article Title: Mitochondrial outer-membrane E3 ligase MUL1 ubiquitinates ULK1 and regulates selenite-induced mitophagy

    doi: 10.1080/15548627.2015.1017180

    Figure Lengend Snippet: ( See previous page ). MUL1 interacts with the ULK1 kinase in mammalian cells. ( A ) HeLa cells transfected with GFP-ULK1 (Green) were treated with 5 μM of selenite for the indicated time followed by staining with MitoTracker Red for mitochondria and DAPI (blue) for the nucleus. ( B ) HeLa cells were treated with 5 μM of selenite for the indicated times. Mitochondria were isolated and subsequently subjected to western blotting analysis of the indicated proteins. ( C ) Immunoprecipitation was performed with an ULK1 antibody. Coimmunoprecipitated endogenous MUL1 was detected through western blotting with an anti-MUL1 antibody. ( D ) ULK1-MYC was transfected into HEK 293T cells, and immunoprecipitation was performed with an anti-MYC antibody. Coimmunoprecipitated endogenous MUL1 was detected through western blotting with an anti-ULK1 antibody. ( E ) MUL1-MYC was transfected into HEK 293T cells, and immunoprecipitation was performed with an anti-MYC antibody. Coimmunoprecipitated endogenous ULK1 was detected through western blotting with an anti-ULK1 antibody. ( F ) HeLa cells transfected with MYC-vector or MUL1-MYC were treated with selenite for the indicated time and immunoprecipitation was performed with an anti-MYC antibody. Coimmunoprecipitated endogenous ULK1 level was detected through western blotting with an anti-ULK1 antibody. ( G ) Purified GST and GST-tagged MUL1 protein were used for the GST affinity isolation of endogenous ULK1, and blotted with an anti-ULK1 antibody. ( H ) GFP-MUL1 and ULK1-MYC were cotransfected with Flag-ATG13 or Flag vector, and immunoprecipitation was performed with an anti-Flag antibody. Coimmunoprecipitated ULK1 and MUL1 were detected through western blotting with anti-GFP and anti-MYC antibodies respectively. ( I ) Truncated forms of ULK1-MYC were constructed based on its functional domains. GFP-MUL1 was cotransfected with full-length or truncated forms of ULK1-MYC, and immunoprecipitation was performed with an anti-MYC antibody. Coimmunoprecipitated MUL1 was detected using an anti-GFP antibody. ( J ) Ulk1 −/− MEFs were transfected with exogenous wild-type ULK1 or truncation ULK1Δ CTD, followed by treatment with selenite for 12 h before western blotting analysis of the indicated proteins.

    Article Snippet: Santa Cruz Biotechnology: MYC (SC-40), GFP (SC-9996), HA (SC-7392), ULK1 (SC-33182).

    Techniques: Polyacrylamide Gel Electrophoresis, Transfection, Staining, Isolation, Western Blot, Immunoprecipitation, Plasmid Preparation, Purification, Construct, Functional Assay

    MUL1 response to selenite-induced ROS stress depending on conserved cysteines 62 and 87. ( A ) HeLa cells treated with selenite as indicated were stained with MitoSox Red and subjected to flow cytometry. ( B ) HeLa cells were pretreated with NAC (10 mM) or GSH-EE (10 mM) for 30 min, followed by treatment with 5 μM of selenite for 12 h. Cell lysates were subjected to western blotting analysis of the indicated proteins. ( C ) HeLa cells transfected with GFP-ULK1 were pretreated with NAC (10 mM) or GSH-EE (10 mM) for 30 mins, followed by treatment of selenite (12 h). The ubiquitination assays were performed as described in Materials and Methods. ( D ) Alignment of the MUL1 amino acids in different species. ( E ) HeLa cells with MUL1 knockdown were transfected with exogenous MYC-tagged wild-type MUL1 or MUL1 with cysteine mutations (C62S, C87S), followed by treatment with selenite (5 μM, 12 h) before western blotting analysis of the indicated proteins (The intensity of the indicated bands was measured with ImageJ software.).

    Journal: Autophagy

    Article Title: Mitochondrial outer-membrane E3 ligase MUL1 ubiquitinates ULK1 and regulates selenite-induced mitophagy

    doi: 10.1080/15548627.2015.1017180

    Figure Lengend Snippet: MUL1 response to selenite-induced ROS stress depending on conserved cysteines 62 and 87. ( A ) HeLa cells treated with selenite as indicated were stained with MitoSox Red and subjected to flow cytometry. ( B ) HeLa cells were pretreated with NAC (10 mM) or GSH-EE (10 mM) for 30 min, followed by treatment with 5 μM of selenite for 12 h. Cell lysates were subjected to western blotting analysis of the indicated proteins. ( C ) HeLa cells transfected with GFP-ULK1 were pretreated with NAC (10 mM) or GSH-EE (10 mM) for 30 mins, followed by treatment of selenite (12 h). The ubiquitination assays were performed as described in Materials and Methods. ( D ) Alignment of the MUL1 amino acids in different species. ( E ) HeLa cells with MUL1 knockdown were transfected with exogenous MYC-tagged wild-type MUL1 or MUL1 with cysteine mutations (C62S, C87S), followed by treatment with selenite (5 μM, 12 h) before western blotting analysis of the indicated proteins (The intensity of the indicated bands was measured with ImageJ software.).

    Article Snippet: Santa Cruz Biotechnology: MYC (SC-40), GFP (SC-9996), HA (SC-7392), ULK1 (SC-33182).

    Techniques: Staining, Flow Cytometry, Cytometry, Western Blot, Transfection, Software

    ( See previous page ). Both ULK1 and ATG5 are required for selenite-induced mitophagy. ( A ) HeLa cells with MUL1 was knocked down were rescued by exogenous wild-type MYC-tagged MUL1, followed by treatment with selenite for 12 h before western blotting analysis of the indicated proteins. ( B ) Wild-type MEFs, Atg5 −/− , Ulk1 −/− and park2 −/− MEFs transfected with GFP-LC3 were treated with or without 5 μM of selenite for 12 h, followed by staining with MitoTracker Red for mitochondria and immunofluorescence microscopy to visualize GFP-LC3 puncta. ( C ) Quantification of GFP-LC3 punctate structures associated with mitochondria (TOMM20) described in ( B ) (mean ±SEM; n = 50 cells from 3 independent experiments, one-way ANOVA, ***P

    Journal: Autophagy

    Article Title: Mitochondrial outer-membrane E3 ligase MUL1 ubiquitinates ULK1 and regulates selenite-induced mitophagy

    doi: 10.1080/15548627.2015.1017180

    Figure Lengend Snippet: ( See previous page ). Both ULK1 and ATG5 are required for selenite-induced mitophagy. ( A ) HeLa cells with MUL1 was knocked down were rescued by exogenous wild-type MYC-tagged MUL1, followed by treatment with selenite for 12 h before western blotting analysis of the indicated proteins. ( B ) Wild-type MEFs, Atg5 −/− , Ulk1 −/− and park2 −/− MEFs transfected with GFP-LC3 were treated with or without 5 μM of selenite for 12 h, followed by staining with MitoTracker Red for mitochondria and immunofluorescence microscopy to visualize GFP-LC3 puncta. ( C ) Quantification of GFP-LC3 punctate structures associated with mitochondria (TOMM20) described in ( B ) (mean ±SEM; n = 50 cells from 3 independent experiments, one-way ANOVA, ***P

    Article Snippet: Santa Cruz Biotechnology: MYC (SC-40), GFP (SC-9996), HA (SC-7392), ULK1 (SC-33182).

    Techniques: Polyacrylamide Gel Electrophoresis, Western Blot, Transfection, Staining, Immunofluorescence, Microscopy

    Cooperative effects of OTX015 with a panel of drugs in ALCL in vitro models A. The combination of OTX015-CEP28122 effectively down-regulated MYC protein levels by Western blot in TS-Supm2 cells (β-actin was used as a loading control), resulting in a pronounced G1 cell cycle arrest with limited effects on cell viability according to FACScan. The effect was maximal after 48 h. B. OTX015 exposure (500 nM) for 30 minutes to 24 h in Karpas299 cells resulted in robust down-regulation of GLI1 mRNA, a known target of GANT61, following an initial rapid upregulation according to qRT-PCR. Exposure of in Karpas299 cells to increasing OTX015 concentrations for 24 h showed that the mRNA down-regulation was dose-dependent. C. The effects on cell viability of the GLI inhibitor GANT61 and OTX015 were measured as single agents and in combination according to the qRT-PCR. Concomitant exposure of OTX015 (500 nM) and GANT61 (2.5 or 5 μM) increased the percentage of dead cells after 24 and 48 h exposure. D. Cells treated with combined ibrutinib (10 μM) and OTX015 (500 nM) had reduced levels of P-STAT3 and P-ITK protein compared to treatment with either single agent, as per Western blot. β-actin was used as a loading control. E. Metabolic readout of ALCL cell lines treated with OTX015 (500 nM) in combination with ibrutinib (1.2 or 5 μM) for 48 h, determined using an ATPlite assay. F. Cell viability was determined after 48 h in multiple cell lines exposed to 500 nM OTX015 in combination with ibrutinib (2, 5, 10 μM), according to the ATPlite assay.

    Journal: Oncotarget

    Article Title: Therapeutic efficacy of the bromodomain inhibitor OTX015/MK-8628 in ALK-positive anaplastic large cell lymphoma: an alternative modality to overcome resistant phenotypes

    doi: 10.18632/oncotarget.12876

    Figure Lengend Snippet: Cooperative effects of OTX015 with a panel of drugs in ALCL in vitro models A. The combination of OTX015-CEP28122 effectively down-regulated MYC protein levels by Western blot in TS-Supm2 cells (β-actin was used as a loading control), resulting in a pronounced G1 cell cycle arrest with limited effects on cell viability according to FACScan. The effect was maximal after 48 h. B. OTX015 exposure (500 nM) for 30 minutes to 24 h in Karpas299 cells resulted in robust down-regulation of GLI1 mRNA, a known target of GANT61, following an initial rapid upregulation according to qRT-PCR. Exposure of in Karpas299 cells to increasing OTX015 concentrations for 24 h showed that the mRNA down-regulation was dose-dependent. C. The effects on cell viability of the GLI inhibitor GANT61 and OTX015 were measured as single agents and in combination according to the qRT-PCR. Concomitant exposure of OTX015 (500 nM) and GANT61 (2.5 or 5 μM) increased the percentage of dead cells after 24 and 48 h exposure. D. Cells treated with combined ibrutinib (10 μM) and OTX015 (500 nM) had reduced levels of P-STAT3 and P-ITK protein compared to treatment with either single agent, as per Western blot. β-actin was used as a loading control. E. Metabolic readout of ALCL cell lines treated with OTX015 (500 nM) in combination with ibrutinib (1.2 or 5 μM) for 48 h, determined using an ATPlite assay. F. Cell viability was determined after 48 h in multiple cell lines exposed to 500 nM OTX015 in combination with ibrutinib (2, 5, 10 μM), according to the ATPlite assay.

    Article Snippet: Western blotting analysis Western blotting for protein expression analysis was performed as previously reported [ ], using the following antibodies: anti-BRD2 (ab37633, AbCam), anti-BRD3 (ab56342, AbCam), anti-BRD4 (ab75898, AbCam), anti-MYC (9B11, Cell Signaling), anti-β-actin (Clone C4, Millipore) or anti-β-tubulin (Clone tub 2.1, Sigma), anti-pSHIP2 (#2007, Cell Signaling, Danvers, MA, USA), anti-pSTAT3 (#9145, Cell Signaling, Danvers, MA, USA), anti-pSRC (#1243, Cell Signaling, Danvers, MA, USA), anti-JunB (#3746, Cell Signaling, Danvers, MA, USA), anti-pITK (#3537, Cell Signaling, Danvers, MA, USA) and anti-Tubulin (#5666, Cell Signaling, Danvers, MA, USA).

    Techniques: In Vitro, Western Blot, Quantitative RT-PCR

    OTX015 modulates MYC and BRD expression A. MYC mRNA and protein levels were reproducibly down-regulated after 72-h OTX015 exposure (100 - 1000 nM) in ALK+ cell lines by qRT-PCR and Western blot respectively. CT, DMSO-treated controls. B. 24 h exposure to OTX015 (250, 500 nM) led to dose-dependent down-regulation of MYC protein levels and G1 cell cycle arrest. β-actin was used as a protein loading control. C. 24 h exposure to OTX015 resulted in down-regulation of BRD2, BRD3 and BRD4 RNA and protein levels by qRT-PCR and Western blot. β-actin was used as a protein loading control.

    Journal: Oncotarget

    Article Title: Therapeutic efficacy of the bromodomain inhibitor OTX015/MK-8628 in ALK-positive anaplastic large cell lymphoma: an alternative modality to overcome resistant phenotypes

    doi: 10.18632/oncotarget.12876

    Figure Lengend Snippet: OTX015 modulates MYC and BRD expression A. MYC mRNA and protein levels were reproducibly down-regulated after 72-h OTX015 exposure (100 - 1000 nM) in ALK+ cell lines by qRT-PCR and Western blot respectively. CT, DMSO-treated controls. B. 24 h exposure to OTX015 (250, 500 nM) led to dose-dependent down-regulation of MYC protein levels and G1 cell cycle arrest. β-actin was used as a protein loading control. C. 24 h exposure to OTX015 resulted in down-regulation of BRD2, BRD3 and BRD4 RNA and protein levels by qRT-PCR and Western blot. β-actin was used as a protein loading control.

    Article Snippet: Western blotting analysis Western blotting for protein expression analysis was performed as previously reported [ ], using the following antibodies: anti-BRD2 (ab37633, AbCam), anti-BRD3 (ab56342, AbCam), anti-BRD4 (ab75898, AbCam), anti-MYC (9B11, Cell Signaling), anti-β-actin (Clone C4, Millipore) or anti-β-tubulin (Clone tub 2.1, Sigma), anti-pSHIP2 (#2007, Cell Signaling, Danvers, MA, USA), anti-pSTAT3 (#9145, Cell Signaling, Danvers, MA, USA), anti-pSRC (#1243, Cell Signaling, Danvers, MA, USA), anti-JunB (#3746, Cell Signaling, Danvers, MA, USA), anti-pITK (#3537, Cell Signaling, Danvers, MA, USA) and anti-Tubulin (#5666, Cell Signaling, Danvers, MA, USA).

    Techniques: Expressing, Quantitative RT-PCR, Western Blot

    TPO interacts with the EGFR protein. A, Mass spectrometric analysis predicted that TPO could interact with EGFR. B, Interaction between transfected TPO and EGFR was verified by co‐immunoprecipitation assays in A549 and H1299 cells. A549 and H1299 cells were collected 48 h after transfection with pCMV6‐Myc‐DDK‐TPO plasmids. Cell lysates were immunoprecipitated with anti‐Myc antibodies (# 2276; Cell Signaling Technology) or control IgG and examined by anti‐EGFR antibody (#4267; Cell Signaling Technology) and anti‐Myc antibodies (# 2278; Cell Signaling Technology). C, Interaction between endogenous TPO and EGFR was verified by co‐immunoprecipitation assays in A549 and H1299 cells. Cell lysates were immunoprecipitated with anti‐TPO antibody (sc‐374045; Santa Cruz Biotechnology) or control IgG and examined by anti‐EGFR antibody (#4267; Cell Signaling Technology) and anti‐TPO antibody (ab196026; Abcam). D, Immunofluorescence staining in A549 and H1299 cells showed that endogenous TPO and EGFR were colocalized in the cytoplasm. Magnification, ×400

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Endogenous thrombopoietin promotes non‐small‐cell lung carcinoma cell proliferation and migration by regulating EGFR signalling, et al. Endogenous thrombopoietin promotes non‐small‐cell lung carcinoma cell proliferation and migration by regulating EGFR signalling

    doi: 10.1111/jcmm.15314

    Figure Lengend Snippet: TPO interacts with the EGFR protein. A, Mass spectrometric analysis predicted that TPO could interact with EGFR. B, Interaction between transfected TPO and EGFR was verified by co‐immunoprecipitation assays in A549 and H1299 cells. A549 and H1299 cells were collected 48 h after transfection with pCMV6‐Myc‐DDK‐TPO plasmids. Cell lysates were immunoprecipitated with anti‐Myc antibodies (# 2276; Cell Signaling Technology) or control IgG and examined by anti‐EGFR antibody (#4267; Cell Signaling Technology) and anti‐Myc antibodies (# 2278; Cell Signaling Technology). C, Interaction between endogenous TPO and EGFR was verified by co‐immunoprecipitation assays in A549 and H1299 cells. Cell lysates were immunoprecipitated with anti‐TPO antibody (sc‐374045; Santa Cruz Biotechnology) or control IgG and examined by anti‐EGFR antibody (#4267; Cell Signaling Technology) and anti‐TPO antibody (ab196026; Abcam). D, Immunofluorescence staining in A549 and H1299 cells showed that endogenous TPO and EGFR were colocalized in the cytoplasm. Magnification, ×400

    Article Snippet: Myc‐tagged fusion proteins were captured by incubation with anti‐Myc antibodies (# 2276; Cell Signaling Technology) and ProteinA + G Agarose beads (P2012; Beyotime Biosciences) overnight at 4°C.

    Techniques: Transfection, Immunoprecipitation, Immunofluorescence, Staining

    IKBKE influences AR mRNA expression via the Hippo pathway. LNCaP cells were reverse transfected in full media (FM) with either N/S or three pooled siRNAs against IKBKE . After 72 h, protein expression was assessed by ( A ) immunoblotting and ( B ) YAP , c-MYC and IKBKE mRNA expression determined by qPCR ( n = 3). ( C ) After 72 h steroid depletion, LNCaP cells were pre-treated with vehicle (DMSO) or increasing concentrations of IKBKE antagonist, CAY10576, for 8 h followed by treatment with vehicle or 1 nM R1881 for a further 24 h. YAP and c-MYC protein levels were assessed by immunoblotting. ( D ) LNCaP cells were reverse transfected in FM with either N/S or pooled siRNAs against IKBKE . After 72 h, MG132 (20 μM) was applied and incubated for a further 16 h. YAP levels were determined by immunoblotting. ( E ) LNCaP cells were reverse transfected in FM with either N/S or pooled siRNAs against IKBKE . After 72 h, nuclear-cytoplasmic fractionation was performed with YAP and c-MYC localization determined by immunoblotting. PARP was used as a nuclear loading control and α-tubulin as a cytoplasmic loading control. Densitometry was performed on YAP expression and normalized to loading controls using ImageJ software. Data is expressed as a % of total cellular YAP. ( F ) LNCaP cells were treated with either vehicle or CAY10576 (5 μM) or CYT387 (5 μM) for 16 h prior to nuclear-cytoplasmic fractionation. Nuclear YAP and c-MYC levels were determined by immunoblotting. ( G ) LNCaP cells were reverse transfected in FM with either N/S or pooled siRNAs against IKBKE and grown for 48 h prior to ChIP analysis using antibodies specific to c-MYC and isotype controls (IgG). Recruitment to the c-MYC binding site within the AR gene was assessed by qPCR. Data are an average of 3 independent experiments ± SEM. One-way ANOVA paired Dunnetts multiple comparison test * P

    Journal: Nucleic Acids Research

    Article Title: IKBKE activity enhances AR levels in advanced prostate cancer via modulation of the Hippo pathway

    doi: 10.1093/nar/gkaa271

    Figure Lengend Snippet: IKBKE influences AR mRNA expression via the Hippo pathway. LNCaP cells were reverse transfected in full media (FM) with either N/S or three pooled siRNAs against IKBKE . After 72 h, protein expression was assessed by ( A ) immunoblotting and ( B ) YAP , c-MYC and IKBKE mRNA expression determined by qPCR ( n = 3). ( C ) After 72 h steroid depletion, LNCaP cells were pre-treated with vehicle (DMSO) or increasing concentrations of IKBKE antagonist, CAY10576, for 8 h followed by treatment with vehicle or 1 nM R1881 for a further 24 h. YAP and c-MYC protein levels were assessed by immunoblotting. ( D ) LNCaP cells were reverse transfected in FM with either N/S or pooled siRNAs against IKBKE . After 72 h, MG132 (20 μM) was applied and incubated for a further 16 h. YAP levels were determined by immunoblotting. ( E ) LNCaP cells were reverse transfected in FM with either N/S or pooled siRNAs against IKBKE . After 72 h, nuclear-cytoplasmic fractionation was performed with YAP and c-MYC localization determined by immunoblotting. PARP was used as a nuclear loading control and α-tubulin as a cytoplasmic loading control. Densitometry was performed on YAP expression and normalized to loading controls using ImageJ software. Data is expressed as a % of total cellular YAP. ( F ) LNCaP cells were treated with either vehicle or CAY10576 (5 μM) or CYT387 (5 μM) for 16 h prior to nuclear-cytoplasmic fractionation. Nuclear YAP and c-MYC levels were determined by immunoblotting. ( G ) LNCaP cells were reverse transfected in FM with either N/S or pooled siRNAs against IKBKE and grown for 48 h prior to ChIP analysis using antibodies specific to c-MYC and isotype controls (IgG). Recruitment to the c-MYC binding site within the AR gene was assessed by qPCR. Data are an average of 3 independent experiments ± SEM. One-way ANOVA paired Dunnetts multiple comparison test * P

    Article Snippet: Antibodies and constructsAR (C-19, sc-815, Santa Cruz Biotechnology and clone G122-434, BD), PSA (A0562, Dako), IKBKE (D20G4, Cell Signalling), α-tubulin (clone DM1A, T9026, Sigma), LATS2 (kpm C-2, sc-515579 Santa Cruz Biotechnology), YAP (G-6, sc-376830 Santa Cruz Biotechnology), c-MYC (ab56, Abcam and N262, sc-764, Santa Cruz Biotechnology), TMPRSS2 (H-4, sc-515727, Santa Cruz Biotechnology), PARP1/2 (clone H250, sc-7150, Santa Cruz Biotechnology), FKBP5 (D-4, sc-271547, Santa Cruz Biotechnology), GFP (ab290, AbCam) Ki67 (clone MM1, Novocastra, Leica Biotechnology).

    Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction, Incubation, Fractionation, Software, Chromatin Immunoprecipitation, Binding Assay

    IKBKE effects on AR expression are phenocopied by c-MYC knockdown and partially rescued by overexpression of constitutively active Yap. ( A ) LNCaP-EV and LNCaP-Yap-S127A were reverse transfected with two individual siRNA sequences targeting IKBKE. RNA was collected after 72 h and assessed for expression of AR and IKBKE by qPCR ( n = 3). ( B ) LNCaP cells were reverse transfected with either N/S or two independent siRNA sequences targeting c-MYC. After 72 hours, RNA was collected and AR mRNA and ( C ) c-MYC expression levels determined by QPCR ( n = 3). One-way ANOVA, Dunnett's multiple comparisons test * P

    Journal: Nucleic Acids Research

    Article Title: IKBKE activity enhances AR levels in advanced prostate cancer via modulation of the Hippo pathway

    doi: 10.1093/nar/gkaa271

    Figure Lengend Snippet: IKBKE effects on AR expression are phenocopied by c-MYC knockdown and partially rescued by overexpression of constitutively active Yap. ( A ) LNCaP-EV and LNCaP-Yap-S127A were reverse transfected with two individual siRNA sequences targeting IKBKE. RNA was collected after 72 h and assessed for expression of AR and IKBKE by qPCR ( n = 3). ( B ) LNCaP cells were reverse transfected with either N/S or two independent siRNA sequences targeting c-MYC. After 72 hours, RNA was collected and AR mRNA and ( C ) c-MYC expression levels determined by QPCR ( n = 3). One-way ANOVA, Dunnett's multiple comparisons test * P

    Article Snippet: Antibodies and constructsAR (C-19, sc-815, Santa Cruz Biotechnology and clone G122-434, BD), PSA (A0562, Dako), IKBKE (D20G4, Cell Signalling), α-tubulin (clone DM1A, T9026, Sigma), LATS2 (kpm C-2, sc-515579 Santa Cruz Biotechnology), YAP (G-6, sc-376830 Santa Cruz Biotechnology), c-MYC (ab56, Abcam and N262, sc-764, Santa Cruz Biotechnology), TMPRSS2 (H-4, sc-515727, Santa Cruz Biotechnology), PARP1/2 (clone H250, sc-7150, Santa Cruz Biotechnology), FKBP5 (D-4, sc-271547, Santa Cruz Biotechnology), GFP (ab290, AbCam) Ki67 (clone MM1, Novocastra, Leica Biotechnology).

    Techniques: Expressing, Over Expression, Transfection, Real-time Polymerase Chain Reaction

    Model of IKBKE mediated AR signalling. ( A ) IKBKE up-regulation in prostate cancer enhances the ubiquitination and turnover of LATS2 in a kinase dependent manner. This is turn promotes nuclear localization of YAP and transcription of YAP regulated genes, including c-MYC. Up-regulated levels of nuclear c-MYC result in up-regulation of c-MYC regulated genes, such as AR. This results in a large pool of AR in cells which in turn, upon ligand mediated activation can also be co-activated by nuclear YAP. ( B ) Upon inhibition of IKBKE kinase activity, LATS2-Ub is impaired allowing the protein to accumulate and phosphorylate YAP. Phosphorylated YAP is held in the cytoplasm and subsequently targeted for proteasomal degradation. This results in c-MYC downregulation which in turn results in AR down-regulation, including constitutively active AR splice variants, and therefore AR regulated gene expression. Ub, ubiquitin; P, phosphate.

    Journal: Nucleic Acids Research

    Article Title: IKBKE activity enhances AR levels in advanced prostate cancer via modulation of the Hippo pathway

    doi: 10.1093/nar/gkaa271

    Figure Lengend Snippet: Model of IKBKE mediated AR signalling. ( A ) IKBKE up-regulation in prostate cancer enhances the ubiquitination and turnover of LATS2 in a kinase dependent manner. This is turn promotes nuclear localization of YAP and transcription of YAP regulated genes, including c-MYC. Up-regulated levels of nuclear c-MYC result in up-regulation of c-MYC regulated genes, such as AR. This results in a large pool of AR in cells which in turn, upon ligand mediated activation can also be co-activated by nuclear YAP. ( B ) Upon inhibition of IKBKE kinase activity, LATS2-Ub is impaired allowing the protein to accumulate and phosphorylate YAP. Phosphorylated YAP is held in the cytoplasm and subsequently targeted for proteasomal degradation. This results in c-MYC downregulation which in turn results in AR down-regulation, including constitutively active AR splice variants, and therefore AR regulated gene expression. Ub, ubiquitin; P, phosphate.

    Article Snippet: Antibodies and constructsAR (C-19, sc-815, Santa Cruz Biotechnology and clone G122-434, BD), PSA (A0562, Dako), IKBKE (D20G4, Cell Signalling), α-tubulin (clone DM1A, T9026, Sigma), LATS2 (kpm C-2, sc-515579 Santa Cruz Biotechnology), YAP (G-6, sc-376830 Santa Cruz Biotechnology), c-MYC (ab56, Abcam and N262, sc-764, Santa Cruz Biotechnology), TMPRSS2 (H-4, sc-515727, Santa Cruz Biotechnology), PARP1/2 (clone H250, sc-7150, Santa Cruz Biotechnology), FKBP5 (D-4, sc-271547, Santa Cruz Biotechnology), GFP (ab290, AbCam) Ki67 (clone MM1, Novocastra, Leica Biotechnology).

    Techniques: Activation Assay, Inhibition, Activity Assay, Expressing

    Interaction of Loqs isoforms with Dicer1 and Dicer2. ( A ) The Loqs-containing complexes were immunoprecipitated from S2 cells using anti-Loqs antibodies and the proteins contained in the immunoprecipitates were visualized by silver staining. The protein bands corresponding to Loqs-PA, Loqs-PB, and Loqs-PC/Loqs-PD are indicated. Two proteins coimmunopurified with Loqs were identified as Dicer1 and Dicer2 by mass spectrometry analyses. (n.i.) Nonimmune IgG used as a negative control. ( B ) Western blot analyses using anti-Dicer1 and anti-Dicer2 antibodies confirmed that the immunoprecipitates obtained using anti-Loqs antibodies (panel A ) contain both Dicer proteins, along with all Loqs isoforms. ( C ) Loqs-PA and Loqs-PB interact with Dicer1, whereas Loqs-PD interacts with Dicer2. Each Loqs isoform was expressed in S2 cells, and the anti-myc immunoprecipitated complexes were probed with anti-Dicer1 and anti-Dicer2 antibodies. (h.c.) Heavy chain of anti-c-myc antibody. ( D ) Deletion mutant analysis of Loqs-PD to determine the domain necessary for interacting with Dicer2. Various deletion mutants of Loqs-PD (m1–m5) were expressed in S2 cells, and their interactions with endogenous Dicer2 were examined. The Loqs-PD-m4 mutant interacted with Dicer2 as efficiently as wt Loqs-PD. The dsRNA-binding domains (light gray boxes) and C-terminal region (dark gray boxes) found exclusively in Loqs-PD but not in other Loqs isoforms, respectively, are indicated ( upper diagram). The lower panel shows immunoblotting results of the protein–protein interaction assays. EGFP was used as a negative control.

    Journal: RNA

    Article Title: Molecular mechanisms that funnel RNA precursors into endogenous small-interfering RNA and microRNA biogenesis pathways in Drosophila

    doi: 10.1261/rna.1952110

    Figure Lengend Snippet: Interaction of Loqs isoforms with Dicer1 and Dicer2. ( A ) The Loqs-containing complexes were immunoprecipitated from S2 cells using anti-Loqs antibodies and the proteins contained in the immunoprecipitates were visualized by silver staining. The protein bands corresponding to Loqs-PA, Loqs-PB, and Loqs-PC/Loqs-PD are indicated. Two proteins coimmunopurified with Loqs were identified as Dicer1 and Dicer2 by mass spectrometry analyses. (n.i.) Nonimmune IgG used as a negative control. ( B ) Western blot analyses using anti-Dicer1 and anti-Dicer2 antibodies confirmed that the immunoprecipitates obtained using anti-Loqs antibodies (panel A ) contain both Dicer proteins, along with all Loqs isoforms. ( C ) Loqs-PA and Loqs-PB interact with Dicer1, whereas Loqs-PD interacts with Dicer2. Each Loqs isoform was expressed in S2 cells, and the anti-myc immunoprecipitated complexes were probed with anti-Dicer1 and anti-Dicer2 antibodies. (h.c.) Heavy chain of anti-c-myc antibody. ( D ) Deletion mutant analysis of Loqs-PD to determine the domain necessary for interacting with Dicer2. Various deletion mutants of Loqs-PD (m1–m5) were expressed in S2 cells, and their interactions with endogenous Dicer2 were examined. The Loqs-PD-m4 mutant interacted with Dicer2 as efficiently as wt Loqs-PD. The dsRNA-binding domains (light gray boxes) and C-terminal region (dark gray boxes) found exclusively in Loqs-PD but not in other Loqs isoforms, respectively, are indicated ( upper diagram). The lower panel shows immunoblotting results of the protein–protein interaction assays. EGFP was used as a negative control.

    Article Snippet: To obtain myc-tagged Loqs proteins, immunoprecipitation was performed using a monoclonal anti-c-myc antibody (9E10, catalog number M4439, Sigma-Aldrich).

    Techniques: Immunoprecipitation, Silver Staining, Mass Spectrometry, Negative Control, Western Blot, Mutagenesis, Binding Assay

    Expression of p300 and MYC in a panel of human B-lymphoma cell lines. Extracts from the indicated human B-lymphoma cell lines were analyzed by Western blotting for p300, MYC, and β-tubulin (as a loading control). In a given panel, all lanes contain

    Journal: Cancer letters

    Article Title: A rearranged EP300 gene in the human B-cell lymphoma cell line RC-K8 encodes a disabled transcriptional co-activator that contributes to cell growth and oncogenicity

    doi: 10.1016/j.canlet.2010.12.018

    Figure Lengend Snippet: Expression of p300 and MYC in a panel of human B-lymphoma cell lines. Extracts from the indicated human B-lymphoma cell lines were analyzed by Western blotting for p300, MYC, and β-tubulin (as a loading control). In a given panel, all lanes contain

    Article Snippet: The following antisera were used: rabbit anti-p300 (1:200; anti-N-terminal, sc-584, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-MYC (1:1000, sc-40, Santa Cruz Biotechnology), and rabbit anti-β-tubulin (1:500; sc-9104, Santa Cruz Biotechnology).

    Techniques: Expressing, Western Blot

    Expression of berghepain-1 in liver stage parasites. HepG2 cells infected with BP1-myc parasites, and merosomes collected from these cultures, were fixed at the indicated timepoints and probed with anti-c-myc antibodies (magenta) to localize berghepain-1, and co-localized with antibodies against A. the cytosolic marker heat-shock protein 70 (HSP70), B. the ER chaperone BIP (green) and C. the membrane maker merozoite surface protein 1 (MSP-1). BP1-myc expression was low 24 h after invasion, but seen in early schizonts after 36 h of development. Host and parasite DNA were stained with DAPI (blue). Scale bars: 10 μm.

    Journal: PLoS Pathogens

    Article Title: Deletion of the rodent malaria ortholog for falcipain-1 highlights differences between hepatic and blood stage merozoites

    doi: 10.1371/journal.ppat.1006586

    Figure Lengend Snippet: Expression of berghepain-1 in liver stage parasites. HepG2 cells infected with BP1-myc parasites, and merosomes collected from these cultures, were fixed at the indicated timepoints and probed with anti-c-myc antibodies (magenta) to localize berghepain-1, and co-localized with antibodies against A. the cytosolic marker heat-shock protein 70 (HSP70), B. the ER chaperone BIP (green) and C. the membrane maker merozoite surface protein 1 (MSP-1). BP1-myc expression was low 24 h after invasion, but seen in early schizonts after 36 h of development. Host and parasite DNA were stained with DAPI (blue). Scale bars: 10 μm.

    Article Snippet: The following antibodies were used, diluted in 1% BSA/PBS: mouse anti-MSP 25.1 diluted 1:500 [ ], polyclonal rabbit anti-UIS4 diluted 1:5000 [ ], mouse anti-CSP at 1 μg/ml [clone 3D11; [ ]], mouse anti-Plasmodium HSP-70 diluted 1:500 [clone 2E6; [ ]], mouse anti-BiP diluted 1:200 [ ], and rabbit anti-c-myc diluted 1:400 (C3956, Sigma).

    Techniques: Expressing, Infection, Marker, Staining

    BTB reduces ER α , c-Myc, and Cyclin D1 protein expression levels in MCF-7, Ishikawa, and SKOV-3 cells but has no effect on ER α gene expression at mRNA level. Western blot analyses of ER α , c-Myc, and Cyclin D1 levels in control and 2.5 uM BTB treated MCF-7, Ishikawa, and SKOV-3 cells in the absence or presence of 10 nM E2. 50 μ g of total protein from cells was applied onto a 10% sodium dodecyl sulfate-polyacrylamide gel and subjected to electrophoresis followed by Western blot using anti-ER α , anti-c-Myc, or anti-Cyclin D1 antibodies. Representative graphs were shown from consistent results collected from three independent experiments.

    Journal: BioMed Research International

    Article Title: The Wedelolactone Derivative Inhibits Estrogen Receptor-Mediated Breast, Endometrial, and Ovarian Cancer Cells Growth

    doi: 10.1155/2014/713263

    Figure Lengend Snippet: BTB reduces ER α , c-Myc, and Cyclin D1 protein expression levels in MCF-7, Ishikawa, and SKOV-3 cells but has no effect on ER α gene expression at mRNA level. Western blot analyses of ER α , c-Myc, and Cyclin D1 levels in control and 2.5 uM BTB treated MCF-7, Ishikawa, and SKOV-3 cells in the absence or presence of 10 nM E2. 50 μ g of total protein from cells was applied onto a 10% sodium dodecyl sulfate-polyacrylamide gel and subjected to electrophoresis followed by Western blot using anti-ER α , anti-c-Myc, or anti-Cyclin D1 antibodies. Representative graphs were shown from consistent results collected from three independent experiments.

    Article Snippet: The blots were probed with primary anti-ERα (Novocastra, 6F11), anti-c-Myc (cell signaling, #9402), anti-Cyclin D1 (#2922), anti-Stat3 (#9132), anti-pStat3 (#9145), anti-Akt (#9272), and anti-pAkt(S-473) (#9271) antibodies with dilutions of 1 : 500 to 1 : 1,000 and incubated at room temperature for 2 hrs.

    Techniques: Expressing, Western Blot, Electrophoresis

    OXSR1 and STK39 are required for pre-TCR induced proliferation and regulation of MYC (A-C) Mean±SEM number of thymocytes at each developmental stage in Stk39 +/+ and Stk39 T243A/T243A mice (A) or in Rag1 -/- radiation chimeras reconstituted with fetal liver cells from Oxsr1 +/+ and Oxsr1 T185A/T185A embryos (B) or Oxsr1 +/+ Stk39 +/+ /RCE, Oxsr1 fl/fl Stk39 +/+ /RCE and Oxsr1 fl/fl Stk39 T243A/T243A /RCE embryos (C). The chimeric mice in C were analyzed 7 d after treatment with tamoxifen. Thymocyte subsets were identified by flow cytometry as shown in Figure 1A, E . (D) Left: flow cytometric analysis of DNA content in DN4 thymocytes from Rag1 -/- radiation chimeras reconstituted with fetal liver cells of the indicated genotypes. Gates show percentage of cells containing more than the diploid amount of DNA (S, G2 and M phases of cell cycle). Right: mean±SEM percentage of cells in S/G2/M phases of the cell cycle based on gate shown on histograms. (E) Left: flow cytometric analysis of MYC levels in DN4 thymocytes from Rag1 -/- radiation chimeras reconstituted with fetal liver cells of the indicated genotypes. Right: mean±SEM levels of MYC in DN4 thymocytes. (F) Mean±SEM number of thymocytes at each developmental stage in Rag1 -/- radiation chimeras reconstituted with liver cells from Slc12a2 +/+ and Slc12a2 -/- embryos. (G) Mean±SEM percentage of cells in each thymocyte population in chimeras reconstituted with Slc12a2 -/- fetal liver normalized to Slc12a2 +/+ control chimeras (set to 100%). * 0.01

    Journal: bioRxiv

    Article Title: Critical role of WNK1 in MYC-dependent early thymocyte development

    doi: 10.1101/2020.06.11.147017

    Figure Lengend Snippet: OXSR1 and STK39 are required for pre-TCR induced proliferation and regulation of MYC (A-C) Mean±SEM number of thymocytes at each developmental stage in Stk39 +/+ and Stk39 T243A/T243A mice (A) or in Rag1 -/- radiation chimeras reconstituted with fetal liver cells from Oxsr1 +/+ and Oxsr1 T185A/T185A embryos (B) or Oxsr1 +/+ Stk39 +/+ /RCE, Oxsr1 fl/fl Stk39 +/+ /RCE and Oxsr1 fl/fl Stk39 T243A/T243A /RCE embryos (C). The chimeric mice in C were analyzed 7 d after treatment with tamoxifen. Thymocyte subsets were identified by flow cytometry as shown in Figure 1A, E . (D) Left: flow cytometric analysis of DNA content in DN4 thymocytes from Rag1 -/- radiation chimeras reconstituted with fetal liver cells of the indicated genotypes. Gates show percentage of cells containing more than the diploid amount of DNA (S, G2 and M phases of cell cycle). Right: mean±SEM percentage of cells in S/G2/M phases of the cell cycle based on gate shown on histograms. (E) Left: flow cytometric analysis of MYC levels in DN4 thymocytes from Rag1 -/- radiation chimeras reconstituted with fetal liver cells of the indicated genotypes. Right: mean±SEM levels of MYC in DN4 thymocytes. (F) Mean±SEM number of thymocytes at each developmental stage in Rag1 -/- radiation chimeras reconstituted with liver cells from Slc12a2 +/+ and Slc12a2 -/- embryos. (G) Mean±SEM percentage of cells in each thymocyte population in chimeras reconstituted with Slc12a2 -/- fetal liver normalized to Slc12a2 +/+ control chimeras (set to 100%). * 0.01

    Article Snippet: Further reagents: anti-active Caspase 3 (C92-605, BD Pharmingen), anti-CXCR4 (ID9, BD Pharmingen), anti-MYC (Y69, Abcam), anti-pS325-OXSR1/pS383-STK39 (MRC-PPU), anti-pS235/pS236-S6 (D57.2.2E, Cell Signaling); 7AAD, Foxp3/Transcription factor staining buffer set (eBioscience), mouse CXCL12, mouse ICAM1-Fc (R & D systems), FxCycle Violet Stain, LIVE/DEAD NearIR (Thermo Fisher), Zombie Aqua (Biolegend).

    Techniques: Mouse Assay, Flow Cytometry

    Overexpression of MXD1 repressed c-Myc and miR-19a/b levels and impeded the malignant phenotypes of gastric cancer cells. ( a ) Western blot of anti-HA and anti-c-Myc using HA-tagged MXD1 over-expressing cells and the vector control cells. ( b and c ) Effects of MXD1 in in vitro migration and invasion assays. ( b ) Representative pictures show effect of MXD1 and si-MXD1 on the migration and invasion of gastric cancer cells. ( c ) Quantification of migration and invasion assays of B (** P ≤0.01 compared with the vector control). Each experiment was repeated at least three times. Error bars correspond to the mean±S.D. ( d ) Effects of MXD1 on in vitro proliferation in gastric cancer (** P ≤0.01, * P ≤0.05, respectively, compared with the vector control). Each experiment was repeated at least three times. Error bars correspond to the mean±S.D. ( e ) Real-time PCR show the expression levels of miR-19a and miR-19b in MXD1 over-expressing cells (** P

    Journal: Cell Death & Disease

    Article Title: MiR-19a/b modulate the metastasis of gastric cancer cells by targeting the tumour suppressor MXD1

    doi: 10.1038/cddis.2014.110

    Figure Lengend Snippet: Overexpression of MXD1 repressed c-Myc and miR-19a/b levels and impeded the malignant phenotypes of gastric cancer cells. ( a ) Western blot of anti-HA and anti-c-Myc using HA-tagged MXD1 over-expressing cells and the vector control cells. ( b and c ) Effects of MXD1 in in vitro migration and invasion assays. ( b ) Representative pictures show effect of MXD1 and si-MXD1 on the migration and invasion of gastric cancer cells. ( c ) Quantification of migration and invasion assays of B (** P ≤0.01 compared with the vector control). Each experiment was repeated at least three times. Error bars correspond to the mean±S.D. ( d ) Effects of MXD1 on in vitro proliferation in gastric cancer (** P ≤0.01, * P ≤0.05, respectively, compared with the vector control). Each experiment was repeated at least three times. Error bars correspond to the mean±S.D. ( e ) Real-time PCR show the expression levels of miR-19a and miR-19b in MXD1 over-expressing cells (** P

    Article Snippet: Membranes were blocked with 10% non-fat milk powder at room temperature for 2 h and incubated overnight with primary antibodies: anti-MXD1 (1 : 1000; Abcam plc, Cambridge, MA, USA), anti-c-Myc (1 : 1000; Abcam plc) or anti-β -actin antibody (1 : 2000; Sigma-Aldrich Co., St. Louis, MO, USA).

    Techniques: Over Expression, Western Blot, Expressing, Plasmid Preparation, In Vitro, Migration, Real-time Polymerase Chain Reaction

    Accumulated LC3-positive structures did not colocalize with phagophore and lysosome markers in DOX-treated TetON-GFPSTX17ΔNTD and TetON-GFPSTX17FL HeLa cells. (A-C) TetON-GFPSTX17ΔNTD and TetON-GFPSTX17FL HeLa cells were cultured with DOX (1.5 μg/ml) for 2 d. Cells were cultured in regular (Gr) or starvation (St) medium for 1 h and analyzed by immunofluorescence microscopy using antibodies against RB1CC1 (A), WIPI2 (B), and LAMP1 (C). Colocalization analysis was performed as described in Materials and Methods. Each correlation plot is derived from 30–61 cells in 3 different fields of view. The mean Pearson's correlation coefficient values ± SEM are shown on the plots and the graphs. The intensities of GFP-STX17 variants are represented on the x-axis. Asterisks indicate significant differences between MYC-tagged STX17 variants at p

    Journal: Autophagy

    Article Title: Accumulation of undegraded autophagosomes by expression of dominant-negative STX17 (syntaxin 17) mutants

    doi: 10.1080/15548627.2017.1327940

    Figure Lengend Snippet: Accumulated LC3-positive structures did not colocalize with phagophore and lysosome markers in DOX-treated TetON-GFPSTX17ΔNTD and TetON-GFPSTX17FL HeLa cells. (A-C) TetON-GFPSTX17ΔNTD and TetON-GFPSTX17FL HeLa cells were cultured with DOX (1.5 μg/ml) for 2 d. Cells were cultured in regular (Gr) or starvation (St) medium for 1 h and analyzed by immunofluorescence microscopy using antibodies against RB1CC1 (A), WIPI2 (B), and LAMP1 (C). Colocalization analysis was performed as described in Materials and Methods. Each correlation plot is derived from 30–61 cells in 3 different fields of view. The mean Pearson's correlation coefficient values ± SEM are shown on the plots and the graphs. The intensities of GFP-STX17 variants are represented on the x-axis. Asterisks indicate significant differences between MYC-tagged STX17 variants at p

    Article Snippet: Mouse monoclonal anti-LC3 (Cosmo Bio Co., clone 1703), anti-MYC (Covance Research Products, 9E10), anti-FLAG (Sigma-Aldrich, clone M2), anti-ACTB/β-actin (Sigma-Aldrich, clone AC74), anti-HSP90AA1 (BD Biosciences, 610419), anti-EEA1 (BD Biosciences, 610456), and anti-TFRC/TfR (Invitrogen, 13–6800) antibodies, rabbit polyclonal anti-LC3, anti-MYC (Protein Tech, 16286–1-AP), anti-SQSTM1 (MBL, PM045), anti-GFP (Thermo Fisher Scientific, A-6455), anti-RB1CC1/FIP200 (Protein Tech, 10043–2-AP), anti-WIPI2 (Sigma-Aldrich, SAB4200400), anti-LAMP1 (abcam, ab24170), anti-STX17 (Sigma-Aldrich, HPA001204), anti-SEC61B (Millipore, 07–205), anti-LMAN1/ERGIC53 (Sigma-Aldrich, E1031), anti-STX5 (Synaptic Systems, 110 053), anti-TOMM20 (Santa Cruz, sc-11415), anti-mCherry (abcam, ab125096), and anti-VPS33A (GeneTex, GTX119416) antibodies, and goat polyclonal anti-LDH (abcam, ab2101) were used.

    Techniques: Cell Culture, Immunofluorescence, Microscopy, Derivative Assay

    Localization of GFP-STX17ΔNTD on autophagosomes is necessary for the dominant-negative effect. (A) WT HeLa cells were transiently transfected with the indicated plasmids. Two d later, cells were cultured in regular or starvation medium for 1 h and analyzed by immunofluorescence microscopy using anti-LC3 antibody. Representative images of 5 randomly selected regions are shown. Scale bars: 20 μm; 2 μm in insets. (B) WT HeLa cells were transiently transfected with the indicated plasmids. Two d later, cells were cultured in regular or starvation medium with or without 100 nM bafilomycin A 1 for 2 h and analyzed by immunoblotting using the indicated antibodies. (C) TetON-GFPSTX17ΔNTD HeLa cells were transiently transfected with a plasmid expressing MYC-SNAP29 or MYC-VAMP8, or stably transfected with a plasmid encoding MYC-VPS33A. Cells were fixed and stained using anti-MYC antibody and analyzed by immunofluorescence microscopy. Representative images of 5 randomly selected regions are shown. Scale bars: 10 μm; 2 μm in insets. (D) HEK293T cells were stably transfected with the indicated plasmids. Cells were lysed and separated into 2 samples, which were immunoprecipitated with (indicated with “+”) and without anti-MYC antibody in combination with protein G-Sepharose, followed by immunoblotting using anti-VPS33A, anti-MYC, and anti-ACTB antibodies.

    Journal: Autophagy

    Article Title: Accumulation of undegraded autophagosomes by expression of dominant-negative STX17 (syntaxin 17) mutants

    doi: 10.1080/15548627.2017.1327940

    Figure Lengend Snippet: Localization of GFP-STX17ΔNTD on autophagosomes is necessary for the dominant-negative effect. (A) WT HeLa cells were transiently transfected with the indicated plasmids. Two d later, cells were cultured in regular or starvation medium for 1 h and analyzed by immunofluorescence microscopy using anti-LC3 antibody. Representative images of 5 randomly selected regions are shown. Scale bars: 20 μm; 2 μm in insets. (B) WT HeLa cells were transiently transfected with the indicated plasmids. Two d later, cells were cultured in regular or starvation medium with or without 100 nM bafilomycin A 1 for 2 h and analyzed by immunoblotting using the indicated antibodies. (C) TetON-GFPSTX17ΔNTD HeLa cells were transiently transfected with a plasmid expressing MYC-SNAP29 or MYC-VAMP8, or stably transfected with a plasmid encoding MYC-VPS33A. Cells were fixed and stained using anti-MYC antibody and analyzed by immunofluorescence microscopy. Representative images of 5 randomly selected regions are shown. Scale bars: 10 μm; 2 μm in insets. (D) HEK293T cells were stably transfected with the indicated plasmids. Cells were lysed and separated into 2 samples, which were immunoprecipitated with (indicated with “+”) and without anti-MYC antibody in combination with protein G-Sepharose, followed by immunoblotting using anti-VPS33A, anti-MYC, and anti-ACTB antibodies.

    Article Snippet: Mouse monoclonal anti-LC3 (Cosmo Bio Co., clone 1703), anti-MYC (Covance Research Products, 9E10), anti-FLAG (Sigma-Aldrich, clone M2), anti-ACTB/β-actin (Sigma-Aldrich, clone AC74), anti-HSP90AA1 (BD Biosciences, 610419), anti-EEA1 (BD Biosciences, 610456), and anti-TFRC/TfR (Invitrogen, 13–6800) antibodies, rabbit polyclonal anti-LC3, anti-MYC (Protein Tech, 16286–1-AP), anti-SQSTM1 (MBL, PM045), anti-GFP (Thermo Fisher Scientific, A-6455), anti-RB1CC1/FIP200 (Protein Tech, 10043–2-AP), anti-WIPI2 (Sigma-Aldrich, SAB4200400), anti-LAMP1 (abcam, ab24170), anti-STX17 (Sigma-Aldrich, HPA001204), anti-SEC61B (Millipore, 07–205), anti-LMAN1/ERGIC53 (Sigma-Aldrich, E1031), anti-STX5 (Synaptic Systems, 110 053), anti-TOMM20 (Santa Cruz, sc-11415), anti-mCherry (abcam, ab125096), and anti-VPS33A (GeneTex, GTX119416) antibodies, and goat polyclonal anti-LDH (abcam, ab2101) were used.

    Techniques: Dominant Negative Mutation, Transfection, Cell Culture, Immunofluorescence, Microscopy, Plasmid Preparation, Expressing, Stable Transfection, Staining, Immunoprecipitation

    LC3-positive puncta accumulate upon overexpression of STX17ΔNTD. (A) Wild-type (WT) HeLa cells were transiently transfected with plasmids expressing the indicated STX17 fragments. Two d later, cells were fixed under growing conditions and analyzed by immuofluorescence microscopy using anti-LC3 and anti-MYC antibodies. Scale bars: 20 μm. (B) Quantification of the ratio of the total area of LC3-positive puncta to the total cellular area expressing the indicated STX17 variants. Cells showing significant MYC signal intensities (10% or more of the highest signal among all of the variants) were selected from 5 or 6 randomly selected regions and 18–46 cells were analyzed. Data represent mean ± SEM. Different letters indicate significant differences between MYC-tagged STX17 variants at p

    Journal: Autophagy

    Article Title: Accumulation of undegraded autophagosomes by expression of dominant-negative STX17 (syntaxin 17) mutants

    doi: 10.1080/15548627.2017.1327940

    Figure Lengend Snippet: LC3-positive puncta accumulate upon overexpression of STX17ΔNTD. (A) Wild-type (WT) HeLa cells were transiently transfected with plasmids expressing the indicated STX17 fragments. Two d later, cells were fixed under growing conditions and analyzed by immuofluorescence microscopy using anti-LC3 and anti-MYC antibodies. Scale bars: 20 μm. (B) Quantification of the ratio of the total area of LC3-positive puncta to the total cellular area expressing the indicated STX17 variants. Cells showing significant MYC signal intensities (10% or more of the highest signal among all of the variants) were selected from 5 or 6 randomly selected regions and 18–46 cells were analyzed. Data represent mean ± SEM. Different letters indicate significant differences between MYC-tagged STX17 variants at p

    Article Snippet: Mouse monoclonal anti-LC3 (Cosmo Bio Co., clone 1703), anti-MYC (Covance Research Products, 9E10), anti-FLAG (Sigma-Aldrich, clone M2), anti-ACTB/β-actin (Sigma-Aldrich, clone AC74), anti-HSP90AA1 (BD Biosciences, 610419), anti-EEA1 (BD Biosciences, 610456), and anti-TFRC/TfR (Invitrogen, 13–6800) antibodies, rabbit polyclonal anti-LC3, anti-MYC (Protein Tech, 16286–1-AP), anti-SQSTM1 (MBL, PM045), anti-GFP (Thermo Fisher Scientific, A-6455), anti-RB1CC1/FIP200 (Protein Tech, 10043–2-AP), anti-WIPI2 (Sigma-Aldrich, SAB4200400), anti-LAMP1 (abcam, ab24170), anti-STX17 (Sigma-Aldrich, HPA001204), anti-SEC61B (Millipore, 07–205), anti-LMAN1/ERGIC53 (Sigma-Aldrich, E1031), anti-STX5 (Synaptic Systems, 110 053), anti-TOMM20 (Santa Cruz, sc-11415), anti-mCherry (abcam, ab125096), and anti-VPS33A (GeneTex, GTX119416) antibodies, and goat polyclonal anti-LDH (abcam, ab2101) were used.

    Techniques: Over Expression, Transfection, Expressing, Microscopy

    Rmi1 -depletion causes a G2/M arrest during RTG. (A) Three Rmi1 -depleted mother-daughter cell pairs from an auxin-treated RMI1 -AID culture (MJL3899) taken 4 hr after shift to rich medium containing auxin. From left to right, differential interference contrast image, detection of DNA (DAPI), beta-tubulin, and Pds1 -Myc. See Materials and Methods for details. The bottom mother-daughter pair was scored as having undergone the metaphase–anaphase transition, based on the absence of Pds1 . (B) Percent of total cells in a control culture with a bud and lacking a bipolar spindle (white) or containing a bipolar spindle and nuclear Pds1 (red) or with a bipolar spindle but lacking nuclear Pds1 (blue). (C) As in (B), but in the presence of auxin. Data are from two experiments, error bars denote range.

    Journal: Genetics

    Article Title: Unresolved Recombination Intermediates Cause a RAD9-Dependent Cell Cycle Arrest in Saccharomyces cerevisiae

    doi: 10.1534/genetics.119.302632

    Figure Lengend Snippet: Rmi1 -depletion causes a G2/M arrest during RTG. (A) Three Rmi1 -depleted mother-daughter cell pairs from an auxin-treated RMI1 -AID culture (MJL3899) taken 4 hr after shift to rich medium containing auxin. From left to right, differential interference contrast image, detection of DNA (DAPI), beta-tubulin, and Pds1 -Myc. See Materials and Methods for details. The bottom mother-daughter pair was scored as having undergone the metaphase–anaphase transition, based on the absence of Pds1 . (B) Percent of total cells in a control culture with a bud and lacking a bipolar spindle (white) or containing a bipolar spindle and nuclear Pds1 (red) or with a bipolar spindle but lacking nuclear Pds1 (blue). (C) As in (B), but in the presence of auxin. Data are from two experiments, error bars denote range.

    Article Snippet: Slides were immunostained overnight at 4° or 4 hr at 30° with a mixture of the two primary antisera diluted in blocking buffer [rat anti-tubulin (ab6160 1:1250; Abcam) and rabbit anti-MYC (sc-789 1:250; Santa Cruz)], washed in PBS (three times, 5 min, room temperature), and then incubated with secondary antisera [Cy3-conjugated donkey anti-rabbit IgG (#711-165-152; Jackson Laboratories) and FITC-conjugated rabbit anti-rat IgG (# F1763; Sigma), both 1:600 in blocking buffer] for 3hr at 30°, followed by three 5-min room temperature washes in PBS.

    Techniques: