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  • 99
    Thermo Fisher rfp tag antibody
    N-terminal phosphorylated Twl peptide is important for conidiation, but not pathogenicity of M . oryzae . (A) Predicted phosphorylated Serine residues (highlighted in red) and acetylated Lysine (marked with a triangle) in the N-terminal 1–150 aa peptide of Twl. Potential p-Ser site(s) are followed immediately by the first P-loop (underlined) in Twl. Serine 34 (denoted by reversed triangle and number 34 on top) was predicted as most likely phosphorylated site by Snf1. (B) Sequence alignment for the synthesized native (WT) or mutated (S34A; M1) 1–150 aa peptide of Twl. Boxed region depicts the predicted phosphor-Serine residues, corresponding to the region highlighted in red in (A) above. The serine at residue 34 that was mutated to alanine in the M1 variant peptide is highlighted in red. (C) Twl 1-150 -mCherry was pulled down with <t>RFP-Trap</t> and probed with anti-RFP antibody. Total protein from untagged wild type was included as a control (Ctrl). (D) Bar chart depicting quantitatively assessed conidiation in wild type, twl Δ and the twl Δ expressing either wild-type or the M1 Twl 1-150 peptide). Mean values (±SE) presented as number of conidia per unit area were derived from three independent experiments (n = 30 colonies for each sample). Assessments were performed on day 2, post photo-induction. (E) The twl Δ expressing either wild-type or M1 <t>GFP-Twl</t> 1-120 peptide was grown in constant dark for three days, before being exposed to light for 12 h. Arrowheads denote the GFP- Twl 1-120 (WT) signal in the nuclei that were co-stained with DAPI. Scale bar = 5μm.
    Rfp Tag Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore anti trim27
    <t>TRIM27</t> is not involved in TNF-α-induced survival signaling. (A) TNFR1 expression. TNFR1 expression in WT and Trim27 −/− MEFs was examined by flow cytometry. (B) Expression of TNF-α signaling components. Expression levels
    Anti Trim27, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ABclonal trim27
    Trim59 promotes WASH K63-linked ubiquitination. a Model of WASH, Arp2/3, E2, E3, and K63 ubiquitin regulation on F-actin polymerization. b Immunoblotting of WASH total Ubiquitin in cotransfected HEK293T cells. HEK293T cells were cotransfected with YFP-WASH and with (+) or without ( − ) FLAG-Trim59FL or FLAG-Vector as well as with HA-Ub. Immunoprecipitation was performed by anti-WASH. Trim59 and WASH were detected by anti-FLAG and anti-WASH antibodies. The polyubiquitination of WASH was detected by anti-HA. c Immunoblotting of WASH K63-linked ubiquitin in cotransfected HEK293T cells. HEK293T cells were cotransfected with YFP-WASH and with (+) or without ( − ) FLAG-Trim59 as well as with HA-Ub, HA-K48-Ub, or HA-K63-Ub. Immunoprecipitation was performed by anti-WASH. Trim59 and WASH were detected by anti-FLAG and anti-WASH antibodies. The polyubiquitination of WASH was detected by anti-HA. d Immunoblotting of WASH K63-linked ubiquitin in cotransfected HEK293T cells. HEK293T cells were cotransfected with YFP-WASH or YFP-K220R and with (+) or without (−) FLAG-Trim59 as well as with HA-K63-Ub. Immunoprecipitation was performed by anti-WASH. Trim59, WASH and WASH variant were detected by anti-FLAG and anti-WASH antibodies. Polyubiquitination of WASH was detected by anti-HA. e Immunoblotting of WASH K63-linked ubiquitin in cotransfected HEK293T cells. HEK293T cells were cotransfected with FLAG-Trim59FL and its fragments (T2 toT5) and with YFP-WASH (+) as well as HA-K63-Ub (+). Immunoprecipitation was performed by anti-WASH. Trim59, Trim59 fragments and WASH were detected by anti-FLAG and anti-WASH antibodies. Polyubiquitination of WASH was detected by anti-HA. f Immunoblotting of total Ub and K63-Ub of endogenous WASH from F1 ESCs after treated with specific Trim59 and <t>Trim27</t> siRNA.Immunoprecipitation was performed by anti-WASH. Trim59, Trim27, and WASH were detected by anti-Trim59, anti-Trim27, and anti-WASH antibodies. Immunoblot analysis of Ub-WASH (top blot), K63-Ub-WASH (below blot) by anti-Ub and anti- K63 antibodies. g Immunofluorescence assay of F-actin assembly in WASH siRNA or siRNA control transfected F1 ESCs. Scale bar, 10 µm. * P
    Trim27, supplied by ABclonal, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam trim27
    <t>TRIM27</t> facilitates the migration and invasion of colorectal cancer cells. A wound-healing assay was performed in (A) LoVo and (B) HCT116 cells following TRIM27 knockdown and overexpression. Quantitative results in the wound-healing assay are shown. A Transwell assay was performed in (C) LoVo and (D) HCT116 cells following TRIM27 knockdown and overexpression. Magnification, ×200. (E) Numbers of migrated LoVo and HCT116 cells through the membrane. (F) Numbers of invaded LoVo and HCT116 cells through the Matrigel. Each assay was performed three times. Data are presented as the mean ± standard deviation; ** P
    Trim27, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Millipore anti trim27 antibody
    Recruitment of <t>TRIM27</t> by SHP2 inhibits VSV-triggered type I IFN production in macrophages. (A) IP and immunoblot analyses of the indicated proteins in HEK293T cells co-transfected with TRIM27 and SHP2 as indicated. (B) Immunoblot analysis of DAP12 and TRIM27 in SHP2-immunoprecipitated products from lysates of VSV-infected (MOI = 10) macrophages. (C) IP and immunoblot analyses of the indicated proteins in HEK293T cells co-transfected with TRIM27 and DAP12, SHP2, or SHP1 as indicated. (D) Immunoblot analysis of TRIM27 in macrophages transfected with TRIM27 siRNA as indicated for 48 h. (E) Macrophages were transfected as in D and then infected with VSV (MOI = 10) or HSV (MOI = 10) for 12 h. Q-PCR analysis was performed to evaluate IFN-β and VSV RNA levels in cells, and ELISA was performed to evaluate IFN-β protein levels in the supernatants. (F) Immunoblot analysis of TRIM27 in RAW264.7 cell clones stably overexpressing TRIM27. (G) RAW264.7 cell clones stably overexpressing TRIM27 were infected with VSV (MOI = 10) or HSV (MOI = 10) for the indicated time. Q-PCR analysis of intracellular IFN-β mRNA and VSV RNA levels and ELISA of IFN-β in the supernatants were then performed. (H) Immunoblot analysis of the indicated molecules in lysates of macrophages transfected with TRIM27 siRNA#1 and infected with VSV (MOI = 10). (I) IFN-β luciferase activity in HEK293T cells transfected with TBK1 or IRF3(5D), and Siglec1 or TRIM27 as indicated. (J) Q-PCR analysis of IFN-β mRNA levels in SHP2-deficient macrophages transfected with Siglec1 or TRIM27 siRNA and infected with VSV. (K) Q-PCR analysis of IFN-β mRNA levels in Siglec1 stably overexpressing-RAW264.7 cell clones that were transfected with TRIM27 siRNA and infected with VSV for 8 h. Data are shown as mean ± SD or representative photographs. * P
    Anti Trim27 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Proteintech rabbit trim27 polyclonal
    Recruitment of <t>TRIM27</t> by SHP2 inhibits VSV-triggered type I IFN production in macrophages. (A) IP and immunoblot analyses of the indicated proteins in HEK293T cells co-transfected with TRIM27 and SHP2 as indicated. (B) Immunoblot analysis of DAP12 and TRIM27 in SHP2-immunoprecipitated products from lysates of VSV-infected (MOI = 10) macrophages. (C) IP and immunoblot analyses of the indicated proteins in HEK293T cells co-transfected with TRIM27 and DAP12, SHP2, or SHP1 as indicated. (D) Immunoblot analysis of TRIM27 in macrophages transfected with TRIM27 siRNA as indicated for 48 h. (E) Macrophages were transfected as in D and then infected with VSV (MOI = 10) or HSV (MOI = 10) for 12 h. Q-PCR analysis was performed to evaluate IFN-β and VSV RNA levels in cells, and ELISA was performed to evaluate IFN-β protein levels in the supernatants. (F) Immunoblot analysis of TRIM27 in RAW264.7 cell clones stably overexpressing TRIM27. (G) RAW264.7 cell clones stably overexpressing TRIM27 were infected with VSV (MOI = 10) or HSV (MOI = 10) for the indicated time. Q-PCR analysis of intracellular IFN-β mRNA and VSV RNA levels and ELISA of IFN-β in the supernatants were then performed. (H) Immunoblot analysis of the indicated molecules in lysates of macrophages transfected with TRIM27 siRNA#1 and infected with VSV (MOI = 10). (I) IFN-β luciferase activity in HEK293T cells transfected with TBK1 or IRF3(5D), and Siglec1 or TRIM27 as indicated. (J) Q-PCR analysis of IFN-β mRNA levels in SHP2-deficient macrophages transfected with Siglec1 or TRIM27 siRNA and infected with VSV. (K) Q-PCR analysis of IFN-β mRNA levels in Siglec1 stably overexpressing-RAW264.7 cell clones that were transfected with TRIM27 siRNA and infected with VSV for 8 h. Data are shown as mean ± SD or representative photographs. * P
    Rabbit Trim27 Polyclonal, supplied by Proteintech, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    GenePharma Company trim27
    <t>TRIM27</t> facilitates the migration and invasion of colorectal cancer cells. A wound-healing assay was performed in (A) LoVo and (B) HCT116 cells following TRIM27 knockdown and overexpression. Quantitative results in the wound-healing assay are shown. A Transwell assay was performed in (C) LoVo and (D) HCT116 cells following TRIM27 knockdown and overexpression. Magnification, ×200. (E) Numbers of migrated LoVo and HCT116 cells through the membrane. (F) Numbers of invaded LoVo and HCT116 cells through the Matrigel. Each assay was performed three times. Data are presented as the mean ± standard deviation; ** P
    Trim27, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 94/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Santa Cruz Biotechnology trim27
    <t>TRIM27</t> promotes the activation of JNK and p38 pathways and suppresses NF-κB activation. ( a,b ) Luciferase assay of of AP-1 (a ) and NF-κB ( b ) activation in U937 cells. Cells were transfected with luciferase siRNA (NC siRNA) or TRIM27 siRNA or TRIM27 siRNA complemented with TRIM27 (ΔRING), and then were infected with WT BCG or BCG (ΔPtpA) or the BCG (ΔPtpA) complemented with PtpA D126A (ΔPtpA+D126A) at a MOI of 10 for 24 h. ( c ) Immunoblot analysis of phosphorylated IκBα, JNK, p38, Erk and GAPDH (loading control) in U937 cells transfected with luciferase siRNA (NC siRNA) or TRIM27 siRNA and infected with BCG at a MOI of 10 for 0–48 h. Non-infected cells served as a control group. Data are shown as the means ± s.e.m. * P
    Trim27, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    IBL America anti trim27
    <t>TRIM27</t> is not involved in TNF-α-induced survival signaling. (A) TNFR1 expression. TNFR1 expression in WT and Trim27 −/− MEFs was examined by flow cytometry. (B) Expression of TNF-α signaling components. Expression levels
    Anti Trim27, supplied by IBL America, used in various techniques. Bioz Stars score: 86/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Millipore rabbit anti trim27
    <t>TRIM27</t> negatively regulates NOD2 signaling. A–C) NF-κB luciferase assays in HEK293T cells to determine the influence of TRIM27 overexpression on MDP-induced NOD2-mediated (A), TNF- (B), or IKK-β-induced (C) NF-κB activation. Normalized luciferase activity (nRLU) of unstimulated (white bars) and stimulated (black bars) samples is shown. Values are given as mean+SD (n = 3). *, P
    Rabbit Anti Trim27, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rfp  (Abcam)
    95
    Abcam rfp
    Cell-to-cell movement analysis of rSYNV sc4, M and G deletion mutants. (A) Schematic representation of rSYNV-GFP and rSYNV-GFP mutants with Δsc4, ΔM or ΔG deletions. Note that the <t>SYNV</t> gene order is shown in the antigenome sense. (B) Cell-to-cell movement of rSYNV-GFP and deletion derivatives. N . benthamiana leaves were agroinfiltrated with the rSYNV-GFP plasmid or the indicated mutant rSYNV-GFP plasmids along with supporting plasmids for expression of the N, P, L core proteins and the VSRs. Leaves were photographed with a fluorescence microscope at 8 and 14 dpi. Scale bar, 100 μm. (C) Complementation of rSYNV-eGFP-Δsc4 cell-to-cell movement by the sc4 protein expressed in trans from the <t>MR-sc4-RFP</t> minireplicon. Agrobacterium strains harboring the rSYNV-eGFP-Δsc4 plasmid, the MR-sc4-RFP plasmid, along with the supporting plasmids indicated in the panel B legend, were mixed and infiltrated into N . benthamiana leaves. Fluorescence images for GFP, RFP and the overlaid images are shown at 8 and 14 dpi. Scale bar, 100 μm.
    Rfp, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 289 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    GE Healthcare anti trim27
    Protein sequence of <t>TRIM27</t> and its expression were determined by RT-PCR and western blotting. A. The human TRIM27 cDNA encodes a protein of 513 amino acids; B. TRIM27 mRNAs are highly expressed in testis and ovary tissues. C. Western blotting shows TRIM27 expressed in both the testes and ovaries.
    Anti Trim27, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Addgene inc rfp coelomocyte rfp marker
    TOMM-40 promotes insulin secretion. ( A ) Adults, carrying an integrated daf-28::gfp transgene ( svIs69) , were analyzed for <t>coelomocyte</t> GFP content. Absent or severely reduced GFP content (a maximum of two faintly fluorescing coelomocytes) was scored as secretion defective. ( B ) Neuronal expression of GFP from the transcriptional reporter transgene P daf-28 ::gfp in tomm-40(peRNAi) and emv(peRNAi) treated animals, imaged with fluorescence optics. ( C ) Fluorescence optics of peRNAi treated animals of the arIs37 strain, carrying a P myo-3 ::ssgfp transgene that expresses ssGFP in body wall muscle, from where it is secreted into the pseudocoelom. Coelomocyte sequestration of GFP indicates functional coelomocyte endocytosis. rme-1(b1045) is a previously characterized endocytosis defective mutant [39] . Arrows indicate GFP labeled coelomocytes. ( D ) Fluorescence micrographs of emv, tomm-20 and tomm-22(RNAi) -treated animals carrying the P hsp-6 ::gfp reporter. ( E–F ) Relative pixel intensity plots of coelomocyte GFP contents in peRNAi or RNAi treated adults carrying ( E ) an Anf::gfp transgene or ( F ) an ins-22::venus transgene. The strongest fluorescing coelomocyte in the posterior most pair was scored. The mean value of the pixel intensity in emv was set to 1 in each experiment. Error bars represent +/− mean standard deviations from three independent experiments. ( G ) Micrographs of RNAi treated sibling animals, carrying either the integrated daf-16::gfp transgene only, or both the integrated daf-16::gfp transgene and an extra chromosomal P daf-28 ::daf-28 transgene for DAF-28 overexpression. Presence of the P daf-28 ::daf-28 transgene is indicated by a coelomocyte <t>RFP</t> co-injection marker (red arrows). White arrowheads indicate nuclear DAF-16::GFP localization in a tomm-40(RNAi) animal. White arrows indicate the absence of DAF-16::GFP in nuclei of intestinal cells in a tomm-40(RNAi); daf-28(++) animal. Scale bars are 25 µm.
    Rfp Coelomocyte Rfp Marker, supplied by Addgene inc, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Novus Biologicals rfp antibody
    TOMM-40 promotes insulin secretion. ( A ) Adults, carrying an integrated daf-28::gfp transgene ( svIs69) , were analyzed for <t>coelomocyte</t> GFP content. Absent or severely reduced GFP content (a maximum of two faintly fluorescing coelomocytes) was scored as secretion defective. ( B ) Neuronal expression of GFP from the transcriptional reporter transgene P daf-28 ::gfp in tomm-40(peRNAi) and emv(peRNAi) treated animals, imaged with fluorescence optics. ( C ) Fluorescence optics of peRNAi treated animals of the arIs37 strain, carrying a P myo-3 ::ssgfp transgene that expresses ssGFP in body wall muscle, from where it is secreted into the pseudocoelom. Coelomocyte sequestration of GFP indicates functional coelomocyte endocytosis. rme-1(b1045) is a previously characterized endocytosis defective mutant [39] . Arrows indicate GFP labeled coelomocytes. ( D ) Fluorescence micrographs of emv, tomm-20 and tomm-22(RNAi) -treated animals carrying the P hsp-6 ::gfp reporter. ( E–F ) Relative pixel intensity plots of coelomocyte GFP contents in peRNAi or RNAi treated adults carrying ( E ) an Anf::gfp transgene or ( F ) an ins-22::venus transgene. The strongest fluorescing coelomocyte in the posterior most pair was scored. The mean value of the pixel intensity in emv was set to 1 in each experiment. Error bars represent +/− mean standard deviations from three independent experiments. ( G ) Micrographs of RNAi treated sibling animals, carrying either the integrated daf-16::gfp transgene only, or both the integrated daf-16::gfp transgene and an extra chromosomal P daf-28 ::daf-28 transgene for DAF-28 overexpression. Presence of the P daf-28 ::daf-28 transgene is indicated by a coelomocyte <t>RFP</t> co-injection marker (red arrows). White arrowheads indicate nuclear DAF-16::GFP localization in a tomm-40(RNAi) animal. White arrows indicate the absence of DAF-16::GFP in nuclei of intestinal cells in a tomm-40(RNAi); daf-28(++) animal. Scale bars are 25 µm.
    Rfp Antibody, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 99/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Abcam anti rfp
    CDS overexpression inhibits LD expansion. HeLa cells were transfected with eCFP-N1 and <t>eCFP-CDS1</t> or mCherry-N1 and CDS2-mCherry for 48 h, followed by the treatment with oleate for 16 h. A, D: The protein lysates from untreated cells were subjected to Western blots against CDS1 (A), <t>RFP</t> (D), and GAPDH. B, E: LDs were stained with Nile Red (B) or BODIPY 493/503 (E) in oleate-treated cells and visualized using a confocal microscope. Bar = 10 μm. Arrows indicate the CDS-overexpressing cells. C, F: LD size distribution in HeLa cells was determined. Data were collected by measuring the diameter of at least 500 LDs using the ImageJ software. G: Cellular TAG level was determined by TLC following neutral lipid extraction and quantified using ImageJ software. Data are expressed as mean ± SD (n = 4). ** P
    Anti Rfp, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 308 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Bioss rfp ptripz rfp ires not1
    CDS overexpression inhibits LD expansion. HeLa cells were transfected with eCFP-N1 and <t>eCFP-CDS1</t> or mCherry-N1 and CDS2-mCherry for 48 h, followed by the treatment with oleate for 16 h. A, D: The protein lysates from untreated cells were subjected to Western blots against CDS1 (A), <t>RFP</t> (D), and GAPDH. B, E: LDs were stained with Nile Red (B) or BODIPY 493/503 (E) in oleate-treated cells and visualized using a confocal microscope. Bar = 10 μm. Arrows indicate the CDS-overexpressing cells. C, F: LD size distribution in HeLa cells was determined. Data were collected by measuring the diameter of at least 500 LDs using the ImageJ software. G: Cellular TAG level was determined by TLC following neutral lipid extraction and quantified using ImageJ software. Data are expressed as mean ± SD (n = 4). ** P
    Rfp Ptripz Rfp Ires Not1, supplied by Bioss, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher rfp
    Co-expression of mutant hSOD1 fused to <t>RFP</t> with mutant hSOD1 fused to YFP. In a matrix approach, all possible combinations for the 6 fusion constructs of mutant <t>SOD1</t> fused to RFP or YFP were examined. In all cases, inclusions contained both proteins in saponin-resistant aggregates. At least three independent transfection experiments were performed and between 200 and 1,000 individual cells were analyzed in compiling these data.
    Rfp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 217 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    BioVision rfp
    Rh1 and TRPL accumulate within culd 1 photoreceptors. (A,B) The left row shows a schematic diagrams of cross-sectional (A) and longitudinal views (B) of photoreceptor cells from a single ommatidium. The rhabdomere (R) is indicated. The right rows show the whole-mount staining of Rh1 in cross-sectional (A) and longitudinal (B) views. Eyes from <t>ninaE-rh1-gfp</t> flies were dissected and stained for Rh1 (red). GFP fluorescence of Rh1–GFP was directly observed (green). (C–E) Rh1 and TRPL accumulated in large vesicles within the cytoplasm of culd 1 photoreceptor cells. Compound eyes from (C) wild-type (wt) ( ninaE-trpl-gfp ), (D) culd 1 ( cn bw; ninaE-trpl-gfp culd 1 ) and (E) rescue ( cn bw; <t>ninaE-culd-rfp</t> culd 1 ) flies were dissected and immunostained for Rh1. The ninaE-trpl-gfp transgene was present in all genotypes and GFP signals were directly observed. Two-day-old flies with white eyes were placed in the dark for 12 h before being exposed to 2000 Lux white light for 2.5 h. Scale bars: 10 µm.
    Rfp, supplied by BioVision, used in various techniques. Bioz Stars score: 91/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Rockland Immunochemicals rfp
    Extrinsic factors are the major determinant of progenitor cell identity in the developing mouse lung. (A) Experimental outline. Epithelial progenitors (tip or stalk) were microdissected from donor E12.5 or E16.5 Tomato + ( Rosa26R mT−mG/+ ) lungs and grafted into the mesenchyme of unlabelled E12.5 hosts. Hosts and grafts were cultured for 8 days without Dx, or with addition of 50 nM Dx at culture day 4 or 5, followed by serial sectioning and staining to determine graft fate. (A′,A″) Grafts integrate into the host lung, grow and form a lumen. (B-G) Sections of grafted lungs with alternate slides stained for: green, <t>LPCAT1</t> (alveolar fate); red, Tomato (graft); white, PDPN (basal and AT1 cells), or: green, SOX2 (bronchiolar fate); red, <t>RFP</t> (Tomato + graft); white, acetylated tubulin (ACT; cilia) to determine graft fate. Examples of tip grafts with alveolar, bronchiolar and mixed broncho-alveolar fate are shown. Note D/D′ and G/G′ are different sections of the same graft. (H,I) Quantitation of graft fate as a percentage of numbers of grafts analysed. Each type of graft was analysed in at least three independent experiments. Scale bars: 25 μm in A″, 100 μm in B-G′.
    Rfp, supplied by Rockland Immunochemicals, used in various techniques. Bioz Stars score: 99/100, based on 470 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam red fluorescent protein rfp
    Extrinsic factors are the major determinant of progenitor cell identity in the developing mouse lung. (A) Experimental outline. Epithelial progenitors (tip or stalk) were microdissected from donor E12.5 or E16.5 Tomato + ( Rosa26R mT−mG/+ ) lungs and grafted into the mesenchyme of unlabelled E12.5 hosts. Hosts and grafts were cultured for 8 days without Dx, or with addition of 50 nM Dx at culture day 4 or 5, followed by serial sectioning and staining to determine graft fate. (A′,A″) Grafts integrate into the host lung, grow and form a lumen. (B-G) Sections of grafted lungs with alternate slides stained for: green, <t>LPCAT1</t> (alveolar fate); red, Tomato (graft); white, PDPN (basal and AT1 cells), or: green, SOX2 (bronchiolar fate); red, <t>RFP</t> (Tomato + graft); white, acetylated tubulin (ACT; cilia) to determine graft fate. Examples of tip grafts with alveolar, bronchiolar and mixed broncho-alveolar fate are shown. Note D/D′ and G/G′ are different sections of the same graft. (H,I) Quantitation of graft fate as a percentage of numbers of grafts analysed. Each type of graft was analysed in at least three independent experiments. Scale bars: 25 μm in A″, 100 μm in B-G′.
    Red Fluorescent Protein Rfp, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam rabbit anti rfp
    IR56d is expressed in two populations of neurons in the labellum. a Immunofluorescence with <t>anti-RFP</t> (magenta), overlaid on bright-field images, on a whole-mount proboscis of a w;UAS-mCD8:RFP;Ir56d-Gal4 animal. The left image corresponds to the maximal projection of the inner face of one labellar palp, and the right image corresponds to the surface of one labellar palp. Scale bar: 25 μm. b Schematic representing the anatomical location of the taste peg neurons (orange) and taste bristle neurons (blue) in the labellum. c Immunofluorescence with anti-GFP (green), <t>anti-IR25a</t> (blue) and anti-RFP (magenta) on a whole-mount proboscis of a w;LexAop-mCD8:GFP-2A-mCD8:GFP/UAS-mCD8:RFP;Ir56d-LexA/Ir76b-Gal4 animal. The images show a close-up of taste peg neurons (arrowheads) to visualise the co-localisation of the three markers. Scale bar: 25 μm. d Immunofluorescence with anti-RFP (magenta) and anti-GFP (green) on a whole-mount proboscis of a w;LexAop-mCD8:GFP-2A-mCD8:GFP/UAS-mCD8:RFP;NompC-LexA/Ir56d-Gal4 animal. The inset in the merged image shows a bright-field view of the imaged tissue (here and in the following panels). Scale bar: 25 μm. e Immunofluorescence with anti-RFP (magenta) and anti-GFP (green) on a whole-mount proboscis of a w;LexAop-mCD8:GFP-2A-mCD8:GFP/E409-Gal4;Ir56d-LexA/UASCD4:tdTomato animal. Scale bar: 25 μm. f Immunofluorescence with anti-RFP (magenta) and anti-GFP (green) on a whole-mount proboscis of a Gr66a-LexA/+; LexAop-rCD2:GFP/UAS-mCD8:RFP;Ir56d-Gal4/(TM6B or TM2) animal. Scale bar: 25 μm. g Immunofluorescence with anti-RFP (magenta) and anti-GFP (green) on a whole-mount proboscis of a w;LexAop-mCD8:GFP-2A-mCD8:GFP/UAS-mCD8:RFP;Gr64f-LexA/Ir56d-Gal4 animal. Scale bar: 25 μm. h Immunofluorescence with anti-RFP (magenta), anti-GFP (green) and nc82 (blue) on a whole-mount brain of a w;LexAop-mCD8:GFP-2A-mCD8:GFP/UAS-mCD8:RFP;Gr64f-LexA/Ir56d-Gal4 animal. Both left and middle panels show the expression of only the Ir56d-Gal4 driver. The left panel shows the maximal projection of the anterior SEZ; the middle panel shows the maximal projection of the most posterior optical slices of the SEZ. The right panel shows the overlay of the Ir56d-Gal4 - and Gr64f-LexA -expressing populations. AMS1 anterior maxillary sensory zone 1, PMS4 posterior maxillary sensory zone 4. Scale bar: 50 μm
    Rabbit Anti Rfp, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 457 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Capital Biosciences adeno rfp
    IR56d is expressed in two populations of neurons in the labellum. a Immunofluorescence with <t>anti-RFP</t> (magenta), overlaid on bright-field images, on a whole-mount proboscis of a w;UAS-mCD8:RFP;Ir56d-Gal4 animal. The left image corresponds to the maximal projection of the inner face of one labellar palp, and the right image corresponds to the surface of one labellar palp. Scale bar: 25 μm. b Schematic representing the anatomical location of the taste peg neurons (orange) and taste bristle neurons (blue) in the labellum. c Immunofluorescence with anti-GFP (green), <t>anti-IR25a</t> (blue) and anti-RFP (magenta) on a whole-mount proboscis of a w;LexAop-mCD8:GFP-2A-mCD8:GFP/UAS-mCD8:RFP;Ir56d-LexA/Ir76b-Gal4 animal. The images show a close-up of taste peg neurons (arrowheads) to visualise the co-localisation of the three markers. Scale bar: 25 μm. d Immunofluorescence with anti-RFP (magenta) and anti-GFP (green) on a whole-mount proboscis of a w;LexAop-mCD8:GFP-2A-mCD8:GFP/UAS-mCD8:RFP;NompC-LexA/Ir56d-Gal4 animal. The inset in the merged image shows a bright-field view of the imaged tissue (here and in the following panels). Scale bar: 25 μm. e Immunofluorescence with anti-RFP (magenta) and anti-GFP (green) on a whole-mount proboscis of a w;LexAop-mCD8:GFP-2A-mCD8:GFP/E409-Gal4;Ir56d-LexA/UASCD4:tdTomato animal. Scale bar: 25 μm. f Immunofluorescence with anti-RFP (magenta) and anti-GFP (green) on a whole-mount proboscis of a Gr66a-LexA/+; LexAop-rCD2:GFP/UAS-mCD8:RFP;Ir56d-Gal4/(TM6B or TM2) animal. Scale bar: 25 μm. g Immunofluorescence with anti-RFP (magenta) and anti-GFP (green) on a whole-mount proboscis of a w;LexAop-mCD8:GFP-2A-mCD8:GFP/UAS-mCD8:RFP;Gr64f-LexA/Ir56d-Gal4 animal. Scale bar: 25 μm. h Immunofluorescence with anti-RFP (magenta), anti-GFP (green) and nc82 (blue) on a whole-mount brain of a w;LexAop-mCD8:GFP-2A-mCD8:GFP/UAS-mCD8:RFP;Gr64f-LexA/Ir56d-Gal4 animal. Both left and middle panels show the expression of only the Ir56d-Gal4 driver. The left panel shows the maximal projection of the anterior SEZ; the middle panel shows the maximal projection of the most posterior optical slices of the SEZ. The right panel shows the overlay of the Ir56d-Gal4 - and Gr64f-LexA -expressing populations. AMS1 anterior maxillary sensory zone 1, PMS4 posterior maxillary sensory zone 4. Scale bar: 50 μm
    Adeno Rfp, supplied by Capital Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addgene inc lamp1 rfp
    TAPE is localized in endolysosomes. A and B , 293 cells were transfected with EGFP-tagged hTAPE and its deletion mutants, together with an endosomal protein Rab5-DsRed ( A ) or a lysosomal protein <t>Lamp1-RFP</t> ( B ). Transfected cells were then passed into a
    Lamp1 Rfp, supplied by Addgene inc, used in various techniques. Bioz Stars score: 99/100, based on 254 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addgene inc lifeact rfp
    Tau-mEOS2 is localized to axons, dendrites and the soma but excluded from the nucleus. Mature DIV18 hippocampal cultures from Tau-mEOS2 mice cotransfected with the spine marker <t>Lifeact-RFP</t> to reveal dendritic spines. (a) Tau-mEOS2 mice show a strong axonal expression of EOS-tagged Tau, with a gradient towards the growth cone of distal axons. (b) Tau-mEOS2 is also present in the somatodendritic domain, ( c ) with very low levels in dendritic spines (zoom-in, white arrow: dendrite; yellow arrow: axon). ( d ) Within the limit of detection, EOS-tagged endogenous Tau cannot be visualized in the nucleus. (e) Time-lapse imaging of photo-converted mEOS2-tagged Tau reveals bidirectional transport of Tau in the axon at about ~0.25 μm/s. Yellow arrows indicate how far photo-converted Tau travelled. Scale bar: 10 μm ( a – d ) and 15 μm ( e ).
    Lifeact Rfp, supplied by Addgene inc, used in various techniques. Bioz Stars score: 94/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genechem nr3c1 rfp
    Tau-mEOS2 is localized to axons, dendrites and the soma but excluded from the nucleus. Mature DIV18 hippocampal cultures from Tau-mEOS2 mice cotransfected with the spine marker <t>Lifeact-RFP</t> to reveal dendritic spines. (a) Tau-mEOS2 mice show a strong axonal expression of EOS-tagged Tau, with a gradient towards the growth cone of distal axons. (b) Tau-mEOS2 is also present in the somatodendritic domain, ( c ) with very low levels in dendritic spines (zoom-in, white arrow: dendrite; yellow arrow: axon). ( d ) Within the limit of detection, EOS-tagged endogenous Tau cannot be visualized in the nucleus. (e) Time-lapse imaging of photo-converted mEOS2-tagged Tau reveals bidirectional transport of Tau in the axon at about ~0.25 μm/s. Yellow arrows indicate how far photo-converted Tau travelled. Scale bar: 10 μm ( a – d ) and 15 μm ( e ).
    Nr3c1 Rfp, supplied by Genechem, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Vector Biolabs reporter rfp
    Genetic complementation of Apc Δ/Δ Cdh1 fl/fl adenoma organoid cells with adenovirus either expressing wild-type or <t>EC1</t> domain mutants of Cdh1 a. Adenovirus transfection of Apc Δ/Δ Cdh1 +/+ and Apc Δ/Δ Cdh1 fl/fl intestinal adenoma organoids with Ad-Cre-GFP, <t>Ad-Cdh1-WT-RFP</t> and Ad-Cdh1-Mut-RFP. Ad-Cre-GFP is expressed in the outer cells of the Apc Δ/Δ Cdh1 +/+ control organoid, and in cells of the disrupted organoid in Apc Δ/Δ Cdh1 fl/fl . Expression of either Ad-Cdh1-WT-RFP or Ad-Cdh1-Mut-RFP results in small contracted organoids in both genotypes. Complementation of Apc Δ/Δ Cdh1 fl/fl Ad-Cre-GFP with Ad-Cdh1-WT-RFP, results in adherent double expressing (yellow) cells in both genotypes. For complementation with Ad-Cdh1-Mut-RFP, double expressing cells (yellow) appear less adherent and separate. b d. Flow cytometry profiles of E-cadherin and β-catenin antibody binding to Apc Δ/Δ Cdh1 +/+ adenoma organoid cells with adenoviral transfection. With Ad-Cre-GFP alone, there was no effect on overall E-cadherin and β-catenin expression, but when combined with either Ad-Cdh1-WT-RFP or Ad-Cdh1-Mut-RFP, results in increased E-cadherin and β-catenin. In Apc Δ/Δ Cdh1 fl/fl cells, loss of E-cadherin and β-catenin occurs following Ad-Cre-GFP transfection, and is rescued by either Ad-Cdh1-WT-RFP or Ad-Cdh1-Mut-RFP. (c) In Apc Δ/Δ Cdh1 +/+ , infection with Ad-Cre-GFP does not result in E-cadherin loss (top row), and Ad-Cdh1-WT-RFP (middle row) or Ad-Cdh1-Mut-RFP (bottom row) expression results in contracted organoids as in a. Note that β-catenin appears cytoplasmic and membrane bound. e. In Apc Δ/Δ Cdh1 fl/fl (top row) Ad-Cre-GFP transfection results in loss of E-cadherin, cytoplasmic and nuclear localisation of β-catenin, and disrupted organoid morphology (large, separate, rounded or elongated) as in a. Complementation of Apc Δ/Δ Cdh1 fl/fl is shown in two example panels. For Ad-Cre-GFP with Ad-Cdh1-WT-RFP (middle row), results in adherent double expressing (yellow) cells, associated with expression of E-cadherin (arrows, left panel) and predominantly membrane bound β-catenin (arrows, right panel). For complementation with Ad-Cdh1-Mut-RFP (yellow cells, bottom row, left panel), this results predominantly in non-adherent cells, with mainly cytoplasmic and nuclear β-catenin expression (white arrow heads, right panel). Scale bar 100μm in (a), otherwise 50μm (c, e).
    Reporter Rfp, supplied by Vector Biolabs, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Addgene inc rfp clc
    TIRF microscopy of clathrin light chain <t>(YFP-CLC</t> or <t>RFP-CLC)</t> reveals an increased lifetime that is accentuated by transferrin labeling (cargo loading) on the basal surface of fibroblasts in MIIB KO cells. (A) Panels on left show kymographs of YFP-CLC in Wt and MIIB KO cells and also in MIIB KO cells following partial rescue by transient expression of the MIIB heavy chain. The MIIB KO shows greater persistence of spots (longer streaks). Images were taken at 5 s intervals. Panels on right show recordings from regions of interest containing a few spots. In the MIIB KO some spots (circled in red) persist for prolonged periods. Time is in seconds. Note that time 0 represents the beginning of the example sequence not the initiation of recording (the spots appeared during the recording). (B) Quantitative analysis of YFP-CLC lifetime. The average lifetime per cell (±S.E.M) is shown along with the average for the group (thick horizontal black line). At minimum, 50 spots were analyzed in each cell. MIIB KO cells show a significant increase in lifetime (t-test; P=0.02, the number of cells is represented by the points on the graph; from 3–4 cultures). This is an underrepresentation of the difference because 2–10% of the YFP-CLC spots in the MIIB KO cells persisted throughout the entire recording and were not included in the analysis. Rescue by transient expression of the MIIB heavy chain in the MIIB KO cells was done blindly (using an adenovirus vector to infect cells) because the vector did not express a fluorescent marker protein. Typical infection rates were 75–80% and most cells showed a shorter lifetime compared to MIIB KO cells. A few cells had longer lifetimes and were negative for MIIB heavy chain expression as determined by post hoc fixation and immunolabeling. (C) Comparison of lifetimes in transferrin-labeled and unlabeled cells. YFP-CLC lifetimes in the MIIB KO cells increase after transferrin labeling. Cells were labeled with Alexa-546 transferrin (100 μg/ml) and then recorded. The difference was not significant in the Wt cells (t-test, P=0.09, N=8 cells each from 3 cultures) but was significant in the MIIB KO cells (t-test, P=0.02, N=8 each from 3 cultures). (D) On left are examples of kymographs for Wt and MIIB KO cells expressing RFP-CLC. On the right are RFP-CLC spots from cells analyzed for lifetime by TIRF microscopy. Co-expression of either GFP-MIIB or GFP-R709C MIIB with RFP-CLC decreased the signal to noise. (E) Quantitative analysis of lifetimes from kymographs. Expression of the Wt MIIB decreased lifetimes MIIB KO cells (rescue), but expression of the mutated MIIB had no detectable affect on lifetimes. Difference were significant, t-test left to right * P≤0.001, Wt, N=12 cells from 4 cultures, MIIB KO N=11 cells from 4 cultures, ** P≤0.001, GFP-MIIB expression in MIIB KO cells N=8 cells from 3 cultures, ***P≤0.001, GFP-R790C expression in MIIB KO cells, N=8 cells from 3 cultures.
    Rfp Clc, supplied by Addgene inc, used in various techniques. Bioz Stars score: 87/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Nikon rfp filter
    TIRF microscopy of clathrin light chain <t>(YFP-CLC</t> or <t>RFP-CLC)</t> reveals an increased lifetime that is accentuated by transferrin labeling (cargo loading) on the basal surface of fibroblasts in MIIB KO cells. (A) Panels on left show kymographs of YFP-CLC in Wt and MIIB KO cells and also in MIIB KO cells following partial rescue by transient expression of the MIIB heavy chain. The MIIB KO shows greater persistence of spots (longer streaks). Images were taken at 5 s intervals. Panels on right show recordings from regions of interest containing a few spots. In the MIIB KO some spots (circled in red) persist for prolonged periods. Time is in seconds. Note that time 0 represents the beginning of the example sequence not the initiation of recording (the spots appeared during the recording). (B) Quantitative analysis of YFP-CLC lifetime. The average lifetime per cell (±S.E.M) is shown along with the average for the group (thick horizontal black line). At minimum, 50 spots were analyzed in each cell. MIIB KO cells show a significant increase in lifetime (t-test; P=0.02, the number of cells is represented by the points on the graph; from 3–4 cultures). This is an underrepresentation of the difference because 2–10% of the YFP-CLC spots in the MIIB KO cells persisted throughout the entire recording and were not included in the analysis. Rescue by transient expression of the MIIB heavy chain in the MIIB KO cells was done blindly (using an adenovirus vector to infect cells) because the vector did not express a fluorescent marker protein. Typical infection rates were 75–80% and most cells showed a shorter lifetime compared to MIIB KO cells. A few cells had longer lifetimes and were negative for MIIB heavy chain expression as determined by post hoc fixation and immunolabeling. (C) Comparison of lifetimes in transferrin-labeled and unlabeled cells. YFP-CLC lifetimes in the MIIB KO cells increase after transferrin labeling. Cells were labeled with Alexa-546 transferrin (100 μg/ml) and then recorded. The difference was not significant in the Wt cells (t-test, P=0.09, N=8 cells each from 3 cultures) but was significant in the MIIB KO cells (t-test, P=0.02, N=8 each from 3 cultures). (D) On left are examples of kymographs for Wt and MIIB KO cells expressing RFP-CLC. On the right are RFP-CLC spots from cells analyzed for lifetime by TIRF microscopy. Co-expression of either GFP-MIIB or GFP-R709C MIIB with RFP-CLC decreased the signal to noise. (E) Quantitative analysis of lifetimes from kymographs. Expression of the Wt MIIB decreased lifetimes MIIB KO cells (rescue), but expression of the mutated MIIB had no detectable affect on lifetimes. Difference were significant, t-test left to right * P≤0.001, Wt, N=12 cells from 4 cultures, MIIB KO N=11 cells from 4 cultures, ** P≤0.001, GFP-MIIB expression in MIIB KO cells N=8 cells from 3 cultures, ***P≤0.001, GFP-R790C expression in MIIB KO cells, N=8 cells from 3 cultures.
    Rfp Filter, supplied by Nikon, used in various techniques. Bioz Stars score: 93/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addgene inc rfp promoter
    TIRF microscopy of clathrin light chain <t>(YFP-CLC</t> or <t>RFP-CLC)</t> reveals an increased lifetime that is accentuated by transferrin labeling (cargo loading) on the basal surface of fibroblasts in MIIB KO cells. (A) Panels on left show kymographs of YFP-CLC in Wt and MIIB KO cells and also in MIIB KO cells following partial rescue by transient expression of the MIIB heavy chain. The MIIB KO shows greater persistence of spots (longer streaks). Images were taken at 5 s intervals. Panels on right show recordings from regions of interest containing a few spots. In the MIIB KO some spots (circled in red) persist for prolonged periods. Time is in seconds. Note that time 0 represents the beginning of the example sequence not the initiation of recording (the spots appeared during the recording). (B) Quantitative analysis of YFP-CLC lifetime. The average lifetime per cell (±S.E.M) is shown along with the average for the group (thick horizontal black line). At minimum, 50 spots were analyzed in each cell. MIIB KO cells show a significant increase in lifetime (t-test; P=0.02, the number of cells is represented by the points on the graph; from 3–4 cultures). This is an underrepresentation of the difference because 2–10% of the YFP-CLC spots in the MIIB KO cells persisted throughout the entire recording and were not included in the analysis. Rescue by transient expression of the MIIB heavy chain in the MIIB KO cells was done blindly (using an adenovirus vector to infect cells) because the vector did not express a fluorescent marker protein. Typical infection rates were 75–80% and most cells showed a shorter lifetime compared to MIIB KO cells. A few cells had longer lifetimes and were negative for MIIB heavy chain expression as determined by post hoc fixation and immunolabeling. (C) Comparison of lifetimes in transferrin-labeled and unlabeled cells. YFP-CLC lifetimes in the MIIB KO cells increase after transferrin labeling. Cells were labeled with Alexa-546 transferrin (100 μg/ml) and then recorded. The difference was not significant in the Wt cells (t-test, P=0.09, N=8 cells each from 3 cultures) but was significant in the MIIB KO cells (t-test, P=0.02, N=8 each from 3 cultures). (D) On left are examples of kymographs for Wt and MIIB KO cells expressing RFP-CLC. On the right are RFP-CLC spots from cells analyzed for lifetime by TIRF microscopy. Co-expression of either GFP-MIIB or GFP-R709C MIIB with RFP-CLC decreased the signal to noise. (E) Quantitative analysis of lifetimes from kymographs. Expression of the Wt MIIB decreased lifetimes MIIB KO cells (rescue), but expression of the mutated MIIB had no detectable affect on lifetimes. Difference were significant, t-test left to right * P≤0.001, Wt, N=12 cells from 4 cultures, MIIB KO N=11 cells from 4 cultures, ** P≤0.001, GFP-MIIB expression in MIIB KO cells N=8 cells from 3 cultures, ***P≤0.001, GFP-R790C expression in MIIB KO cells, N=8 cells from 3 cultures.
    Rfp Promoter, supplied by Addgene inc, used in various techniques. Bioz Stars score: 94/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam mouse anti rfp
    Trafficking of proteasomes versus synaptobrevin in motor neurons. (A) Top, the set up used to record movies of axonal transport of Syb–GFP (left) and <t>Prosβ5–RFP</t> (right) in axons of motor neurons projecting from the ventral nerve cord (VNC) of the Drosophila third-instar larvae. Bottom, representative kymographs from live-imaging of Syb–GFP (left kymograph) and Prosβ5–RFP (right kymograph) particles in Drosophila third-instar larvae motor neurons ( OK6-Gal4/UAS-syb-GFP , OK6-Gal4,UAS-prosβ5-RFP;UAS-prosβ5-RFP ). Kymographs represent cumulative movement (displacement on the x -axis) over time ( y -axis). Mobile particles appear as diagonal lines moving either in the anterograde (to the right) or retrograde (to the left) direction. (B) Average proportion of Syb–GFP (light gray, n =429 particles) and Prosβ5–RFP (dark gray, n =325 particles) particles moving in anterograde direction, retrograde direction, and that are stationary and reversing. (C,D) Mean run length (C) and mean segmental velocity (D) of Syb–GFP (light gray, n =197 particles) and Prosβ5–RFP (dark gray, n =138 particles) particles moving in the anterograde and retrograde direction as determined from analysis of live imaging; 10–20 animals per genotype were analyzed. (E,F) Normalized frequency distribution of mean anterograde (E) and retrograde (F) segmental velocities of Syb–GFP (light gray) and Prosβ5–RFP (dark gray) during axonal transport as determined from analysis of live imaging. Error bars indicate s.e.m. * P
    Mouse Anti Rfp, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    TaKaRa pqcxix rfp
    Trafficking of proteasomes versus synaptobrevin in motor neurons. (A) Top, the set up used to record movies of axonal transport of Syb–GFP (left) and <t>Prosβ5–RFP</t> (right) in axons of motor neurons projecting from the ventral nerve cord (VNC) of the Drosophila third-instar larvae. Bottom, representative kymographs from live-imaging of Syb–GFP (left kymograph) and Prosβ5–RFP (right kymograph) particles in Drosophila third-instar larvae motor neurons ( OK6-Gal4/UAS-syb-GFP , OK6-Gal4,UAS-prosβ5-RFP;UAS-prosβ5-RFP ). Kymographs represent cumulative movement (displacement on the x -axis) over time ( y -axis). Mobile particles appear as diagonal lines moving either in the anterograde (to the right) or retrograde (to the left) direction. (B) Average proportion of Syb–GFP (light gray, n =429 particles) and Prosβ5–RFP (dark gray, n =325 particles) particles moving in anterograde direction, retrograde direction, and that are stationary and reversing. (C,D) Mean run length (C) and mean segmental velocity (D) of Syb–GFP (light gray, n =197 particles) and Prosβ5–RFP (dark gray, n =138 particles) particles moving in the anterograde and retrograde direction as determined from analysis of live imaging; 10–20 animals per genotype were analyzed. (E,F) Normalized frequency distribution of mean anterograde (E) and retrograde (F) segmental velocities of Syb–GFP (light gray) and Prosβ5–RFP (dark gray) during axonal transport as determined from analysis of live imaging. Error bars indicate s.e.m. * P
    Pqcxix Rfp, supplied by TaKaRa, used in various techniques. Bioz Stars score: 89/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    N-terminal phosphorylated Twl peptide is important for conidiation, but not pathogenicity of M . oryzae . (A) Predicted phosphorylated Serine residues (highlighted in red) and acetylated Lysine (marked with a triangle) in the N-terminal 1–150 aa peptide of Twl. Potential p-Ser site(s) are followed immediately by the first P-loop (underlined) in Twl. Serine 34 (denoted by reversed triangle and number 34 on top) was predicted as most likely phosphorylated site by Snf1. (B) Sequence alignment for the synthesized native (WT) or mutated (S34A; M1) 1–150 aa peptide of Twl. Boxed region depicts the predicted phosphor-Serine residues, corresponding to the region highlighted in red in (A) above. The serine at residue 34 that was mutated to alanine in the M1 variant peptide is highlighted in red. (C) Twl 1-150 -mCherry was pulled down with RFP-Trap and probed with anti-RFP antibody. Total protein from untagged wild type was included as a control (Ctrl). (D) Bar chart depicting quantitatively assessed conidiation in wild type, twl Δ and the twl Δ expressing either wild-type or the M1 Twl 1-150 peptide). Mean values (±SE) presented as number of conidia per unit area were derived from three independent experiments (n = 30 colonies for each sample). Assessments were performed on day 2, post photo-induction. (E) The twl Δ expressing either wild-type or M1 GFP-Twl 1-120 peptide was grown in constant dark for three days, before being exposed to light for 12 h. Arrowheads denote the GFP- Twl 1-120 (WT) signal in the nuclei that were co-stained with DAPI. Scale bar = 5μm.

    Journal: PLoS Pathogens

    Article Title: Twilight, a Novel Circadian-Regulated Gene, Integrates Phototropism with Nutrient and Redox Homeostasis during Fungal Development

    doi: 10.1371/journal.ppat.1004972

    Figure Lengend Snippet: N-terminal phosphorylated Twl peptide is important for conidiation, but not pathogenicity of M . oryzae . (A) Predicted phosphorylated Serine residues (highlighted in red) and acetylated Lysine (marked with a triangle) in the N-terminal 1–150 aa peptide of Twl. Potential p-Ser site(s) are followed immediately by the first P-loop (underlined) in Twl. Serine 34 (denoted by reversed triangle and number 34 on top) was predicted as most likely phosphorylated site by Snf1. (B) Sequence alignment for the synthesized native (WT) or mutated (S34A; M1) 1–150 aa peptide of Twl. Boxed region depicts the predicted phosphor-Serine residues, corresponding to the region highlighted in red in (A) above. The serine at residue 34 that was mutated to alanine in the M1 variant peptide is highlighted in red. (C) Twl 1-150 -mCherry was pulled down with RFP-Trap and probed with anti-RFP antibody. Total protein from untagged wild type was included as a control (Ctrl). (D) Bar chart depicting quantitatively assessed conidiation in wild type, twl Δ and the twl Δ expressing either wild-type or the M1 Twl 1-150 peptide). Mean values (±SE) presented as number of conidia per unit area were derived from three independent experiments (n = 30 colonies for each sample). Assessments were performed on day 2, post photo-induction. (E) The twl Δ expressing either wild-type or M1 GFP-Twl 1-120 peptide was grown in constant dark for three days, before being exposed to light for 12 h. Arrowheads denote the GFP- Twl 1-120 (WT) signal in the nuclei that were co-stained with DAPI. Scale bar = 5μm.

    Article Snippet: Primary antibodies used include: anti-GFP, Invitrogen- Molecular Probes, A6455; anti-RFP, Invitrogen- Molecular Probes, R10367; anti-Porin, Invitrogen, 459500; anti-PhoSer, Santa Cruz, sc-81514; anti-ACK, Acetyl Lysine, ab21623, at recommended dilutions.

    Techniques: Sequencing, Synthesized, Variant Assay, Expressing, Derivative Assay, Staining

    Snf1 kinase dependent phosphorylation drives the translocation of GFP-Twl into the nucleus in response to phototropic cues. (A) WT or snf1 Δ mutant expressing GFP-Twl and hH1-RFP (as a marker of nuclei), were grown in constant dark or under constant illumination (12 h) and subjected to confocal microscopy. GFP-Twl co-localizes with nuclei marked with hH1-RFP in the WT in response to light exposure, but not in the snf1 Δ mutant. Scale bar = 10μm. Arrows denote the overlap between GFP-Twl and the nuclei. Arrowheads denote nuclei (hH1-RFP) without GFP-Twl. (B) GFP-Twl is phosphorylated by Snf1 during phototropic growth. The GFP-Twl strain or snf1 Δ expressing GFP-Twl was grown in the dark or subjected to photo-induction for 12 h before total protein extraction. GFP-Trap samples from the total lysates were probed with anti-PhoSer antibody to detect phosphorylation on Serine residue(s), and then stripped and re-probed with anti-GFP as a loading control. The same membrane was stripped and re-probed with anti-AcetLysine (anti-AcK) to detect possible acetylation on Twl.

    Journal: PLoS Pathogens

    Article Title: Twilight, a Novel Circadian-Regulated Gene, Integrates Phototropism with Nutrient and Redox Homeostasis during Fungal Development

    doi: 10.1371/journal.ppat.1004972

    Figure Lengend Snippet: Snf1 kinase dependent phosphorylation drives the translocation of GFP-Twl into the nucleus in response to phototropic cues. (A) WT or snf1 Δ mutant expressing GFP-Twl and hH1-RFP (as a marker of nuclei), were grown in constant dark or under constant illumination (12 h) and subjected to confocal microscopy. GFP-Twl co-localizes with nuclei marked with hH1-RFP in the WT in response to light exposure, but not in the snf1 Δ mutant. Scale bar = 10μm. Arrows denote the overlap between GFP-Twl and the nuclei. Arrowheads denote nuclei (hH1-RFP) without GFP-Twl. (B) GFP-Twl is phosphorylated by Snf1 during phototropic growth. The GFP-Twl strain or snf1 Δ expressing GFP-Twl was grown in the dark or subjected to photo-induction for 12 h before total protein extraction. GFP-Trap samples from the total lysates were probed with anti-PhoSer antibody to detect phosphorylation on Serine residue(s), and then stripped and re-probed with anti-GFP as a loading control. The same membrane was stripped and re-probed with anti-AcetLysine (anti-AcK) to detect possible acetylation on Twl.

    Article Snippet: Primary antibodies used include: anti-GFP, Invitrogen- Molecular Probes, A6455; anti-RFP, Invitrogen- Molecular Probes, R10367; anti-Porin, Invitrogen, 459500; anti-PhoSer, Santa Cruz, sc-81514; anti-ACK, Acetyl Lysine, ab21623, at recommended dilutions.

    Techniques: Translocation Assay, Mutagenesis, Expressing, Marker, Confocal Microscopy, Protein Extraction

    TRIM27 is not involved in TNF-α-induced survival signaling. (A) TNFR1 expression. TNFR1 expression in WT and Trim27 −/− MEFs was examined by flow cytometry. (B) Expression of TNF-α signaling components. Expression levels

    Journal: Molecular and Cellular Biology

    Article Title: Ubiquitination-Deubiquitination by the TRIM27-USP7 Complex Regulates Tumor Necrosis Factor Alpha-Induced Apoptosis

    doi: 10.1128/MCB.00465-13

    Figure Lengend Snippet: TRIM27 is not involved in TNF-α-induced survival signaling. (A) TNFR1 expression. TNFR1 expression in WT and Trim27 −/− MEFs was examined by flow cytometry. (B) Expression of TNF-α signaling components. Expression levels

    Article Snippet: Anti-cleaved caspase-3 (Cell Signaling), anti-TNFR1 (R & D Systems), anti-TRADD (Santa Cruz), anti-TRAF2 (Santa Cruz), anti-RIP1 (BD Bioscience), anti-FADD (Santa Cruz), anti-CYLD (Santa Cruz), anti-USP7 (Bethyl Laboratories), anti-TRIM27 (IBL Chemicals), and anti-α-tubulin (Sigma) antibodies were utilized to check the respective endogenous protein levels by Western blotting.

    Techniques: Expressing, Flow Cytometry, Cytometry

    USP7 is required for the TRIM27-induced deubiquitination of RIP1. (A) Formation of a complex between TRIM27 and USP7. The TRIM27 complex was purified from HeLa cells expressing FLAG- and HA-tagged TRIM27 (FH-TRIM27) or control HeLa cells (mock) using

    Journal: Molecular and Cellular Biology

    Article Title: Ubiquitination-Deubiquitination by the TRIM27-USP7 Complex Regulates Tumor Necrosis Factor Alpha-Induced Apoptosis

    doi: 10.1128/MCB.00465-13

    Figure Lengend Snippet: USP7 is required for the TRIM27-induced deubiquitination of RIP1. (A) Formation of a complex between TRIM27 and USP7. The TRIM27 complex was purified from HeLa cells expressing FLAG- and HA-tagged TRIM27 (FH-TRIM27) or control HeLa cells (mock) using

    Article Snippet: Anti-cleaved caspase-3 (Cell Signaling), anti-TNFR1 (R & D Systems), anti-TRADD (Santa Cruz), anti-TRAF2 (Santa Cruz), anti-RIP1 (BD Bioscience), anti-FADD (Santa Cruz), anti-CYLD (Santa Cruz), anti-USP7 (Bethyl Laboratories), anti-TRIM27 (IBL Chemicals), and anti-α-tubulin (Sigma) antibodies were utilized to check the respective endogenous protein levels by Western blotting.

    Techniques: Purification, Expressing

    Localization of TRIM27 in mitochondria. (A) Subcellular localization of TRIM27. HepG2 cells were transfected with a FLAG-TRIM27 expression vector, incubated with Mitotracker, fixed with 1% paraformaldehyde, and incubated with an anti-FLAG antibody. After

    Journal: Molecular and Cellular Biology

    Article Title: Ubiquitination-Deubiquitination by the TRIM27-USP7 Complex Regulates Tumor Necrosis Factor Alpha-Induced Apoptosis

    doi: 10.1128/MCB.00465-13

    Figure Lengend Snippet: Localization of TRIM27 in mitochondria. (A) Subcellular localization of TRIM27. HepG2 cells were transfected with a FLAG-TRIM27 expression vector, incubated with Mitotracker, fixed with 1% paraformaldehyde, and incubated with an anti-FLAG antibody. After

    Article Snippet: Anti-cleaved caspase-3 (Cell Signaling), anti-TNFR1 (R & D Systems), anti-TRADD (Santa Cruz), anti-TRAF2 (Santa Cruz), anti-RIP1 (BD Bioscience), anti-FADD (Santa Cruz), anti-CYLD (Santa Cruz), anti-USP7 (Bethyl Laboratories), anti-TRIM27 (IBL Chemicals), and anti-α-tubulin (Sigma) antibodies were utilized to check the respective endogenous protein levels by Western blotting.

    Techniques: Transfection, Expressing, Plasmid Preparation, Incubation

    TRIM27 positively regulates TNF-α-induced apoptosis. (A) Resistance of wild-type (WT) and Trim27 −/− mice to TNF-α cytotoxicity. WT ( n = 6) and Trim27 −/− ( n = 10) mice were treated with TNF-α (20

    Journal: Molecular and Cellular Biology

    Article Title: Ubiquitination-Deubiquitination by the TRIM27-USP7 Complex Regulates Tumor Necrosis Factor Alpha-Induced Apoptosis

    doi: 10.1128/MCB.00465-13

    Figure Lengend Snippet: TRIM27 positively regulates TNF-α-induced apoptosis. (A) Resistance of wild-type (WT) and Trim27 −/− mice to TNF-α cytotoxicity. WT ( n = 6) and Trim27 −/− ( n = 10) mice were treated with TNF-α (20

    Article Snippet: Anti-cleaved caspase-3 (Cell Signaling), anti-TNFR1 (R & D Systems), anti-TRADD (Santa Cruz), anti-TRAF2 (Santa Cruz), anti-RIP1 (BD Bioscience), anti-FADD (Santa Cruz), anti-CYLD (Santa Cruz), anti-USP7 (Bethyl Laboratories), anti-TRIM27 (IBL Chemicals), and anti-α-tubulin (Sigma) antibodies were utilized to check the respective endogenous protein levels by Western blotting.

    Techniques: Mouse Assay

    Model describing the role of TRIM27-USP7 in TNF-α-induced apoptosis. Upon binding to TNF-α, TNFR1 binds to TRADD and triggers the formation of complex I and II. Complex I, containing TRADD, TRAF2, TRAF5, RIP1, cIAP1, and cIAP2, activates

    Journal: Molecular and Cellular Biology

    Article Title: Ubiquitination-Deubiquitination by the TRIM27-USP7 Complex Regulates Tumor Necrosis Factor Alpha-Induced Apoptosis

    doi: 10.1128/MCB.00465-13

    Figure Lengend Snippet: Model describing the role of TRIM27-USP7 in TNF-α-induced apoptosis. Upon binding to TNF-α, TNFR1 binds to TRADD and triggers the formation of complex I and II. Complex I, containing TRADD, TRAF2, TRAF5, RIP1, cIAP1, and cIAP2, activates

    Article Snippet: Anti-cleaved caspase-3 (Cell Signaling), anti-TNFR1 (R & D Systems), anti-TRADD (Santa Cruz), anti-TRAF2 (Santa Cruz), anti-RIP1 (BD Bioscience), anti-FADD (Santa Cruz), anti-CYLD (Santa Cruz), anti-USP7 (Bethyl Laboratories), anti-TRIM27 (IBL Chemicals), and anti-α-tubulin (Sigma) antibodies were utilized to check the respective endogenous protein levels by Western blotting.

    Techniques: Binding Assay

    TRIM27-USP7 regulation of RIP1 ubiquitination in the presence of TNF-α and CHX. (A) Knockdown of USP7 using siRNA in immortalized MEF. Three different sequences of mouse USP7 siRNA were transfected, and cell lysates were analyzed by Western blotting

    Journal: Molecular and Cellular Biology

    Article Title: Ubiquitination-Deubiquitination by the TRIM27-USP7 Complex Regulates Tumor Necrosis Factor Alpha-Induced Apoptosis

    doi: 10.1128/MCB.00465-13

    Figure Lengend Snippet: TRIM27-USP7 regulation of RIP1 ubiquitination in the presence of TNF-α and CHX. (A) Knockdown of USP7 using siRNA in immortalized MEF. Three different sequences of mouse USP7 siRNA were transfected, and cell lysates were analyzed by Western blotting

    Article Snippet: Anti-cleaved caspase-3 (Cell Signaling), anti-TNFR1 (R & D Systems), anti-TRADD (Santa Cruz), anti-TRAF2 (Santa Cruz), anti-RIP1 (BD Bioscience), anti-FADD (Santa Cruz), anti-CYLD (Santa Cruz), anti-USP7 (Bethyl Laboratories), anti-TRIM27 (IBL Chemicals), and anti-α-tubulin (Sigma) antibodies were utilized to check the respective endogenous protein levels by Western blotting.

    Techniques: Transfection, Western Blot

    TRIM27 deubiquitinates RIP1. (A) Deubiquitination of RIP1 by TRIM27. HEK293T cells were transfected with the FLAG-RIP1 expression vector or control empty vector, together with the Myc-Ub expression vector and increasing amounts of the TRIM27 expression

    Journal: Molecular and Cellular Biology

    Article Title: Ubiquitination-Deubiquitination by the TRIM27-USP7 Complex Regulates Tumor Necrosis Factor Alpha-Induced Apoptosis

    doi: 10.1128/MCB.00465-13

    Figure Lengend Snippet: TRIM27 deubiquitinates RIP1. (A) Deubiquitination of RIP1 by TRIM27. HEK293T cells were transfected with the FLAG-RIP1 expression vector or control empty vector, together with the Myc-Ub expression vector and increasing amounts of the TRIM27 expression

    Article Snippet: Anti-cleaved caspase-3 (Cell Signaling), anti-TNFR1 (R & D Systems), anti-TRADD (Santa Cruz), anti-TRAF2 (Santa Cruz), anti-RIP1 (BD Bioscience), anti-FADD (Santa Cruz), anti-CYLD (Santa Cruz), anti-USP7 (Bethyl Laboratories), anti-TRIM27 (IBL Chemicals), and anti-α-tubulin (Sigma) antibodies were utilized to check the respective endogenous protein levels by Western blotting.

    Techniques: Transfection, Expressing, Plasmid Preparation

    TRIM27 induces RIP1 deubiquitination by ubiquitinating and activating USP7. (A) Domain structure of TRIM27. (B) TRIM27 ubiquitinates USP7 via its B-box domain. HEK293T cells were cotransfected with FLAG-USP7 and Myc-ubiquitin expression vectors and a

    Journal: Molecular and Cellular Biology

    Article Title: Ubiquitination-Deubiquitination by the TRIM27-USP7 Complex Regulates Tumor Necrosis Factor Alpha-Induced Apoptosis

    doi: 10.1128/MCB.00465-13

    Figure Lengend Snippet: TRIM27 induces RIP1 deubiquitination by ubiquitinating and activating USP7. (A) Domain structure of TRIM27. (B) TRIM27 ubiquitinates USP7 via its B-box domain. HEK293T cells were cotransfected with FLAG-USP7 and Myc-ubiquitin expression vectors and a

    Article Snippet: Anti-cleaved caspase-3 (Cell Signaling), anti-TNFR1 (R & D Systems), anti-TRADD (Santa Cruz), anti-TRAF2 (Santa Cruz), anti-RIP1 (BD Bioscience), anti-FADD (Santa Cruz), anti-CYLD (Santa Cruz), anti-USP7 (Bethyl Laboratories), anti-TRIM27 (IBL Chemicals), and anti-α-tubulin (Sigma) antibodies were utilized to check the respective endogenous protein levels by Western blotting.

    Techniques: Expressing

    Trim59 promotes WASH K63-linked ubiquitination. a Model of WASH, Arp2/3, E2, E3, and K63 ubiquitin regulation on F-actin polymerization. b Immunoblotting of WASH total Ubiquitin in cotransfected HEK293T cells. HEK293T cells were cotransfected with YFP-WASH and with (+) or without ( − ) FLAG-Trim59FL or FLAG-Vector as well as with HA-Ub. Immunoprecipitation was performed by anti-WASH. Trim59 and WASH were detected by anti-FLAG and anti-WASH antibodies. The polyubiquitination of WASH was detected by anti-HA. c Immunoblotting of WASH K63-linked ubiquitin in cotransfected HEK293T cells. HEK293T cells were cotransfected with YFP-WASH and with (+) or without ( − ) FLAG-Trim59 as well as with HA-Ub, HA-K48-Ub, or HA-K63-Ub. Immunoprecipitation was performed by anti-WASH. Trim59 and WASH were detected by anti-FLAG and anti-WASH antibodies. The polyubiquitination of WASH was detected by anti-HA. d Immunoblotting of WASH K63-linked ubiquitin in cotransfected HEK293T cells. HEK293T cells were cotransfected with YFP-WASH or YFP-K220R and with (+) or without (−) FLAG-Trim59 as well as with HA-K63-Ub. Immunoprecipitation was performed by anti-WASH. Trim59, WASH and WASH variant were detected by anti-FLAG and anti-WASH antibodies. Polyubiquitination of WASH was detected by anti-HA. e Immunoblotting of WASH K63-linked ubiquitin in cotransfected HEK293T cells. HEK293T cells were cotransfected with FLAG-Trim59FL and its fragments (T2 toT5) and with YFP-WASH (+) as well as HA-K63-Ub (+). Immunoprecipitation was performed by anti-WASH. Trim59, Trim59 fragments and WASH were detected by anti-FLAG and anti-WASH antibodies. Polyubiquitination of WASH was detected by anti-HA. f Immunoblotting of total Ub and K63-Ub of endogenous WASH from F1 ESCs after treated with specific Trim59 and Trim27 siRNA.Immunoprecipitation was performed by anti-WASH. Trim59, Trim27, and WASH were detected by anti-Trim59, anti-Trim27, and anti-WASH antibodies. Immunoblot analysis of Ub-WASH (top blot), K63-Ub-WASH (below blot) by anti-Ub and anti- K63 antibodies. g Immunofluorescence assay of F-actin assembly in WASH siRNA or siRNA control transfected F1 ESCs. Scale bar, 10 µm. * P

    Journal: Cell Death & Disease

    Article Title: Embryonic lethality in mice lacking Trim59 due to impaired gastrulation development

    doi: 10.1038/s41419-018-0370-y

    Figure Lengend Snippet: Trim59 promotes WASH K63-linked ubiquitination. a Model of WASH, Arp2/3, E2, E3, and K63 ubiquitin regulation on F-actin polymerization. b Immunoblotting of WASH total Ubiquitin in cotransfected HEK293T cells. HEK293T cells were cotransfected with YFP-WASH and with (+) or without ( − ) FLAG-Trim59FL or FLAG-Vector as well as with HA-Ub. Immunoprecipitation was performed by anti-WASH. Trim59 and WASH were detected by anti-FLAG and anti-WASH antibodies. The polyubiquitination of WASH was detected by anti-HA. c Immunoblotting of WASH K63-linked ubiquitin in cotransfected HEK293T cells. HEK293T cells were cotransfected with YFP-WASH and with (+) or without ( − ) FLAG-Trim59 as well as with HA-Ub, HA-K48-Ub, or HA-K63-Ub. Immunoprecipitation was performed by anti-WASH. Trim59 and WASH were detected by anti-FLAG and anti-WASH antibodies. The polyubiquitination of WASH was detected by anti-HA. d Immunoblotting of WASH K63-linked ubiquitin in cotransfected HEK293T cells. HEK293T cells were cotransfected with YFP-WASH or YFP-K220R and with (+) or without (−) FLAG-Trim59 as well as with HA-K63-Ub. Immunoprecipitation was performed by anti-WASH. Trim59, WASH and WASH variant were detected by anti-FLAG and anti-WASH antibodies. Polyubiquitination of WASH was detected by anti-HA. e Immunoblotting of WASH K63-linked ubiquitin in cotransfected HEK293T cells. HEK293T cells were cotransfected with FLAG-Trim59FL and its fragments (T2 toT5) and with YFP-WASH (+) as well as HA-K63-Ub (+). Immunoprecipitation was performed by anti-WASH. Trim59, Trim59 fragments and WASH were detected by anti-FLAG and anti-WASH antibodies. Polyubiquitination of WASH was detected by anti-HA. f Immunoblotting of total Ub and K63-Ub of endogenous WASH from F1 ESCs after treated with specific Trim59 and Trim27 siRNA.Immunoprecipitation was performed by anti-WASH. Trim59, Trim27, and WASH were detected by anti-Trim59, anti-Trim27, and anti-WASH antibodies. Immunoblot analysis of Ub-WASH (top blot), K63-Ub-WASH (below blot) by anti-Ub and anti- K63 antibodies. g Immunofluorescence assay of F-actin assembly in WASH siRNA or siRNA control transfected F1 ESCs. Scale bar, 10 µm. * P

    Article Snippet: Anti- Trim59 (ab69639, Abcam), anti-Trim27 (A6405, Abclonal), anti-WASH (SAB-4200552, Sigma), anti-Oct4 (ab18976, Abcam), anti-FLAG (M20008, Abmart), anti-HA (#T501-1, Signalway Antibody), anti-Ub (YT4793, Immunoway), anti-K48 (#4289, Cell Signaling Technology), anti-K63 (BML-PW0600, Enzo), anti-ACTR2 (ab134082, Abcam), anti-MYH9 (ab138498, Abcam), anti-CAPZA1 (ab166892, Abcam), anti-CAPZB (ab175212, Abcam), anti-β-actin (SC-81178, Santa), Phalloidin-iFluor 488 (ab176753, Abcam), DAPI (#4083, Cell Signaling Technology), Alexa Fluor 488 Conjugate (#4416, #4408, Cell Signaling Technology), and Alexa Fluor 594 Conjugate (#8890, #8889, Cell Signaling Technology) antibodies were purchased.

    Techniques: Plasmid Preparation, Immunoprecipitation, Variant Assay, Immunofluorescence, Transfection

    TRIM27 facilitates the migration and invasion of colorectal cancer cells. A wound-healing assay was performed in (A) LoVo and (B) HCT116 cells following TRIM27 knockdown and overexpression. Quantitative results in the wound-healing assay are shown. A Transwell assay was performed in (C) LoVo and (D) HCT116 cells following TRIM27 knockdown and overexpression. Magnification, ×200. (E) Numbers of migrated LoVo and HCT116 cells through the membrane. (F) Numbers of invaded LoVo and HCT116 cells through the Matrigel. Each assay was performed three times. Data are presented as the mean ± standard deviation; ** P

    Journal: International Journal of Oncology

    Article Title: TRIM27 functions as an oncogene by activating epithelial-mesenchymal transition and p-AKT in colorectal cancer

    doi: 10.3892/ijo.2018.4408

    Figure Lengend Snippet: TRIM27 facilitates the migration and invasion of colorectal cancer cells. A wound-healing assay was performed in (A) LoVo and (B) HCT116 cells following TRIM27 knockdown and overexpression. Quantitative results in the wound-healing assay are shown. A Transwell assay was performed in (C) LoVo and (D) HCT116 cells following TRIM27 knockdown and overexpression. Magnification, ×200. (E) Numbers of migrated LoVo and HCT116 cells through the membrane. (F) Numbers of invaded LoVo and HCT116 cells through the Matrigel. Each assay was performed three times. Data are presented as the mean ± standard deviation; ** P

    Article Snippet: The specific primary and secondary antibodies were as follows: TRIM27 (diluted 1:1,000, polyclonal, rabbit, cat. no. ab78393), E-cadherin (diluted 1:500, monoclonal, mouse, cat. no. ab1416), N-cadherin (diluted 1:1,000, poly-clonal, rabbit, cat. no. ab18203), vimentin (diluted 1:2,000, monoclonal, rabbit, cat. no. ab92547), AKT (diluted 1:500, polyclonal, rabbit, cat. no. ab8805), p-AKT (diluted 1:500, polyclonal, rabbit, cat. no. ab38449) (all from Abcam), anti-rabbit secondary antibodies (diluted 1:5,000, polyclonal, cat. no. GAB007), anti-mouse secondary antibodies (diluted 1:5,000, polyclonal, cat. no. GAM007) (both from Hangzhou Multi Sciences Biotech Co., Ltd., Hangzhou, China).

    Techniques: Migration, Wound Healing Assay, Over Expression, Transwell Assay, Standard Deviation

    TRIM27 promotes proliferation and colony formation in colorectal cancer cells. The cell counting kit-8 assay detected the cell viability of (A) LoVo and (B) HCT116 cells following TRIM27 knockdown or overexpression. Colony-forming abilities of (C) LoVo and (D) HCT116 cells were detected following TRIM27 knockdown or overexpression. Data are presented as the mean ± standard deviation from three independent experiments; * P

    Journal: International Journal of Oncology

    Article Title: TRIM27 functions as an oncogene by activating epithelial-mesenchymal transition and p-AKT in colorectal cancer

    doi: 10.3892/ijo.2018.4408

    Figure Lengend Snippet: TRIM27 promotes proliferation and colony formation in colorectal cancer cells. The cell counting kit-8 assay detected the cell viability of (A) LoVo and (B) HCT116 cells following TRIM27 knockdown or overexpression. Colony-forming abilities of (C) LoVo and (D) HCT116 cells were detected following TRIM27 knockdown or overexpression. Data are presented as the mean ± standard deviation from three independent experiments; * P

    Article Snippet: The specific primary and secondary antibodies were as follows: TRIM27 (diluted 1:1,000, polyclonal, rabbit, cat. no. ab78393), E-cadherin (diluted 1:500, monoclonal, mouse, cat. no. ab1416), N-cadherin (diluted 1:1,000, poly-clonal, rabbit, cat. no. ab18203), vimentin (diluted 1:2,000, monoclonal, rabbit, cat. no. ab92547), AKT (diluted 1:500, polyclonal, rabbit, cat. no. ab8805), p-AKT (diluted 1:500, polyclonal, rabbit, cat. no. ab38449) (all from Abcam), anti-rabbit secondary antibodies (diluted 1:5,000, polyclonal, cat. no. GAB007), anti-mouse secondary antibodies (diluted 1:5,000, polyclonal, cat. no. GAM007) (both from Hangzhou Multi Sciences Biotech Co., Ltd., Hangzhou, China).

    Techniques: Cell Counting, Over Expression, Standard Deviation

    TRIM27 is upregulated in CRC cell lines and can be regulated by siRNA and plasmid transfection. (A) mRNA expression of TRIM27 in different CRC cell lines compared with NCM460 normal colon epithelial cells. (B) Relative protein levels of TRIM27 in different CRC cell lines compared with NCM460 normal colon epithelial cells. (C) Relative mRNA expression of TRIM27 in LoVo cells following siRNA transfection. (D) Relative protein levels of TRIM27 in LoVo cells following siRNA transfection. (E) Relative mRNA expression of TRIM27 in HCT116 cells following plasmid transfection. (F) Relative protein levels of TRIM27 in HCT116 cells following plasmid transfection. Data are presented as the mean ± standard deviation from three independent experiments; * P

    Journal: International Journal of Oncology

    Article Title: TRIM27 functions as an oncogene by activating epithelial-mesenchymal transition and p-AKT in colorectal cancer

    doi: 10.3892/ijo.2018.4408

    Figure Lengend Snippet: TRIM27 is upregulated in CRC cell lines and can be regulated by siRNA and plasmid transfection. (A) mRNA expression of TRIM27 in different CRC cell lines compared with NCM460 normal colon epithelial cells. (B) Relative protein levels of TRIM27 in different CRC cell lines compared with NCM460 normal colon epithelial cells. (C) Relative mRNA expression of TRIM27 in LoVo cells following siRNA transfection. (D) Relative protein levels of TRIM27 in LoVo cells following siRNA transfection. (E) Relative mRNA expression of TRIM27 in HCT116 cells following plasmid transfection. (F) Relative protein levels of TRIM27 in HCT116 cells following plasmid transfection. Data are presented as the mean ± standard deviation from three independent experiments; * P

    Article Snippet: The specific primary and secondary antibodies were as follows: TRIM27 (diluted 1:1,000, polyclonal, rabbit, cat. no. ab78393), E-cadherin (diluted 1:500, monoclonal, mouse, cat. no. ab1416), N-cadherin (diluted 1:1,000, poly-clonal, rabbit, cat. no. ab18203), vimentin (diluted 1:2,000, monoclonal, rabbit, cat. no. ab92547), AKT (diluted 1:500, polyclonal, rabbit, cat. no. ab8805), p-AKT (diluted 1:500, polyclonal, rabbit, cat. no. ab38449) (all from Abcam), anti-rabbit secondary antibodies (diluted 1:5,000, polyclonal, cat. no. GAB007), anti-mouse secondary antibodies (diluted 1:5,000, polyclonal, cat. no. GAM007) (both from Hangzhou Multi Sciences Biotech Co., Ltd., Hangzhou, China).

    Techniques: Plasmid Preparation, Transfection, Expressing, Standard Deviation

    Expression of TRIM27 is upregulated in human CRC tissues and predicts poor prognosis. (A) mRNA expression levels of TRIM27 in CRC and normal tissues (n=80) were detected by reverse transcription-quantitative polymerase chain reaction. mRNA levels of TRIM27 were normalized to GAPDH . (B) Immunohistochemical results of TRIM27 in CRC tissues and normal adjacent tissues. (C) Percentage of specimens exhibiting strong or weak expression of TRIM27 in immunohistochemistry. (D) Kaplan-Meier survival analysis of 80 patients with CRC based on the expression level of TRIM27. *** P

    Journal: International Journal of Oncology

    Article Title: TRIM27 functions as an oncogene by activating epithelial-mesenchymal transition and p-AKT in colorectal cancer

    doi: 10.3892/ijo.2018.4408

    Figure Lengend Snippet: Expression of TRIM27 is upregulated in human CRC tissues and predicts poor prognosis. (A) mRNA expression levels of TRIM27 in CRC and normal tissues (n=80) were detected by reverse transcription-quantitative polymerase chain reaction. mRNA levels of TRIM27 were normalized to GAPDH . (B) Immunohistochemical results of TRIM27 in CRC tissues and normal adjacent tissues. (C) Percentage of specimens exhibiting strong or weak expression of TRIM27 in immunohistochemistry. (D) Kaplan-Meier survival analysis of 80 patients with CRC based on the expression level of TRIM27. *** P

    Article Snippet: The specific primary and secondary antibodies were as follows: TRIM27 (diluted 1:1,000, polyclonal, rabbit, cat. no. ab78393), E-cadherin (diluted 1:500, monoclonal, mouse, cat. no. ab1416), N-cadherin (diluted 1:1,000, poly-clonal, rabbit, cat. no. ab18203), vimentin (diluted 1:2,000, monoclonal, rabbit, cat. no. ab92547), AKT (diluted 1:500, polyclonal, rabbit, cat. no. ab8805), p-AKT (diluted 1:500, polyclonal, rabbit, cat. no. ab38449) (all from Abcam), anti-rabbit secondary antibodies (diluted 1:5,000, polyclonal, cat. no. GAB007), anti-mouse secondary antibodies (diluted 1:5,000, polyclonal, cat. no. GAM007) (both from Hangzhou Multi Sciences Biotech Co., Ltd., Hangzhou, China).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunohistochemistry

    Knockdown of TRIM27 inhibits the proliferation and metastasis of colorectal cancer cells in vivo . (A) Nude mice 4 weeks following tumor implantation in the shTRIM27 group (left armpit) and control group (right armpit). (B) Representative images of isolated tumors in the control group (first row) and shTRIM27-treated group (second row). (C) Average weights of the isolated tumors in the two groups. (D) Representative images of hematoxylin and eosin staining results of the liver tissues from mice in the shTRIM27-treated group and control group. Magnification, ×200. (E) Comparative analysis of the number of liver metastases in mice. ** P

    Journal: International Journal of Oncology

    Article Title: TRIM27 functions as an oncogene by activating epithelial-mesenchymal transition and p-AKT in colorectal cancer

    doi: 10.3892/ijo.2018.4408

    Figure Lengend Snippet: Knockdown of TRIM27 inhibits the proliferation and metastasis of colorectal cancer cells in vivo . (A) Nude mice 4 weeks following tumor implantation in the shTRIM27 group (left armpit) and control group (right armpit). (B) Representative images of isolated tumors in the control group (first row) and shTRIM27-treated group (second row). (C) Average weights of the isolated tumors in the two groups. (D) Representative images of hematoxylin and eosin staining results of the liver tissues from mice in the shTRIM27-treated group and control group. Magnification, ×200. (E) Comparative analysis of the number of liver metastases in mice. ** P

    Article Snippet: The specific primary and secondary antibodies were as follows: TRIM27 (diluted 1:1,000, polyclonal, rabbit, cat. no. ab78393), E-cadherin (diluted 1:500, monoclonal, mouse, cat. no. ab1416), N-cadherin (diluted 1:1,000, poly-clonal, rabbit, cat. no. ab18203), vimentin (diluted 1:2,000, monoclonal, rabbit, cat. no. ab92547), AKT (diluted 1:500, polyclonal, rabbit, cat. no. ab8805), p-AKT (diluted 1:500, polyclonal, rabbit, cat. no. ab38449) (all from Abcam), anti-rabbit secondary antibodies (diluted 1:5,000, polyclonal, cat. no. GAB007), anti-mouse secondary antibodies (diluted 1:5,000, polyclonal, cat. no. GAM007) (both from Hangzhou Multi Sciences Biotech Co., Ltd., Hangzhou, China).

    Techniques: In Vivo, Mouse Assay, Tumor Implantation, Isolation, Staining

    TRIM27 promotes the activation of p-AKT and the epiethlium-mesenchymal transition process in colorectal cancer cells. (A) Expression levels of p-AKT, AKT, E-cadherin, N-cadherin and vimentin in LoVo and HCT116 cells following TRIM27 knockdown and overexpression were detected using western blot analysis. (B) Relative protein levels of p-AKT, E-cadherin, N-cadherin and vimentin were quantified in LoVo and HCT116 cells. Data are presented as the mean ± standard deviation from three independent experiments; ** P

    Journal: International Journal of Oncology

    Article Title: TRIM27 functions as an oncogene by activating epithelial-mesenchymal transition and p-AKT in colorectal cancer

    doi: 10.3892/ijo.2018.4408

    Figure Lengend Snippet: TRIM27 promotes the activation of p-AKT and the epiethlium-mesenchymal transition process in colorectal cancer cells. (A) Expression levels of p-AKT, AKT, E-cadherin, N-cadherin and vimentin in LoVo and HCT116 cells following TRIM27 knockdown and overexpression were detected using western blot analysis. (B) Relative protein levels of p-AKT, E-cadherin, N-cadherin and vimentin were quantified in LoVo and HCT116 cells. Data are presented as the mean ± standard deviation from three independent experiments; ** P

    Article Snippet: The specific primary and secondary antibodies were as follows: TRIM27 (diluted 1:1,000, polyclonal, rabbit, cat. no. ab78393), E-cadherin (diluted 1:500, monoclonal, mouse, cat. no. ab1416), N-cadherin (diluted 1:1,000, poly-clonal, rabbit, cat. no. ab18203), vimentin (diluted 1:2,000, monoclonal, rabbit, cat. no. ab92547), AKT (diluted 1:500, polyclonal, rabbit, cat. no. ab8805), p-AKT (diluted 1:500, polyclonal, rabbit, cat. no. ab38449) (all from Abcam), anti-rabbit secondary antibodies (diluted 1:5,000, polyclonal, cat. no. GAB007), anti-mouse secondary antibodies (diluted 1:5,000, polyclonal, cat. no. GAM007) (both from Hangzhou Multi Sciences Biotech Co., Ltd., Hangzhou, China).

    Techniques: Activation Assay, Expressing, Over Expression, Western Blot, Standard Deviation

    TRIM27 induces cell apoptosis resistance and accelerated cell cycle in colorectal cancer cells. (A) Cell cycles of LoVo and HCT116 cells were detected using flow cytometry, and the comparative analysis of cell numbers in the G0/G1 and S phase are shown. (B) Total cell apoptosis of LoVo and HCT116 cells was detected by flow cytometry, and the comparative analysis of apoptotic cells is shown. Data are presented as the mean ± standard deviation from three independent experiments; * P

    Journal: International Journal of Oncology

    Article Title: TRIM27 functions as an oncogene by activating epithelial-mesenchymal transition and p-AKT in colorectal cancer

    doi: 10.3892/ijo.2018.4408

    Figure Lengend Snippet: TRIM27 induces cell apoptosis resistance and accelerated cell cycle in colorectal cancer cells. (A) Cell cycles of LoVo and HCT116 cells were detected using flow cytometry, and the comparative analysis of cell numbers in the G0/G1 and S phase are shown. (B) Total cell apoptosis of LoVo and HCT116 cells was detected by flow cytometry, and the comparative analysis of apoptotic cells is shown. Data are presented as the mean ± standard deviation from three independent experiments; * P

    Article Snippet: The specific primary and secondary antibodies were as follows: TRIM27 (diluted 1:1,000, polyclonal, rabbit, cat. no. ab78393), E-cadherin (diluted 1:500, monoclonal, mouse, cat. no. ab1416), N-cadherin (diluted 1:1,000, poly-clonal, rabbit, cat. no. ab18203), vimentin (diluted 1:2,000, monoclonal, rabbit, cat. no. ab92547), AKT (diluted 1:500, polyclonal, rabbit, cat. no. ab8805), p-AKT (diluted 1:500, polyclonal, rabbit, cat. no. ab38449) (all from Abcam), anti-rabbit secondary antibodies (diluted 1:5,000, polyclonal, cat. no. GAB007), anti-mouse secondary antibodies (diluted 1:5,000, polyclonal, cat. no. GAM007) (both from Hangzhou Multi Sciences Biotech Co., Ltd., Hangzhou, China).

    Techniques: Flow Cytometry, Cytometry, Standard Deviation

    TRIM27 facilitates the migration and invasion of colorectal cancer cells. A wound-healing assay was performed in (A) LoVo and (B) HCT116 cells following TRIM27 knockdown and overexpression. Quantitative results in the wound-healing assay are shown. A Transwell assay was performed in (C) LoVo and (D) HCT116 cells following TRIM27 knockdown and overexpression. Magnification, ×200. (E) Numbers of migrated LoVo and HCT116 cells through the membrane. (F) Numbers of invaded LoVo and HCT116 cells through the Matrigel. Each assay was performed three times. Data are presented as the mean ± standard deviation; ** P

    Journal: International Journal of Oncology

    Article Title: TRIM27 functions as an oncogene by activating epithelial-mesenchymal transition and p-AKT in colorectal cancer

    doi: 10.3892/ijo.2018.4408

    Figure Lengend Snippet: TRIM27 facilitates the migration and invasion of colorectal cancer cells. A wound-healing assay was performed in (A) LoVo and (B) HCT116 cells following TRIM27 knockdown and overexpression. Quantitative results in the wound-healing assay are shown. A Transwell assay was performed in (C) LoVo and (D) HCT116 cells following TRIM27 knockdown and overexpression. Magnification, ×200. (E) Numbers of migrated LoVo and HCT116 cells through the membrane. (F) Numbers of invaded LoVo and HCT116 cells through the Matrigel. Each assay was performed three times. Data are presented as the mean ± standard deviation; ** P

    Article Snippet: Finally, the tissues were reacted with anti-TRIM27 antibodies (diluted 1:1,000, polyclonal, rabbit, cat. no. ab78393; Abcam, Cambridge, MA, USA) overnight at 4°C, followed by incubation with anti-rabbit antibodies (horseradish peroxidase-tagged, diluted 1:1,000, polyclonal, cat. no. ab6721; Abcam) at room temperature for 50 min, and with the color agent diaminobenzidine.

    Techniques: Migration, Wound Healing Assay, Over Expression, Transwell Assay, Standard Deviation

    TRIM27 promotes proliferation and colony formation in colorectal cancer cells. The cell counting kit-8 assay detected the cell viability of (A) LoVo and (B) HCT116 cells following TRIM27 knockdown or overexpression. Colony-forming abilities of (C) LoVo and (D) HCT116 cells were detected following TRIM27 knockdown or overexpression. Data are presented as the mean ± standard deviation from three independent experiments; * P

    Journal: International Journal of Oncology

    Article Title: TRIM27 functions as an oncogene by activating epithelial-mesenchymal transition and p-AKT in colorectal cancer

    doi: 10.3892/ijo.2018.4408

    Figure Lengend Snippet: TRIM27 promotes proliferation and colony formation in colorectal cancer cells. The cell counting kit-8 assay detected the cell viability of (A) LoVo and (B) HCT116 cells following TRIM27 knockdown or overexpression. Colony-forming abilities of (C) LoVo and (D) HCT116 cells were detected following TRIM27 knockdown or overexpression. Data are presented as the mean ± standard deviation from three independent experiments; * P

    Article Snippet: Finally, the tissues were reacted with anti-TRIM27 antibodies (diluted 1:1,000, polyclonal, rabbit, cat. no. ab78393; Abcam, Cambridge, MA, USA) overnight at 4°C, followed by incubation with anti-rabbit antibodies (horseradish peroxidase-tagged, diluted 1:1,000, polyclonal, cat. no. ab6721; Abcam) at room temperature for 50 min, and with the color agent diaminobenzidine.

    Techniques: Cell Counting, Over Expression, Standard Deviation

    TRIM27 is upregulated in CRC cell lines and can be regulated by siRNA and plasmid transfection. (A) mRNA expression of TRIM27 in different CRC cell lines compared with NCM460 normal colon epithelial cells. (B) Relative protein levels of TRIM27 in different CRC cell lines compared with NCM460 normal colon epithelial cells. (C) Relative mRNA expression of TRIM27 in LoVo cells following siRNA transfection. (D) Relative protein levels of TRIM27 in LoVo cells following siRNA transfection. (E) Relative mRNA expression of TRIM27 in HCT116 cells following plasmid transfection. (F) Relative protein levels of TRIM27 in HCT116 cells following plasmid transfection. Data are presented as the mean ± standard deviation from three independent experiments; * P

    Journal: International Journal of Oncology

    Article Title: TRIM27 functions as an oncogene by activating epithelial-mesenchymal transition and p-AKT in colorectal cancer

    doi: 10.3892/ijo.2018.4408

    Figure Lengend Snippet: TRIM27 is upregulated in CRC cell lines and can be regulated by siRNA and plasmid transfection. (A) mRNA expression of TRIM27 in different CRC cell lines compared with NCM460 normal colon epithelial cells. (B) Relative protein levels of TRIM27 in different CRC cell lines compared with NCM460 normal colon epithelial cells. (C) Relative mRNA expression of TRIM27 in LoVo cells following siRNA transfection. (D) Relative protein levels of TRIM27 in LoVo cells following siRNA transfection. (E) Relative mRNA expression of TRIM27 in HCT116 cells following plasmid transfection. (F) Relative protein levels of TRIM27 in HCT116 cells following plasmid transfection. Data are presented as the mean ± standard deviation from three independent experiments; * P

    Article Snippet: Finally, the tissues were reacted with anti-TRIM27 antibodies (diluted 1:1,000, polyclonal, rabbit, cat. no. ab78393; Abcam, Cambridge, MA, USA) overnight at 4°C, followed by incubation with anti-rabbit antibodies (horseradish peroxidase-tagged, diluted 1:1,000, polyclonal, cat. no. ab6721; Abcam) at room temperature for 50 min, and with the color agent diaminobenzidine.

    Techniques: Plasmid Preparation, Transfection, Expressing, Standard Deviation

    Knockdown of TRIM27 inhibits the proliferation and metastasis of colorectal cancer cells in vivo . (A) Nude mice 4 weeks following tumor implantation in the shTRIM27 group (left armpit) and control group (right armpit). (B) Representative images of isolated tumors in the control group (first row) and shTRIM27-treated group (second row). (C) Average weights of the isolated tumors in the two groups. (D) Representative images of hematoxylin and eosin staining results of the liver tissues from mice in the shTRIM27-treated group and control group. Magnification, ×200. (E) Comparative analysis of the number of liver metastases in mice. ** P

    Journal: International Journal of Oncology

    Article Title: TRIM27 functions as an oncogene by activating epithelial-mesenchymal transition and p-AKT in colorectal cancer

    doi: 10.3892/ijo.2018.4408

    Figure Lengend Snippet: Knockdown of TRIM27 inhibits the proliferation and metastasis of colorectal cancer cells in vivo . (A) Nude mice 4 weeks following tumor implantation in the shTRIM27 group (left armpit) and control group (right armpit). (B) Representative images of isolated tumors in the control group (first row) and shTRIM27-treated group (second row). (C) Average weights of the isolated tumors in the two groups. (D) Representative images of hematoxylin and eosin staining results of the liver tissues from mice in the shTRIM27-treated group and control group. Magnification, ×200. (E) Comparative analysis of the number of liver metastases in mice. ** P

    Article Snippet: Finally, the tissues were reacted with anti-TRIM27 antibodies (diluted 1:1,000, polyclonal, rabbit, cat. no. ab78393; Abcam, Cambridge, MA, USA) overnight at 4°C, followed by incubation with anti-rabbit antibodies (horseradish peroxidase-tagged, diluted 1:1,000, polyclonal, cat. no. ab6721; Abcam) at room temperature for 50 min, and with the color agent diaminobenzidine.

    Techniques: In Vivo, Mouse Assay, Tumor Implantation, Isolation, Staining

    TRIM27 promotes the activation of p-AKT and the epiethlium-mesenchymal transition process in colorectal cancer cells. (A) Expression levels of p-AKT, AKT, E-cadherin, N-cadherin and vimentin in LoVo and HCT116 cells following TRIM27 knockdown and overexpression were detected using western blot analysis. (B) Relative protein levels of p-AKT, E-cadherin, N-cadherin and vimentin were quantified in LoVo and HCT116 cells. Data are presented as the mean ± standard deviation from three independent experiments; ** P

    Journal: International Journal of Oncology

    Article Title: TRIM27 functions as an oncogene by activating epithelial-mesenchymal transition and p-AKT in colorectal cancer

    doi: 10.3892/ijo.2018.4408

    Figure Lengend Snippet: TRIM27 promotes the activation of p-AKT and the epiethlium-mesenchymal transition process in colorectal cancer cells. (A) Expression levels of p-AKT, AKT, E-cadherin, N-cadherin and vimentin in LoVo and HCT116 cells following TRIM27 knockdown and overexpression were detected using western blot analysis. (B) Relative protein levels of p-AKT, E-cadherin, N-cadherin and vimentin were quantified in LoVo and HCT116 cells. Data are presented as the mean ± standard deviation from three independent experiments; ** P

    Article Snippet: Finally, the tissues were reacted with anti-TRIM27 antibodies (diluted 1:1,000, polyclonal, rabbit, cat. no. ab78393; Abcam, Cambridge, MA, USA) overnight at 4°C, followed by incubation with anti-rabbit antibodies (horseradish peroxidase-tagged, diluted 1:1,000, polyclonal, cat. no. ab6721; Abcam) at room temperature for 50 min, and with the color agent diaminobenzidine.

    Techniques: Activation Assay, Expressing, Over Expression, Western Blot, Standard Deviation

    TRIM27 induces cell apoptosis resistance and accelerated cell cycle in colorectal cancer cells. (A) Cell cycles of LoVo and HCT116 cells were detected using flow cytometry, and the comparative analysis of cell numbers in the G0/G1 and S phase are shown. (B) Total cell apoptosis of LoVo and HCT116 cells was detected by flow cytometry, and the comparative analysis of apoptotic cells is shown. Data are presented as the mean ± standard deviation from three independent experiments; * P

    Journal: International Journal of Oncology

    Article Title: TRIM27 functions as an oncogene by activating epithelial-mesenchymal transition and p-AKT in colorectal cancer

    doi: 10.3892/ijo.2018.4408

    Figure Lengend Snippet: TRIM27 induces cell apoptosis resistance and accelerated cell cycle in colorectal cancer cells. (A) Cell cycles of LoVo and HCT116 cells were detected using flow cytometry, and the comparative analysis of cell numbers in the G0/G1 and S phase are shown. (B) Total cell apoptosis of LoVo and HCT116 cells was detected by flow cytometry, and the comparative analysis of apoptotic cells is shown. Data are presented as the mean ± standard deviation from three independent experiments; * P

    Article Snippet: Finally, the tissues were reacted with anti-TRIM27 antibodies (diluted 1:1,000, polyclonal, rabbit, cat. no. ab78393; Abcam, Cambridge, MA, USA) overnight at 4°C, followed by incubation with anti-rabbit antibodies (horseradish peroxidase-tagged, diluted 1:1,000, polyclonal, cat. no. ab6721; Abcam) at room temperature for 50 min, and with the color agent diaminobenzidine.

    Techniques: Flow Cytometry, Cytometry, Standard Deviation

    Expression of mouse Trim27 and Slx2 detected by RT-PCR and western blotting. (A)Trim27 and Slx2 expression were investigated in multiple developmental stages in mouse testis tissue by RT-PCR, and expression was high in various different stages. (B) Trim27

    Journal: Cell Cycle

    Article Title: Trim27 interacts with Slx2, is associated with meiotic processes during spermatogenesis

    doi: 10.1080/15384101.2016.1174796

    Figure Lengend Snippet: Expression of mouse Trim27 and Slx2 detected by RT-PCR and western blotting. (A)Trim27 and Slx2 expression were investigated in multiple developmental stages in mouse testis tissue by RT-PCR, and expression was high in various different stages. (B) Trim27

    Article Snippet: Slides were stained using anti-Scp3 (1:200, Novus Biologicals), anti-γH2AX (1:200, Abcam), and anti-Trim27 (1:300 Abcam).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

    Interaction between Trim27and Slx2, identified using yeast 2-hybrid analysis and confirmed using co-immunoprecipitation (CoIP). (A and B). CoIP using lysates from HEK293T cells co-transfected with Trim27-GFP and FLAG- Slx2. (C). The in vivo interaction

    Journal: Cell Cycle

    Article Title: Trim27 interacts with Slx2, is associated with meiotic processes during spermatogenesis

    doi: 10.1080/15384101.2016.1174796

    Figure Lengend Snippet: Interaction between Trim27and Slx2, identified using yeast 2-hybrid analysis and confirmed using co-immunoprecipitation (CoIP). (A and B). CoIP using lysates from HEK293T cells co-transfected with Trim27-GFP and FLAG- Slx2. (C). The in vivo interaction

    Article Snippet: Slides were stained using anti-Scp3 (1:200, Novus Biologicals), anti-γH2AX (1:200, Abcam), and anti-Trim27 (1:300 Abcam).

    Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, In Vivo

    Recruitment of TRIM27 by SHP2 inhibits VSV-triggered type I IFN production in macrophages. (A) IP and immunoblot analyses of the indicated proteins in HEK293T cells co-transfected with TRIM27 and SHP2 as indicated. (B) Immunoblot analysis of DAP12 and TRIM27 in SHP2-immunoprecipitated products from lysates of VSV-infected (MOI = 10) macrophages. (C) IP and immunoblot analyses of the indicated proteins in HEK293T cells co-transfected with TRIM27 and DAP12, SHP2, or SHP1 as indicated. (D) Immunoblot analysis of TRIM27 in macrophages transfected with TRIM27 siRNA as indicated for 48 h. (E) Macrophages were transfected as in D and then infected with VSV (MOI = 10) or HSV (MOI = 10) for 12 h. Q-PCR analysis was performed to evaluate IFN-β and VSV RNA levels in cells, and ELISA was performed to evaluate IFN-β protein levels in the supernatants. (F) Immunoblot analysis of TRIM27 in RAW264.7 cell clones stably overexpressing TRIM27. (G) RAW264.7 cell clones stably overexpressing TRIM27 were infected with VSV (MOI = 10) or HSV (MOI = 10) for the indicated time. Q-PCR analysis of intracellular IFN-β mRNA and VSV RNA levels and ELISA of IFN-β in the supernatants were then performed. (H) Immunoblot analysis of the indicated molecules in lysates of macrophages transfected with TRIM27 siRNA#1 and infected with VSV (MOI = 10). (I) IFN-β luciferase activity in HEK293T cells transfected with TBK1 or IRF3(5D), and Siglec1 or TRIM27 as indicated. (J) Q-PCR analysis of IFN-β mRNA levels in SHP2-deficient macrophages transfected with Siglec1 or TRIM27 siRNA and infected with VSV. (K) Q-PCR analysis of IFN-β mRNA levels in Siglec1 stably overexpressing-RAW264.7 cell clones that were transfected with TRIM27 siRNA and infected with VSV for 8 h. Data are shown as mean ± SD or representative photographs. * P

    Journal: Cell Research

    Article Title: Siglec1 suppresses antiviral innate immune response by inducing TBK1 degradation via the ubiquitin ligase TRIM27

    doi: 10.1038/cr.2015.108

    Figure Lengend Snippet: Recruitment of TRIM27 by SHP2 inhibits VSV-triggered type I IFN production in macrophages. (A) IP and immunoblot analyses of the indicated proteins in HEK293T cells co-transfected with TRIM27 and SHP2 as indicated. (B) Immunoblot analysis of DAP12 and TRIM27 in SHP2-immunoprecipitated products from lysates of VSV-infected (MOI = 10) macrophages. (C) IP and immunoblot analyses of the indicated proteins in HEK293T cells co-transfected with TRIM27 and DAP12, SHP2, or SHP1 as indicated. (D) Immunoblot analysis of TRIM27 in macrophages transfected with TRIM27 siRNA as indicated for 48 h. (E) Macrophages were transfected as in D and then infected with VSV (MOI = 10) or HSV (MOI = 10) for 12 h. Q-PCR analysis was performed to evaluate IFN-β and VSV RNA levels in cells, and ELISA was performed to evaluate IFN-β protein levels in the supernatants. (F) Immunoblot analysis of TRIM27 in RAW264.7 cell clones stably overexpressing TRIM27. (G) RAW264.7 cell clones stably overexpressing TRIM27 were infected with VSV (MOI = 10) or HSV (MOI = 10) for the indicated time. Q-PCR analysis of intracellular IFN-β mRNA and VSV RNA levels and ELISA of IFN-β in the supernatants were then performed. (H) Immunoblot analysis of the indicated molecules in lysates of macrophages transfected with TRIM27 siRNA#1 and infected with VSV (MOI = 10). (I) IFN-β luciferase activity in HEK293T cells transfected with TBK1 or IRF3(5D), and Siglec1 or TRIM27 as indicated. (J) Q-PCR analysis of IFN-β mRNA levels in SHP2-deficient macrophages transfected with Siglec1 or TRIM27 siRNA and infected with VSV. (K) Q-PCR analysis of IFN-β mRNA levels in Siglec1 stably overexpressing-RAW264.7 cell clones that were transfected with TRIM27 siRNA and infected with VSV for 8 h. Data are shown as mean ± SD or representative photographs. * P

    Article Snippet: Antibodies against TRIM27 (AV34701) and the Flag-tags (F7425) as well as the agarose used for IP were from Sigma-Aldrich.

    Techniques: Transfection, Immunoprecipitation, Infection, Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Clone Assay, Stable Transfection, Luciferase, Activity Assay

    Lys251 and Lys372 of TBK1 are critical sites for TRIM27-mediated degradation of TBK1. (A, B) IP and immunoblot analyses of the indicated proteins in HEK293T cells co-transfected with various TBK1 truncated fragments (KD, 35 KD; KD+ULD, 40 KD; CC, 44 KD; ULD+CC, 49 KD), TRIM27, and K48-linked ubiquitin as indicated. (C, D) IP and immunoblot analyses of the indicated proteins in HEK293T cells transfected with TRIM27 and K48-linked ubiquitin together with various TBK1 mutants as indicated. (E) IFN-β luciferase activity in HEK293T cells transfected with various TBK1 mutants and TRIM27. (F) IFN-β luciferase activity in HEK293T cells transfected with WT or mutant TBK1 and Siglec1 as indicated. (G) Q-PCR analysis of VSV RNA levels in RAW264.7 cells that were transfected with WT or mutant TBK1 and Siglec1 or TRIM27 and then infected with VSV (MOI = 10) for 12 h. (H, I) Working model for the mechanism by which viral infection-induced upregulation of Siglec1 negatively regulates type I IFN production in a feedback manner via recruiting TRIM27 to degrade TBK1 and impair IRF3 signaling in the initiation of type I IFN production. This proposed negative feedback pathway is independent of the phosphorylation and the phosphatase activity of SHP2. Data are shown as mean ± SD or representative photographs. ** P .

    Journal: Cell Research

    Article Title: Siglec1 suppresses antiviral innate immune response by inducing TBK1 degradation via the ubiquitin ligase TRIM27

    doi: 10.1038/cr.2015.108

    Figure Lengend Snippet: Lys251 and Lys372 of TBK1 are critical sites for TRIM27-mediated degradation of TBK1. (A, B) IP and immunoblot analyses of the indicated proteins in HEK293T cells co-transfected with various TBK1 truncated fragments (KD, 35 KD; KD+ULD, 40 KD; CC, 44 KD; ULD+CC, 49 KD), TRIM27, and K48-linked ubiquitin as indicated. (C, D) IP and immunoblot analyses of the indicated proteins in HEK293T cells transfected with TRIM27 and K48-linked ubiquitin together with various TBK1 mutants as indicated. (E) IFN-β luciferase activity in HEK293T cells transfected with various TBK1 mutants and TRIM27. (F) IFN-β luciferase activity in HEK293T cells transfected with WT or mutant TBK1 and Siglec1 as indicated. (G) Q-PCR analysis of VSV RNA levels in RAW264.7 cells that were transfected with WT or mutant TBK1 and Siglec1 or TRIM27 and then infected with VSV (MOI = 10) for 12 h. (H, I) Working model for the mechanism by which viral infection-induced upregulation of Siglec1 negatively regulates type I IFN production in a feedback manner via recruiting TRIM27 to degrade TBK1 and impair IRF3 signaling in the initiation of type I IFN production. This proposed negative feedback pathway is independent of the phosphorylation and the phosphatase activity of SHP2. Data are shown as mean ± SD or representative photographs. ** P .

    Article Snippet: Antibodies against TRIM27 (AV34701) and the Flag-tags (F7425) as well as the agarose used for IP were from Sigma-Aldrich.

    Techniques: Transfection, Luciferase, Activity Assay, Mutagenesis, Polymerase Chain Reaction, Infection

    TRIM27-mediated K48-linked ubiquitination and degradation of TBK1. (A) IP and immunoblot analyses of the indicated proteins in HEK293T cells co-transfected with TRIM27 and TBK1 as indicated. (B) Immunoblot analysis of TBK1 in RAW264.7 cells transfected with increasing doses of TRIM27 plasmids. (C) RT-PCR analysis of TBK1 mRNA levels in RAW264.7 cells transfected with increasing doses of TRIM27 plasmids. GAPDH mRNA serves as a loading control. (D) Immunoblot analysis of extracts from HEK293T cells transfected with TBK1 and TRIM27 plasmids and treated with MG-132 or dimethyl sulfoxide (DMSO). (E) IP and immunoblot analyses of extracts from HEK293T cells transfected with TBK1 with or without TRIM27 as well as WT, K48-linked, or K63-linked ubiquitin as indicated. (F) IP and immunoblot analyses of the indicated proteins in HEK293T cells transfected with various TRIM27 truncated fragments along with TBK1. ▴R, lacking RING domain; ▴R+BB, lacking RING and B-Box domains; ▴RFP, lacking PRY-SPRY domain; RFP, PRY-SPRY domain. Numbers above the domain names indicate amino acid positions. (G) .

    Journal: Cell Research

    Article Title: Siglec1 suppresses antiviral innate immune response by inducing TBK1 degradation via the ubiquitin ligase TRIM27

    doi: 10.1038/cr.2015.108

    Figure Lengend Snippet: TRIM27-mediated K48-linked ubiquitination and degradation of TBK1. (A) IP and immunoblot analyses of the indicated proteins in HEK293T cells co-transfected with TRIM27 and TBK1 as indicated. (B) Immunoblot analysis of TBK1 in RAW264.7 cells transfected with increasing doses of TRIM27 plasmids. (C) RT-PCR analysis of TBK1 mRNA levels in RAW264.7 cells transfected with increasing doses of TRIM27 plasmids. GAPDH mRNA serves as a loading control. (D) Immunoblot analysis of extracts from HEK293T cells transfected with TBK1 and TRIM27 plasmids and treated with MG-132 or dimethyl sulfoxide (DMSO). (E) IP and immunoblot analyses of extracts from HEK293T cells transfected with TBK1 with or without TRIM27 as well as WT, K48-linked, or K63-linked ubiquitin as indicated. (F) IP and immunoblot analyses of the indicated proteins in HEK293T cells transfected with various TRIM27 truncated fragments along with TBK1. ▴R, lacking RING domain; ▴R+BB, lacking RING and B-Box domains; ▴RFP, lacking PRY-SPRY domain; RFP, PRY-SPRY domain. Numbers above the domain names indicate amino acid positions. (G) .

    Article Snippet: Antibodies against TRIM27 (AV34701) and the Flag-tags (F7425) as well as the agarose used for IP were from Sigma-Aldrich.

    Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction

    TRIM27 is associated with ubc9, p53 and Mdm2 ( a) 293T cells were co-transfected with Flag-TRIM27 and/or HA-Ubc9. Cell lysates were incubated with anti-Flag M2 agarose. Immunoprecipitated proteins (IP) and whole cell lysates were analyzed by western blot. ( b ) 293T cells were co-transfected with Flag-TRIM27 and either HA-Mdm2 (left) or HA-p53 (right). Protein interactions were examined as in (a). *, a cleaved product of p53. ( c ) TRIM27 directly binds to p53. GST, GST-p53, and GST-Mdm2 immobilized on glutathione beads were incubated with Flag-TRIM27 purified from 293T cells. Top: The bound proteins and 10% of the input were analyzed by Western blotting with anti-Flag antibody. Bottom: GST proteins were analyzed by Coomassie staining. GST, full-length GST-p53 and GST-Mdm2 proteins are labeled by asterisks. The sizes of the molecular weight standards in the left lane are labeled (in kDa).

    Journal: Oncogene

    Article Title: SUMO E3 ligase activity of TRIM proteins

    doi: 10.1038/onc.2010.462

    Figure Lengend Snippet: TRIM27 is associated with ubc9, p53 and Mdm2 ( a) 293T cells were co-transfected with Flag-TRIM27 and/or HA-Ubc9. Cell lysates were incubated with anti-Flag M2 agarose. Immunoprecipitated proteins (IP) and whole cell lysates were analyzed by western blot. ( b ) 293T cells were co-transfected with Flag-TRIM27 and either HA-Mdm2 (left) or HA-p53 (right). Protein interactions were examined as in (a). *, a cleaved product of p53. ( c ) TRIM27 directly binds to p53. GST, GST-p53, and GST-Mdm2 immobilized on glutathione beads were incubated with Flag-TRIM27 purified from 293T cells. Top: The bound proteins and 10% of the input were analyzed by Western blotting with anti-Flag antibody. Bottom: GST proteins were analyzed by Coomassie staining. GST, full-length GST-p53 and GST-Mdm2 proteins are labeled by asterisks. The sizes of the molecular weight standards in the left lane are labeled (in kDa).

    Article Snippet: Antibodies against p53 (Ab-6) and Mdm2 (Ab-1) were purchased from EMD Chemicals (Gibbstown, NJ), TRIM27 antibody (18791) from Immuno-Biological Lab (Minneapolis, MN), and Flag peptides and anti-Flag antibody (M2) from Sigma (St. Louis, MO).

    Techniques: Transfection, Incubation, Immunoprecipitation, Western Blot, Purification, Staining, Labeling, Molecular Weight

    TRIM27 stabilizes Mdm2 through SUMOylation ( a ) Pml −/− MEF cells were transfected with His-SUMO1, HA-Mdm2, TRIM27, PML or PIASy as indicated. His-SUMO1 conjugates and WCL were analyzed by western blot. ( b ) Bacterially expressed p53 was incubated with SUMO E1, His-SUMO1/His-SUMO2, in the presence or absence of Ubc9 and Flag-TRIM27. Reaction mixes were analyzed by western blot using anti-p53 antibody. ( c ) In vitro Mdm2 SUMOylation reactions were performed with SUMO E1, ATP, His-SUMO1/SUMO2, in vitro -translated Mdm2, in the presence or absence of Ubc9 and Flag-TRIM27 purified from 293T cells. Proteins conjugated to His-SUMO were captured by Ni 2+ -NTA beads pull down and analyzed by western blot with anti-Mdm2 antibody. ( d ) His-p53 was incubated with ATP and Ubc9, E1, and bacterial recombinant GST or GST-TRIM27 as indicated. The reaction mixes were analyzed by western blot using anti-p53 antibody. ( e and f ) HA-Mdm2 were transfected into HeLa cells together with Flag-TRIM27 and/or His-SUMO1. Cells were treated with cycloheximide for the indicated durations. (e) Cell lysates were analyzed for the levels of Mdm2. (f) The same samples were analyzed by western blot with different exposure times for each blot to achieve comparable band intensity at time 0. ( g ) Ubc9 down-regulation reduces Mdm2 level. U2OS cells treated with Ubc9 siRNA control or control siRNA were transfected with HA-Mdm2, Flag-TRIM27, His-SUMO1 as indicated.

    Journal: Oncogene

    Article Title: SUMO E3 ligase activity of TRIM proteins

    doi: 10.1038/onc.2010.462

    Figure Lengend Snippet: TRIM27 stabilizes Mdm2 through SUMOylation ( a ) Pml −/− MEF cells were transfected with His-SUMO1, HA-Mdm2, TRIM27, PML or PIASy as indicated. His-SUMO1 conjugates and WCL were analyzed by western blot. ( b ) Bacterially expressed p53 was incubated with SUMO E1, His-SUMO1/His-SUMO2, in the presence or absence of Ubc9 and Flag-TRIM27. Reaction mixes were analyzed by western blot using anti-p53 antibody. ( c ) In vitro Mdm2 SUMOylation reactions were performed with SUMO E1, ATP, His-SUMO1/SUMO2, in vitro -translated Mdm2, in the presence or absence of Ubc9 and Flag-TRIM27 purified from 293T cells. Proteins conjugated to His-SUMO were captured by Ni 2+ -NTA beads pull down and analyzed by western blot with anti-Mdm2 antibody. ( d ) His-p53 was incubated with ATP and Ubc9, E1, and bacterial recombinant GST or GST-TRIM27 as indicated. The reaction mixes were analyzed by western blot using anti-p53 antibody. ( e and f ) HA-Mdm2 were transfected into HeLa cells together with Flag-TRIM27 and/or His-SUMO1. Cells were treated with cycloheximide for the indicated durations. (e) Cell lysates were analyzed for the levels of Mdm2. (f) The same samples were analyzed by western blot with different exposure times for each blot to achieve comparable band intensity at time 0. ( g ) Ubc9 down-regulation reduces Mdm2 level. U2OS cells treated with Ubc9 siRNA control or control siRNA were transfected with HA-Mdm2, Flag-TRIM27, His-SUMO1 as indicated.

    Article Snippet: Antibodies against p53 (Ab-6) and Mdm2 (Ab-1) were purchased from EMD Chemicals (Gibbstown, NJ), TRIM27 antibody (18791) from Immuno-Biological Lab (Minneapolis, MN), and Flag peptides and anti-Flag antibody (M2) from Sigma (St. Louis, MO).

    Techniques: Transfection, Western Blot, Incubation, In Vitro, Purification, Recombinant

    TRIM27 facilitates the migration and invasion of colorectal cancer cells. A wound-healing assay was performed in (A) LoVo and (B) HCT116 cells following TRIM27 knockdown and overexpression. Quantitative results in the wound-healing assay are shown. A Transwell assay was performed in (C) LoVo and (D) HCT116 cells following TRIM27 knockdown and overexpression. Magnification, ×200. (E) Numbers of migrated LoVo and HCT116 cells through the membrane. (F) Numbers of invaded LoVo and HCT116 cells through the Matrigel. Each assay was performed three times. Data are presented as the mean ± standard deviation; ** P

    Journal: International Journal of Oncology

    Article Title: TRIM27 functions as an oncogene by activating epithelial-mesenchymal transition and p-AKT in colorectal cancer

    doi: 10.3892/ijo.2018.4408

    Figure Lengend Snippet: TRIM27 facilitates the migration and invasion of colorectal cancer cells. A wound-healing assay was performed in (A) LoVo and (B) HCT116 cells following TRIM27 knockdown and overexpression. Quantitative results in the wound-healing assay are shown. A Transwell assay was performed in (C) LoVo and (D) HCT116 cells following TRIM27 knockdown and overexpression. Magnification, ×200. (E) Numbers of migrated LoVo and HCT116 cells through the membrane. (F) Numbers of invaded LoVo and HCT116 cells through the Matrigel. Each assay was performed three times. Data are presented as the mean ± standard deviation; ** P

    Article Snippet: Knockdown and overexpression of TRIM27 Small interference RNA (siRNA) targeting TRIM27 and a negative control sequence (NC) were designed by GenePharma Corporation (Shanghai, China).

    Techniques: Migration, Wound Healing Assay, Over Expression, Transwell Assay, Standard Deviation

    TRIM27 promotes proliferation and colony formation in colorectal cancer cells. The cell counting kit-8 assay detected the cell viability of (A) LoVo and (B) HCT116 cells following TRIM27 knockdown or overexpression. Colony-forming abilities of (C) LoVo and (D) HCT116 cells were detected following TRIM27 knockdown or overexpression. Data are presented as the mean ± standard deviation from three independent experiments; * P

    Journal: International Journal of Oncology

    Article Title: TRIM27 functions as an oncogene by activating epithelial-mesenchymal transition and p-AKT in colorectal cancer

    doi: 10.3892/ijo.2018.4408

    Figure Lengend Snippet: TRIM27 promotes proliferation and colony formation in colorectal cancer cells. The cell counting kit-8 assay detected the cell viability of (A) LoVo and (B) HCT116 cells following TRIM27 knockdown or overexpression. Colony-forming abilities of (C) LoVo and (D) HCT116 cells were detected following TRIM27 knockdown or overexpression. Data are presented as the mean ± standard deviation from three independent experiments; * P

    Article Snippet: Knockdown and overexpression of TRIM27 Small interference RNA (siRNA) targeting TRIM27 and a negative control sequence (NC) were designed by GenePharma Corporation (Shanghai, China).

    Techniques: Cell Counting, Over Expression, Standard Deviation

    TRIM27 is upregulated in CRC cell lines and can be regulated by siRNA and plasmid transfection. (A) mRNA expression of TRIM27 in different CRC cell lines compared with NCM460 normal colon epithelial cells. (B) Relative protein levels of TRIM27 in different CRC cell lines compared with NCM460 normal colon epithelial cells. (C) Relative mRNA expression of TRIM27 in LoVo cells following siRNA transfection. (D) Relative protein levels of TRIM27 in LoVo cells following siRNA transfection. (E) Relative mRNA expression of TRIM27 in HCT116 cells following plasmid transfection. (F) Relative protein levels of TRIM27 in HCT116 cells following plasmid transfection. Data are presented as the mean ± standard deviation from three independent experiments; * P

    Journal: International Journal of Oncology

    Article Title: TRIM27 functions as an oncogene by activating epithelial-mesenchymal transition and p-AKT in colorectal cancer

    doi: 10.3892/ijo.2018.4408

    Figure Lengend Snippet: TRIM27 is upregulated in CRC cell lines and can be regulated by siRNA and plasmid transfection. (A) mRNA expression of TRIM27 in different CRC cell lines compared with NCM460 normal colon epithelial cells. (B) Relative protein levels of TRIM27 in different CRC cell lines compared with NCM460 normal colon epithelial cells. (C) Relative mRNA expression of TRIM27 in LoVo cells following siRNA transfection. (D) Relative protein levels of TRIM27 in LoVo cells following siRNA transfection. (E) Relative mRNA expression of TRIM27 in HCT116 cells following plasmid transfection. (F) Relative protein levels of TRIM27 in HCT116 cells following plasmid transfection. Data are presented as the mean ± standard deviation from three independent experiments; * P

    Article Snippet: Knockdown and overexpression of TRIM27 Small interference RNA (siRNA) targeting TRIM27 and a negative control sequence (NC) were designed by GenePharma Corporation (Shanghai, China).

    Techniques: Plasmid Preparation, Transfection, Expressing, Standard Deviation

    Expression of TRIM27 is upregulated in human CRC tissues and predicts poor prognosis. (A) mRNA expression levels of TRIM27 in CRC and normal tissues (n=80) were detected by reverse transcription-quantitative polymerase chain reaction. mRNA levels of TRIM27 were normalized to GAPDH . (B) Immunohistochemical results of TRIM27 in CRC tissues and normal adjacent tissues. (C) Percentage of specimens exhibiting strong or weak expression of TRIM27 in immunohistochemistry. (D) Kaplan-Meier survival analysis of 80 patients with CRC based on the expression level of TRIM27. *** P

    Journal: International Journal of Oncology

    Article Title: TRIM27 functions as an oncogene by activating epithelial-mesenchymal transition and p-AKT in colorectal cancer

    doi: 10.3892/ijo.2018.4408

    Figure Lengend Snippet: Expression of TRIM27 is upregulated in human CRC tissues and predicts poor prognosis. (A) mRNA expression levels of TRIM27 in CRC and normal tissues (n=80) were detected by reverse transcription-quantitative polymerase chain reaction. mRNA levels of TRIM27 were normalized to GAPDH . (B) Immunohistochemical results of TRIM27 in CRC tissues and normal adjacent tissues. (C) Percentage of specimens exhibiting strong or weak expression of TRIM27 in immunohistochemistry. (D) Kaplan-Meier survival analysis of 80 patients with CRC based on the expression level of TRIM27. *** P

    Article Snippet: Knockdown and overexpression of TRIM27 Small interference RNA (siRNA) targeting TRIM27 and a negative control sequence (NC) were designed by GenePharma Corporation (Shanghai, China).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunohistochemistry

    Knockdown of TRIM27 inhibits the proliferation and metastasis of colorectal cancer cells in vivo . (A) Nude mice 4 weeks following tumor implantation in the shTRIM27 group (left armpit) and control group (right armpit). (B) Representative images of isolated tumors in the control group (first row) and shTRIM27-treated group (second row). (C) Average weights of the isolated tumors in the two groups. (D) Representative images of hematoxylin and eosin staining results of the liver tissues from mice in the shTRIM27-treated group and control group. Magnification, ×200. (E) Comparative analysis of the number of liver metastases in mice. ** P

    Journal: International Journal of Oncology

    Article Title: TRIM27 functions as an oncogene by activating epithelial-mesenchymal transition and p-AKT in colorectal cancer

    doi: 10.3892/ijo.2018.4408

    Figure Lengend Snippet: Knockdown of TRIM27 inhibits the proliferation and metastasis of colorectal cancer cells in vivo . (A) Nude mice 4 weeks following tumor implantation in the shTRIM27 group (left armpit) and control group (right armpit). (B) Representative images of isolated tumors in the control group (first row) and shTRIM27-treated group (second row). (C) Average weights of the isolated tumors in the two groups. (D) Representative images of hematoxylin and eosin staining results of the liver tissues from mice in the shTRIM27-treated group and control group. Magnification, ×200. (E) Comparative analysis of the number of liver metastases in mice. ** P

    Article Snippet: Knockdown and overexpression of TRIM27 Small interference RNA (siRNA) targeting TRIM27 and a negative control sequence (NC) were designed by GenePharma Corporation (Shanghai, China).

    Techniques: In Vivo, Mouse Assay, Tumor Implantation, Isolation, Staining

    TRIM27 promotes the activation of p-AKT and the epiethlium-mesenchymal transition process in colorectal cancer cells. (A) Expression levels of p-AKT, AKT, E-cadherin, N-cadherin and vimentin in LoVo and HCT116 cells following TRIM27 knockdown and overexpression were detected using western blot analysis. (B) Relative protein levels of p-AKT, E-cadherin, N-cadherin and vimentin were quantified in LoVo and HCT116 cells. Data are presented as the mean ± standard deviation from three independent experiments; ** P

    Journal: International Journal of Oncology

    Article Title: TRIM27 functions as an oncogene by activating epithelial-mesenchymal transition and p-AKT in colorectal cancer

    doi: 10.3892/ijo.2018.4408

    Figure Lengend Snippet: TRIM27 promotes the activation of p-AKT and the epiethlium-mesenchymal transition process in colorectal cancer cells. (A) Expression levels of p-AKT, AKT, E-cadherin, N-cadherin and vimentin in LoVo and HCT116 cells following TRIM27 knockdown and overexpression were detected using western blot analysis. (B) Relative protein levels of p-AKT, E-cadherin, N-cadherin and vimentin were quantified in LoVo and HCT116 cells. Data are presented as the mean ± standard deviation from three independent experiments; ** P

    Article Snippet: Knockdown and overexpression of TRIM27 Small interference RNA (siRNA) targeting TRIM27 and a negative control sequence (NC) were designed by GenePharma Corporation (Shanghai, China).

    Techniques: Activation Assay, Expressing, Over Expression, Western Blot, Standard Deviation

    TRIM27 induces cell apoptosis resistance and accelerated cell cycle in colorectal cancer cells. (A) Cell cycles of LoVo and HCT116 cells were detected using flow cytometry, and the comparative analysis of cell numbers in the G0/G1 and S phase are shown. (B) Total cell apoptosis of LoVo and HCT116 cells was detected by flow cytometry, and the comparative analysis of apoptotic cells is shown. Data are presented as the mean ± standard deviation from three independent experiments; * P

    Journal: International Journal of Oncology

    Article Title: TRIM27 functions as an oncogene by activating epithelial-mesenchymal transition and p-AKT in colorectal cancer

    doi: 10.3892/ijo.2018.4408

    Figure Lengend Snippet: TRIM27 induces cell apoptosis resistance and accelerated cell cycle in colorectal cancer cells. (A) Cell cycles of LoVo and HCT116 cells were detected using flow cytometry, and the comparative analysis of cell numbers in the G0/G1 and S phase are shown. (B) Total cell apoptosis of LoVo and HCT116 cells was detected by flow cytometry, and the comparative analysis of apoptotic cells is shown. Data are presented as the mean ± standard deviation from three independent experiments; * P

    Article Snippet: Knockdown and overexpression of TRIM27 Small interference RNA (siRNA) targeting TRIM27 and a negative control sequence (NC) were designed by GenePharma Corporation (Shanghai, China).

    Techniques: Flow Cytometry, Cytometry, Standard Deviation

    TRIM27 promotes the activation of JNK and p38 pathways and suppresses NF-κB activation. ( a,b ) Luciferase assay of of AP-1 (a ) and NF-κB ( b ) activation in U937 cells. Cells were transfected with luciferase siRNA (NC siRNA) or TRIM27 siRNA or TRIM27 siRNA complemented with TRIM27 (ΔRING), and then were infected with WT BCG or BCG (ΔPtpA) or the BCG (ΔPtpA) complemented with PtpA D126A (ΔPtpA+D126A) at a MOI of 10 for 24 h. ( c ) Immunoblot analysis of phosphorylated IκBα, JNK, p38, Erk and GAPDH (loading control) in U937 cells transfected with luciferase siRNA (NC siRNA) or TRIM27 siRNA and infected with BCG at a MOI of 10 for 0–48 h. Non-infected cells served as a control group. Data are shown as the means ± s.e.m. * P

    Journal: Scientific Reports

    Article Title: The ubiquitin ligase TRIM27 functions as a host restriction factor antagonized by Mycobacterium tuberculosis PtpA during mycobacterial infection

    doi: 10.1038/srep34827

    Figure Lengend Snippet: TRIM27 promotes the activation of JNK and p38 pathways and suppresses NF-κB activation. ( a,b ) Luciferase assay of of AP-1 (a ) and NF-κB ( b ) activation in U937 cells. Cells were transfected with luciferase siRNA (NC siRNA) or TRIM27 siRNA or TRIM27 siRNA complemented with TRIM27 (ΔRING), and then were infected with WT BCG or BCG (ΔPtpA) or the BCG (ΔPtpA) complemented with PtpA D126A (ΔPtpA+D126A) at a MOI of 10 for 24 h. ( c ) Immunoblot analysis of phosphorylated IκBα, JNK, p38, Erk and GAPDH (loading control) in U937 cells transfected with luciferase siRNA (NC siRNA) or TRIM27 siRNA and infected with BCG at a MOI of 10 for 0–48 h. Non-infected cells served as a control group. Data are shown as the means ± s.e.m. * P

    Article Snippet: After 24 h, cells were infected with BCG for 4 h at a MOI of 20 and washed three times, fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 and blocked with BSA for 1 h, and labeled with antibodies against PtpA (prepared as described previously ) and TRIM27 (sc-47513, Santa cruz).

    Techniques: Activation Assay, Luciferase, Transfection, Infection

    TRIM27 restricts the intracellular survival of mycobacteria. ( a ) The protein levels of TRIM27 were analyzed by immunoblot analysis in U937 cells transfected with luciferase siRNA (NC) or TRIM27 siRNA. ( b,c ) Survival of BCG ( b ) and M. smegmatis ( c ) in U937 cells infected with BCG or M. smegmatis at a MOI of 10 for 0–48 h. Non-infected cells were used as a control. ( d,e ) The levels of TRIM27 were examined in U937 cells infected with BCG ( d ) or M. smegmatis ( e ) at a MOI of 10 for 0–24 h. Non-infected cells and cells treated with latex beads served as control groups. Data are shown as the means ± s.e.m. * P

    Journal: Scientific Reports

    Article Title: The ubiquitin ligase TRIM27 functions as a host restriction factor antagonized by Mycobacterium tuberculosis PtpA during mycobacterial infection

    doi: 10.1038/srep34827

    Figure Lengend Snippet: TRIM27 restricts the intracellular survival of mycobacteria. ( a ) The protein levels of TRIM27 were analyzed by immunoblot analysis in U937 cells transfected with luciferase siRNA (NC) or TRIM27 siRNA. ( b,c ) Survival of BCG ( b ) and M. smegmatis ( c ) in U937 cells infected with BCG or M. smegmatis at a MOI of 10 for 0–48 h. Non-infected cells were used as a control. ( d,e ) The levels of TRIM27 were examined in U937 cells infected with BCG ( d ) or M. smegmatis ( e ) at a MOI of 10 for 0–24 h. Non-infected cells and cells treated with latex beads served as control groups. Data are shown as the means ± s.e.m. * P

    Article Snippet: After 24 h, cells were infected with BCG for 4 h at a MOI of 20 and washed three times, fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 and blocked with BSA for 1 h, and labeled with antibodies against PtpA (prepared as described previously ) and TRIM27 (sc-47513, Santa cruz).

    Techniques: Transfection, Luciferase, Infection

    Mtb PtpA antagonizes TRIM27-promoted cell apoptosis. ( a ) Immunoblot analysis of cleaved caspase 3 in U937 cells. Cells were transfected with NC or TRIM27 siRNA or TRIM27 siRNA complemented with TRIM27 (ΔRING), and were then infected with WT BCG or BCGΔPtpA or BCG (ΔPtpA+D126A) stain at a MOI of 10 for 0–48 h. Non-infected cells served as a control group. GAPDH was used as a loading control. ( b ) U937 cells were treated as in a and were infected with BCG at a MOI of 10. After 24 h, the cells were stained with PI and annexin V, followed by flow cytometry analysis (top) and statistical analysis (bottom). Data are shown as the means ± s.e.m. * P

    Journal: Scientific Reports

    Article Title: The ubiquitin ligase TRIM27 functions as a host restriction factor antagonized by Mycobacterium tuberculosis PtpA during mycobacterial infection

    doi: 10.1038/srep34827

    Figure Lengend Snippet: Mtb PtpA antagonizes TRIM27-promoted cell apoptosis. ( a ) Immunoblot analysis of cleaved caspase 3 in U937 cells. Cells were transfected with NC or TRIM27 siRNA or TRIM27 siRNA complemented with TRIM27 (ΔRING), and were then infected with WT BCG or BCGΔPtpA or BCG (ΔPtpA+D126A) stain at a MOI of 10 for 0–48 h. Non-infected cells served as a control group. GAPDH was used as a loading control. ( b ) U937 cells were treated as in a and were infected with BCG at a MOI of 10. After 24 h, the cells were stained with PI and annexin V, followed by flow cytometry analysis (top) and statistical analysis (bottom). Data are shown as the means ± s.e.m. * P

    Article Snippet: After 24 h, cells were infected with BCG for 4 h at a MOI of 20 and washed three times, fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 and blocked with BSA for 1 h, and labeled with antibodies against PtpA (prepared as described previously ) and TRIM27 (sc-47513, Santa cruz).

    Techniques: Transfection, Infection, Staining, Flow Cytometry, Cytometry

    Mtb PtpA antagonizes TRIM27-promoted cytokine production and TRIM27-inhibited mycobacterial intracellular survival. ( a,b ) Quantitative PCR analysis of il1b mRNA ( a ) and il8 mRNA ( b ) in U937 cells. Cells were transfected with NC siRNA or TRIM27 siRNA or TRIM27 siRNA complemented with TRIM27 (ΔRING), and then were infected with WT BCG or BCGΔPtpA or BCG (ΔPtpA+D126A) at a MOI of 10 for 0–48 h. Non-infected cells served as a control group. Analysis of each gene was normalized to GAPDH. ( c ) Survival of BCG in U937 cells treated as in ( a,b ). Data are shown as the means ± s.e.m. * P

    Journal: Scientific Reports

    Article Title: The ubiquitin ligase TRIM27 functions as a host restriction factor antagonized by Mycobacterium tuberculosis PtpA during mycobacterial infection

    doi: 10.1038/srep34827

    Figure Lengend Snippet: Mtb PtpA antagonizes TRIM27-promoted cytokine production and TRIM27-inhibited mycobacterial intracellular survival. ( a,b ) Quantitative PCR analysis of il1b mRNA ( a ) and il8 mRNA ( b ) in U937 cells. Cells were transfected with NC siRNA or TRIM27 siRNA or TRIM27 siRNA complemented with TRIM27 (ΔRING), and then were infected with WT BCG or BCGΔPtpA or BCG (ΔPtpA+D126A) at a MOI of 10 for 0–48 h. Non-infected cells served as a control group. Analysis of each gene was normalized to GAPDH. ( c ) Survival of BCG in U937 cells treated as in ( a,b ). Data are shown as the means ± s.e.m. * P

    Article Snippet: After 24 h, cells were infected with BCG for 4 h at a MOI of 20 and washed three times, fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 and blocked with BSA for 1 h, and labeled with antibodies against PtpA (prepared as described previously ) and TRIM27 (sc-47513, Santa cruz).

    Techniques: Real-time Polymerase Chain Reaction, Transfection, Infection

    TRIM27-interacting Mtb proteins visualized by Cytoscape. ( a ) TRIM27-interacting total Mtb proteins visualized by Cytoscape with fold change data incorporated (red = high fold change; green = low fold change). ( b ) TRIM27-interacting secreted Mtb proteins visualized by Cytoscape with fold change data incorporated (red: large fold change; green: small fold change).

    Journal: Scientific Reports

    Article Title: The ubiquitin ligase TRIM27 functions as a host restriction factor antagonized by Mycobacterium tuberculosis PtpA during mycobacterial infection

    doi: 10.1038/srep34827

    Figure Lengend Snippet: TRIM27-interacting Mtb proteins visualized by Cytoscape. ( a ) TRIM27-interacting total Mtb proteins visualized by Cytoscape with fold change data incorporated (red = high fold change; green = low fold change). ( b ) TRIM27-interacting secreted Mtb proteins visualized by Cytoscape with fold change data incorporated (red: large fold change; green: small fold change).

    Article Snippet: After 24 h, cells were infected with BCG for 4 h at a MOI of 20 and washed three times, fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 and blocked with BSA for 1 h, and labeled with antibodies against PtpA (prepared as described previously ) and TRIM27 (sc-47513, Santa cruz).

    Techniques:

    Identification of TRIM27 as a Mycobacterium tuberculosis (Mtb) PtpA-interacting host protein. ( a ) Mtb PtpA interacts with TRIM27 in the yeast two-hybrid assay. Yeast strains were transformed with indicated vectors in which TAK1 and TAB2 interaction served as a positive control. Left, high-stringency. Right, low-stringency. ( b ) Immunoblot analysis (IB) of proteins immunoprecipitated (IP) with immunoglobulin G (IgG; control), anti-Flag or anti-Myc from lysates of HEK293T cells transfected with vectors encoding Flag-tagged PtpA and Myc-tagged TRIM27 (top), and immunoblot analysis without immunoprecipitation (Input; below). ( c ) Immunoblot analysis of proteins immunoprecipitated with anti-PtpA from lysates of U937 cells infected with wild-type (WT) BCG or PtpA deleted BCG (BCGΔPtpA) for 24 h. Non-infected cells were used as a control. ( d ) Immunoblot analysis of proteins immunoprecipitated with anti-Myc from lysates of HEK293T cells transfected with vectors encoding Flag-tagged PtpA and Myc-tagged full-length (FL) TRIM27 or its truncated forms (R, RING; BCC, B-Box region and coiled-coil region; RFP, RFP region). ( e ) Confocal microscopy of U937 cells infected with BCG at a MOI of 20. Mtb PtpA (Green) colocalizes with TRIM27 (Red) in U937 cells. Bacteria (Blue) were stained with Alexa 350 carboxylic acid succinimidyl eater. Scale bars, 10 μm.

    Journal: Scientific Reports

    Article Title: The ubiquitin ligase TRIM27 functions as a host restriction factor antagonized by Mycobacterium tuberculosis PtpA during mycobacterial infection

    doi: 10.1038/srep34827

    Figure Lengend Snippet: Identification of TRIM27 as a Mycobacterium tuberculosis (Mtb) PtpA-interacting host protein. ( a ) Mtb PtpA interacts with TRIM27 in the yeast two-hybrid assay. Yeast strains were transformed with indicated vectors in which TAK1 and TAB2 interaction served as a positive control. Left, high-stringency. Right, low-stringency. ( b ) Immunoblot analysis (IB) of proteins immunoprecipitated (IP) with immunoglobulin G (IgG; control), anti-Flag or anti-Myc from lysates of HEK293T cells transfected with vectors encoding Flag-tagged PtpA and Myc-tagged TRIM27 (top), and immunoblot analysis without immunoprecipitation (Input; below). ( c ) Immunoblot analysis of proteins immunoprecipitated with anti-PtpA from lysates of U937 cells infected with wild-type (WT) BCG or PtpA deleted BCG (BCGΔPtpA) for 24 h. Non-infected cells were used as a control. ( d ) Immunoblot analysis of proteins immunoprecipitated with anti-Myc from lysates of HEK293T cells transfected with vectors encoding Flag-tagged PtpA and Myc-tagged full-length (FL) TRIM27 or its truncated forms (R, RING; BCC, B-Box region and coiled-coil region; RFP, RFP region). ( e ) Confocal microscopy of U937 cells infected with BCG at a MOI of 20. Mtb PtpA (Green) colocalizes with TRIM27 (Red) in U937 cells. Bacteria (Blue) were stained with Alexa 350 carboxylic acid succinimidyl eater. Scale bars, 10 μm.

    Article Snippet: After 24 h, cells were infected with BCG for 4 h at a MOI of 20 and washed three times, fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 and blocked with BSA for 1 h, and labeled with antibodies against PtpA (prepared as described previously ) and TRIM27 (sc-47513, Santa cruz).

    Techniques: Y2H Assay, Transformation Assay, Positive Control, Immunoprecipitation, Transfection, Infection, Confocal Microscopy, Staining

    TRIM27 is not involved in TNF-α-induced survival signaling. (A) TNFR1 expression. TNFR1 expression in WT and Trim27 −/− MEFs was examined by flow cytometry. (B) Expression of TNF-α signaling components. Expression levels

    Journal: Molecular and Cellular Biology

    Article Title: Ubiquitination-Deubiquitination by the TRIM27-USP7 Complex Regulates Tumor Necrosis Factor Alpha-Induced Apoptosis

    doi: 10.1128/MCB.00465-13

    Figure Lengend Snippet: TRIM27 is not involved in TNF-α-induced survival signaling. (A) TNFR1 expression. TNFR1 expression in WT and Trim27 −/− MEFs was examined by flow cytometry. (B) Expression of TNF-α signaling components. Expression levels

    Article Snippet: Anti-TRIM27 (IBL-America) or anti-USP7 (Bethyl Laboratories) antibody was used for immunoblotting.

    Techniques: Expressing, Flow Cytometry, Cytometry

    USP7 is required for the TRIM27-induced deubiquitination of RIP1. (A) Formation of a complex between TRIM27 and USP7. The TRIM27 complex was purified from HeLa cells expressing FLAG- and HA-tagged TRIM27 (FH-TRIM27) or control HeLa cells (mock) using

    Journal: Molecular and Cellular Biology

    Article Title: Ubiquitination-Deubiquitination by the TRIM27-USP7 Complex Regulates Tumor Necrosis Factor Alpha-Induced Apoptosis

    doi: 10.1128/MCB.00465-13

    Figure Lengend Snippet: USP7 is required for the TRIM27-induced deubiquitination of RIP1. (A) Formation of a complex between TRIM27 and USP7. The TRIM27 complex was purified from HeLa cells expressing FLAG- and HA-tagged TRIM27 (FH-TRIM27) or control HeLa cells (mock) using

    Article Snippet: Anti-TRIM27 (IBL-America) or anti-USP7 (Bethyl Laboratories) antibody was used for immunoblotting.

    Techniques: Purification, Expressing

    Localization of TRIM27 in mitochondria. (A) Subcellular localization of TRIM27. HepG2 cells were transfected with a FLAG-TRIM27 expression vector, incubated with Mitotracker, fixed with 1% paraformaldehyde, and incubated with an anti-FLAG antibody. After

    Journal: Molecular and Cellular Biology

    Article Title: Ubiquitination-Deubiquitination by the TRIM27-USP7 Complex Regulates Tumor Necrosis Factor Alpha-Induced Apoptosis

    doi: 10.1128/MCB.00465-13

    Figure Lengend Snippet: Localization of TRIM27 in mitochondria. (A) Subcellular localization of TRIM27. HepG2 cells were transfected with a FLAG-TRIM27 expression vector, incubated with Mitotracker, fixed with 1% paraformaldehyde, and incubated with an anti-FLAG antibody. After

    Article Snippet: Anti-TRIM27 (IBL-America) or anti-USP7 (Bethyl Laboratories) antibody was used for immunoblotting.

    Techniques: Transfection, Expressing, Plasmid Preparation, Incubation

    TRIM27 positively regulates TNF-α-induced apoptosis. (A) Resistance of wild-type (WT) and Trim27 −/− mice to TNF-α cytotoxicity. WT ( n = 6) and Trim27 −/− ( n = 10) mice were treated with TNF-α (20

    Journal: Molecular and Cellular Biology

    Article Title: Ubiquitination-Deubiquitination by the TRIM27-USP7 Complex Regulates Tumor Necrosis Factor Alpha-Induced Apoptosis

    doi: 10.1128/MCB.00465-13

    Figure Lengend Snippet: TRIM27 positively regulates TNF-α-induced apoptosis. (A) Resistance of wild-type (WT) and Trim27 −/− mice to TNF-α cytotoxicity. WT ( n = 6) and Trim27 −/− ( n = 10) mice were treated with TNF-α (20

    Article Snippet: Anti-TRIM27 (IBL-America) or anti-USP7 (Bethyl Laboratories) antibody was used for immunoblotting.

    Techniques: Mouse Assay

    Model describing the role of TRIM27-USP7 in TNF-α-induced apoptosis. Upon binding to TNF-α, TNFR1 binds to TRADD and triggers the formation of complex I and II. Complex I, containing TRADD, TRAF2, TRAF5, RIP1, cIAP1, and cIAP2, activates

    Journal: Molecular and Cellular Biology

    Article Title: Ubiquitination-Deubiquitination by the TRIM27-USP7 Complex Regulates Tumor Necrosis Factor Alpha-Induced Apoptosis

    doi: 10.1128/MCB.00465-13

    Figure Lengend Snippet: Model describing the role of TRIM27-USP7 in TNF-α-induced apoptosis. Upon binding to TNF-α, TNFR1 binds to TRADD and triggers the formation of complex I and II. Complex I, containing TRADD, TRAF2, TRAF5, RIP1, cIAP1, and cIAP2, activates

    Article Snippet: Anti-TRIM27 (IBL-America) or anti-USP7 (Bethyl Laboratories) antibody was used for immunoblotting.

    Techniques: Binding Assay

    TRIM27-USP7 regulation of RIP1 ubiquitination in the presence of TNF-α and CHX. (A) Knockdown of USP7 using siRNA in immortalized MEF. Three different sequences of mouse USP7 siRNA were transfected, and cell lysates were analyzed by Western blotting

    Journal: Molecular and Cellular Biology

    Article Title: Ubiquitination-Deubiquitination by the TRIM27-USP7 Complex Regulates Tumor Necrosis Factor Alpha-Induced Apoptosis

    doi: 10.1128/MCB.00465-13

    Figure Lengend Snippet: TRIM27-USP7 regulation of RIP1 ubiquitination in the presence of TNF-α and CHX. (A) Knockdown of USP7 using siRNA in immortalized MEF. Three different sequences of mouse USP7 siRNA were transfected, and cell lysates were analyzed by Western blotting

    Article Snippet: Anti-TRIM27 (IBL-America) or anti-USP7 (Bethyl Laboratories) antibody was used for immunoblotting.

    Techniques: Transfection, Western Blot

    TRIM27 deubiquitinates RIP1. (A) Deubiquitination of RIP1 by TRIM27. HEK293T cells were transfected with the FLAG-RIP1 expression vector or control empty vector, together with the Myc-Ub expression vector and increasing amounts of the TRIM27 expression

    Journal: Molecular and Cellular Biology

    Article Title: Ubiquitination-Deubiquitination by the TRIM27-USP7 Complex Regulates Tumor Necrosis Factor Alpha-Induced Apoptosis

    doi: 10.1128/MCB.00465-13

    Figure Lengend Snippet: TRIM27 deubiquitinates RIP1. (A) Deubiquitination of RIP1 by TRIM27. HEK293T cells were transfected with the FLAG-RIP1 expression vector or control empty vector, together with the Myc-Ub expression vector and increasing amounts of the TRIM27 expression

    Article Snippet: Anti-TRIM27 (IBL-America) or anti-USP7 (Bethyl Laboratories) antibody was used for immunoblotting.

    Techniques: Transfection, Expressing, Plasmid Preparation

    TRIM27 induces RIP1 deubiquitination by ubiquitinating and activating USP7. (A) Domain structure of TRIM27. (B) TRIM27 ubiquitinates USP7 via its B-box domain. HEK293T cells were cotransfected with FLAG-USP7 and Myc-ubiquitin expression vectors and a

    Journal: Molecular and Cellular Biology

    Article Title: Ubiquitination-Deubiquitination by the TRIM27-USP7 Complex Regulates Tumor Necrosis Factor Alpha-Induced Apoptosis

    doi: 10.1128/MCB.00465-13

    Figure Lengend Snippet: TRIM27 induces RIP1 deubiquitination by ubiquitinating and activating USP7. (A) Domain structure of TRIM27. (B) TRIM27 ubiquitinates USP7 via its B-box domain. HEK293T cells were cotransfected with FLAG-USP7 and Myc-ubiquitin expression vectors and a

    Article Snippet: Anti-TRIM27 (IBL-America) or anti-USP7 (Bethyl Laboratories) antibody was used for immunoblotting.

    Techniques: Expressing

    Generation of BMMCs from TRIM27 +/+ and TRIM27 −/− mice. (A) Lysates of TRIM27 +/+ and TRIM27 −/− BMMCs immunoblotted with antibodies to TRIM27 and PI3KC2β. (B) FACS analysis demonstrating similar levels of expression

    Journal: Molecular and Cellular Biology

    Article Title: Phosphatidylinositol-3-Kinase C2? and TRIM27 Function To Positively and Negatively Regulate IgE Receptor Activation of Mast Cells

    doi: 10.1128/MCB.00019-12

    Figure Lengend Snippet: Generation of BMMCs from TRIM27 +/+ and TRIM27 −/− mice. (A) Lysates of TRIM27 +/+ and TRIM27 −/− BMMCs immunoblotted with antibodies to TRIM27 and PI3KC2β. (B) FACS analysis demonstrating similar levels of expression

    Article Snippet: Anti-TRIM27 antibodies were purchased from IBL America.

    Techniques: Mouse Assay, FACS, Expressing

    Systemic anaphylaxis in TRIM27 +/+ and TRIM27 −/− mice. (A) Mean decrease in body temperature (°C) of TRIM27 +/+ and TRIM27 −/− mice following induction of passive systemic anaphylaxis ( n = 5 mice in each group). (B)

    Journal: Molecular and Cellular Biology

    Article Title: Phosphatidylinositol-3-Kinase C2? and TRIM27 Function To Positively and Negatively Regulate IgE Receptor Activation of Mast Cells

    doi: 10.1128/MCB.00019-12

    Figure Lengend Snippet: Systemic anaphylaxis in TRIM27 +/+ and TRIM27 −/− mice. (A) Mean decrease in body temperature (°C) of TRIM27 +/+ and TRIM27 −/− mice following induction of passive systemic anaphylaxis ( n = 5 mice in each group). (B)

    Article Snippet: Anti-TRIM27 antibodies were purchased from IBL America.

    Techniques: Mouse Assay

    Increased KCa3.1 channel activity and FcεRI-stimulated Ca 2+ influx in TRIM27 −/− BMMCs. (A) TRIM27 +/+ and TRIM27 −/− BMMCs were sensitized with anti-DNP IgE, and KCa3.1 channel activity was assessed before (a) and

    Journal: Molecular and Cellular Biology

    Article Title: Phosphatidylinositol-3-Kinase C2? and TRIM27 Function To Positively and Negatively Regulate IgE Receptor Activation of Mast Cells

    doi: 10.1128/MCB.00019-12

    Figure Lengend Snippet: Increased KCa3.1 channel activity and FcεRI-stimulated Ca 2+ influx in TRIM27 −/− BMMCs. (A) TRIM27 +/+ and TRIM27 −/− BMMCs were sensitized with anti-DNP IgE, and KCa3.1 channel activity was assessed before (a) and

    Article Snippet: Anti-TRIM27 antibodies were purchased from IBL America.

    Techniques: Activity Assay

    PI3KC2β is necessary for FcεRI-stimulated activation of KCa3.1 and Ca 2+ influx of BMMCs. (A) Real-time PCR of PI3KC2β in TRIM27 +/+ BMMCs transfected with a control or siRNA to PI3KC2β. (B) TRIM27 +/+ BMMCs transfected with

    Journal: Molecular and Cellular Biology

    Article Title: Phosphatidylinositol-3-Kinase C2? and TRIM27 Function To Positively and Negatively Regulate IgE Receptor Activation of Mast Cells

    doi: 10.1128/MCB.00019-12

    Figure Lengend Snippet: PI3KC2β is necessary for FcεRI-stimulated activation of KCa3.1 and Ca 2+ influx of BMMCs. (A) Real-time PCR of PI3KC2β in TRIM27 +/+ BMMCs transfected with a control or siRNA to PI3KC2β. (B) TRIM27 +/+ BMMCs transfected with

    Article Snippet: Anti-TRIM27 antibodies were purchased from IBL America.

    Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Transfection

    β-Hexosaminidase release and cytokine production are increased in TRIM27 −/− BMMCs. (A) TRIM27 +/+ and TRIM27 −/− BMMCs (1 × 10 6 ) were plated in 96-well plates, sensitized with anti-DNP IgE, and then stimulated

    Journal: Molecular and Cellular Biology

    Article Title: Phosphatidylinositol-3-Kinase C2? and TRIM27 Function To Positively and Negatively Regulate IgE Receptor Activation of Mast Cells

    doi: 10.1128/MCB.00019-12

    Figure Lengend Snippet: β-Hexosaminidase release and cytokine production are increased in TRIM27 −/− BMMCs. (A) TRIM27 +/+ and TRIM27 −/− BMMCs (1 × 10 6 ) were plated in 96-well plates, sensitized with anti-DNP IgE, and then stimulated

    Article Snippet: Anti-TRIM27 antibodies were purchased from IBL America.

    Techniques:

    FcεRI-stimulated tyrosine phosphorylation of proximal signaling molecules and the ERK MAP kinase pathway are similar between TRIM27 +/+ and TRIM27 −/− BMMCs. Lysates from TRIM27 +/+ and TRIM27 −/− BMMCs were stimulated

    Journal: Molecular and Cellular Biology

    Article Title: Phosphatidylinositol-3-Kinase C2? and TRIM27 Function To Positively and Negatively Regulate IgE Receptor Activation of Mast Cells

    doi: 10.1128/MCB.00019-12

    Figure Lengend Snippet: FcεRI-stimulated tyrosine phosphorylation of proximal signaling molecules and the ERK MAP kinase pathway are similar between TRIM27 +/+ and TRIM27 −/− BMMCs. Lysates from TRIM27 +/+ and TRIM27 −/− BMMCs were stimulated

    Article Snippet: Anti-TRIM27 antibodies were purchased from IBL America.

    Techniques:

    TRIM27 negatively regulates NOD2 signaling. A–C) NF-κB luciferase assays in HEK293T cells to determine the influence of TRIM27 overexpression on MDP-induced NOD2-mediated (A), TNF- (B), or IKK-β-induced (C) NF-κB activation. Normalized luciferase activity (nRLU) of unstimulated (white bars) and stimulated (black bars) samples is shown. Values are given as mean+SD (n = 3). *, P

    Journal: PLoS ONE

    Article Title: TRIM27 Negatively Regulates NOD2 by Ubiquitination and Proteasomal Degradation

    doi: 10.1371/journal.pone.0041255

    Figure Lengend Snippet: TRIM27 negatively regulates NOD2 signaling. A–C) NF-κB luciferase assays in HEK293T cells to determine the influence of TRIM27 overexpression on MDP-induced NOD2-mediated (A), TNF- (B), or IKK-β-induced (C) NF-κB activation. Normalized luciferase activity (nRLU) of unstimulated (white bars) and stimulated (black bars) samples is shown. Values are given as mean+SD (n = 3). *, P

    Article Snippet: Primary antibodies: rabbit anti-TRIM27 (1∶500; IBL) and mouse anti-myc (1∶1000; 9E10, Sigma).

    Techniques: Luciferase, Over Expression, Activation Assay, Activity Assay

    TRIM27 contributes to proteasomal degradation of NOD2. A) HEK293T cells transfected with low amounts of Flag-NOD2 and myc-TRIM27, E3 or CTR as indicated were treated with 30 µg/ml cycloheximid (CHX) and immunoblots of total cell lysates (top) were performed using the indicated antibodies. GAPDH served as loading control. B) HEK293T cells expressing the indicated proteins were treated with 100 nM bortezomib. Lysates were subjected to immunoprecipitation as described in Figure 1A . Actin served as loading control. C) HEK293T cells transfected for 48 h with siCTR, siTRIM27-1 or -3 and subsequently with Flag-NOD2 were treated with 30 µg/ml CHX, as indicated. Immunoblots of total cell lysates were performed using the indicated antibodies. GAPDH served as loading control. Representative data of at least three independent experiments are shown (see also Fig. S3B ).

    Journal: PLoS ONE

    Article Title: TRIM27 Negatively Regulates NOD2 by Ubiquitination and Proteasomal Degradation

    doi: 10.1371/journal.pone.0041255

    Figure Lengend Snippet: TRIM27 contributes to proteasomal degradation of NOD2. A) HEK293T cells transfected with low amounts of Flag-NOD2 and myc-TRIM27, E3 or CTR as indicated were treated with 30 µg/ml cycloheximid (CHX) and immunoblots of total cell lysates (top) were performed using the indicated antibodies. GAPDH served as loading control. B) HEK293T cells expressing the indicated proteins were treated with 100 nM bortezomib. Lysates were subjected to immunoprecipitation as described in Figure 1A . Actin served as loading control. C) HEK293T cells transfected for 48 h with siCTR, siTRIM27-1 or -3 and subsequently with Flag-NOD2 were treated with 30 µg/ml CHX, as indicated. Immunoblots of total cell lysates were performed using the indicated antibodies. GAPDH served as loading control. Representative data of at least three independent experiments are shown (see also Fig. S3B ).

    Article Snippet: Primary antibodies: rabbit anti-TRIM27 (1∶500; IBL) and mouse anti-myc (1∶1000; 9E10, Sigma).

    Techniques: Transfection, Western Blot, Expressing, Immunoprecipitation

    NOD2 is ubiquitinated. A) To determine NOD1 and NOD2 ubiquitination, denatured lysates of HEK293T cells expressing the indicated proteins were subjected to immunoprecipitation. Immunoprecipitates were immunoblotted using the indicated antibodies B) To determine the type of ubiquitin-linkage, lysates of HEK293T cells expressing the NOD1 or NOD2 were subjected to immunoprecipitation. Endogenous ubiquitin was revealed using a K48-link specific antibody. C) HEK293T cells were transfected for 72 h with siCTR, siTRIM27-1 or -3 after expression of Flag-NOD2 and HA-Ubiquitin. Immunoblots of immunoprecipitates (IP) and total lysates (Input) were performed using the indicated antibodies. D) TRIM27 and a E3 mutant of TRIM27 were expressed together with NOD2 and HA-ubiquitin in HEK293T cells. Immunoprecipitates were probed with the indicated antibodies. E) HEK293T cells expressing the indicated proteins were stimulated with 10 µM MDP for the indicated time. Lysates were subjected to immunoprecipitation as described in (C). Densitometric analysis of the TRIM27 signal in the immunoprecipitation normalized to the input signal is shown (bottom). Representative data of at least three independent experiments are shown.

    Journal: PLoS ONE

    Article Title: TRIM27 Negatively Regulates NOD2 by Ubiquitination and Proteasomal Degradation

    doi: 10.1371/journal.pone.0041255

    Figure Lengend Snippet: NOD2 is ubiquitinated. A) To determine NOD1 and NOD2 ubiquitination, denatured lysates of HEK293T cells expressing the indicated proteins were subjected to immunoprecipitation. Immunoprecipitates were immunoblotted using the indicated antibodies B) To determine the type of ubiquitin-linkage, lysates of HEK293T cells expressing the NOD1 or NOD2 were subjected to immunoprecipitation. Endogenous ubiquitin was revealed using a K48-link specific antibody. C) HEK293T cells were transfected for 72 h with siCTR, siTRIM27-1 or -3 after expression of Flag-NOD2 and HA-Ubiquitin. Immunoblots of immunoprecipitates (IP) and total lysates (Input) were performed using the indicated antibodies. D) TRIM27 and a E3 mutant of TRIM27 were expressed together with NOD2 and HA-ubiquitin in HEK293T cells. Immunoprecipitates were probed with the indicated antibodies. E) HEK293T cells expressing the indicated proteins were stimulated with 10 µM MDP for the indicated time. Lysates were subjected to immunoprecipitation as described in (C). Densitometric analysis of the TRIM27 signal in the immunoprecipitation normalized to the input signal is shown (bottom). Representative data of at least three independent experiments are shown.

    Article Snippet: Primary antibodies: rabbit anti-TRIM27 (1∶500; IBL) and mouse anti-myc (1∶1000; 9E10, Sigma).

    Techniques: Expressing, Immunoprecipitation, Transfection, Western Blot, Mutagenesis

    NOD2 physically interacts with TRIM27 via the NBD domain. A–C) Lysates of HEK293T cells expressing the indicated proteins were subjected to immunoprecipitation using anti-Flag (A and C) or anti-myc beads (B). Immunoblots of immunoprecipitates (IP) and total lysates (Input) were performed using the indicated antibodies. D) Immunoprecipitation of endogenous NOD2 from SW480 cells using the NOD2- specific monoclonal antibody 6F6. Cells were treated with 10 µM MDP for 3 h as indicated. Immunoblots of immunoprecipitates (IP) and total lysates (Input) were performed using the indicated antibodies. E) Protein-protein binding assays using in vitro transcribed and translated [35S]-methionine-labeled NOD2 and recombinant GST or GST-TRIM27 (WT, ΔRING or ΔRING+B-Box) bound to glutathione-Sepharose beads. The coomassie-stained gel (bottom) and the autoradiograph (top) of co-precipitated NOD2 are shown.

    Journal: PLoS ONE

    Article Title: TRIM27 Negatively Regulates NOD2 by Ubiquitination and Proteasomal Degradation

    doi: 10.1371/journal.pone.0041255

    Figure Lengend Snippet: NOD2 physically interacts with TRIM27 via the NBD domain. A–C) Lysates of HEK293T cells expressing the indicated proteins were subjected to immunoprecipitation using anti-Flag (A and C) or anti-myc beads (B). Immunoblots of immunoprecipitates (IP) and total lysates (Input) were performed using the indicated antibodies. D) Immunoprecipitation of endogenous NOD2 from SW480 cells using the NOD2- specific monoclonal antibody 6F6. Cells were treated with 10 µM MDP for 3 h as indicated. Immunoblots of immunoprecipitates (IP) and total lysates (Input) were performed using the indicated antibodies. E) Protein-protein binding assays using in vitro transcribed and translated [35S]-methionine-labeled NOD2 and recombinant GST or GST-TRIM27 (WT, ΔRING or ΔRING+B-Box) bound to glutathione-Sepharose beads. The coomassie-stained gel (bottom) and the autoradiograph (top) of co-precipitated NOD2 are shown.

    Article Snippet: Primary antibodies: rabbit anti-TRIM27 (1∶500; IBL) and mouse anti-myc (1∶1000; 9E10, Sigma).

    Techniques: Expressing, Immunoprecipitation, Western Blot, Protein Binding, In Vitro, Labeling, Recombinant, Staining, Autoradiography

    Mapping of the interaction domains in NOD2 and TRIM27. The depicted NOD2 (A) or TRIM27 (B) deletion constructs (upper panels) were used for the domain mapping. Lysates were subjected to immunoprecipitation using anti-Flag beads and immunoblotted with the indicated antibodies.

    Journal: PLoS ONE

    Article Title: TRIM27 Negatively Regulates NOD2 by Ubiquitination and Proteasomal Degradation

    doi: 10.1371/journal.pone.0041255

    Figure Lengend Snippet: Mapping of the interaction domains in NOD2 and TRIM27. The depicted NOD2 (A) or TRIM27 (B) deletion constructs (upper panels) were used for the domain mapping. Lysates were subjected to immunoprecipitation using anti-Flag beads and immunoblotted with the indicated antibodies.

    Article Snippet: Primary antibodies: rabbit anti-TRIM27 (1∶500; IBL) and mouse anti-myc (1∶1000; 9E10, Sigma).

    Techniques: Construct, Immunoprecipitation

    NOD2 WT localizes to the nucleus whereas NOD2 K305R or NOD1 do not. A) Indirect immunofluorescence micrographs of HeLa cells grown on coverslips and expressing myc-NOD2 WT (top) or myc-NOD2 K305R (bottom) were treated with 50 nM leptomycine B (LMB) for 4 h or left untreated. Images with signals for DAPI, TRIM27, myc-NOD2 and an overlay (blue: DAPI, red: TRIM27, green: NOD2) are shown. B) Quantification of NOD2 subcellular localization in HeLa cells expressing Flag-NOD2 WT or K305R, as indicated (n = 100). Data is representative of two independent experiments. C) Cellular fractions of HEK293T cells transfected with Flag-NOD1, -NOD2 WT or -NOD2 K305R, as indicated, and treated as described in A were generated. Immunoblots of precipitated protein and total lysates are shown. For densitometric analysis, cytosolic and nuclear signals were normalized to GAPDH and Lamin A/C, respectively. The ratio of nuclear to cytosolic protein is shown in fold. Representative data of at least three independent experiments are shown. D) Indirect immunofluorescence micrographs of HeLa cells expressing untagged-NOD2 WT treated with 50 nM leptomycine B (LMB) for 4 h or left untreated. Bars, 10 µm.

    Journal: PLoS ONE

    Article Title: TRIM27 Negatively Regulates NOD2 by Ubiquitination and Proteasomal Degradation

    doi: 10.1371/journal.pone.0041255

    Figure Lengend Snippet: NOD2 WT localizes to the nucleus whereas NOD2 K305R or NOD1 do not. A) Indirect immunofluorescence micrographs of HeLa cells grown on coverslips and expressing myc-NOD2 WT (top) or myc-NOD2 K305R (bottom) were treated with 50 nM leptomycine B (LMB) for 4 h or left untreated. Images with signals for DAPI, TRIM27, myc-NOD2 and an overlay (blue: DAPI, red: TRIM27, green: NOD2) are shown. B) Quantification of NOD2 subcellular localization in HeLa cells expressing Flag-NOD2 WT or K305R, as indicated (n = 100). Data is representative of two independent experiments. C) Cellular fractions of HEK293T cells transfected with Flag-NOD1, -NOD2 WT or -NOD2 K305R, as indicated, and treated as described in A were generated. Immunoblots of precipitated protein and total lysates are shown. For densitometric analysis, cytosolic and nuclear signals were normalized to GAPDH and Lamin A/C, respectively. The ratio of nuclear to cytosolic protein is shown in fold. Representative data of at least three independent experiments are shown. D) Indirect immunofluorescence micrographs of HeLa cells expressing untagged-NOD2 WT treated with 50 nM leptomycine B (LMB) for 4 h or left untreated. Bars, 10 µm.

    Article Snippet: Primary antibodies: rabbit anti-TRIM27 (1∶500; IBL) and mouse anti-myc (1∶1000; 9E10, Sigma).

    Techniques: Immunofluorescence, Expressing, Transfection, Generated, Western Blot

    Cell-to-cell movement analysis of rSYNV sc4, M and G deletion mutants. (A) Schematic representation of rSYNV-GFP and rSYNV-GFP mutants with Δsc4, ΔM or ΔG deletions. Note that the SYNV gene order is shown in the antigenome sense. (B) Cell-to-cell movement of rSYNV-GFP and deletion derivatives. N . benthamiana leaves were agroinfiltrated with the rSYNV-GFP plasmid or the indicated mutant rSYNV-GFP plasmids along with supporting plasmids for expression of the N, P, L core proteins and the VSRs. Leaves were photographed with a fluorescence microscope at 8 and 14 dpi. Scale bar, 100 μm. (C) Complementation of rSYNV-eGFP-Δsc4 cell-to-cell movement by the sc4 protein expressed in trans from the MR-sc4-RFP minireplicon. Agrobacterium strains harboring the rSYNV-eGFP-Δsc4 plasmid, the MR-sc4-RFP plasmid, along with the supporting plasmids indicated in the panel B legend, were mixed and infiltrated into N . benthamiana leaves. Fluorescence images for GFP, RFP and the overlaid images are shown at 8 and 14 dpi. Scale bar, 100 μm.

    Journal: PLoS Pathogens

    Article Title: Rescue of a Plant Negative-Strand RNA Virus from Cloned cDNA: Insights into Enveloped Plant Virus Movement and Morphogenesis

    doi: 10.1371/journal.ppat.1005223

    Figure Lengend Snippet: Cell-to-cell movement analysis of rSYNV sc4, M and G deletion mutants. (A) Schematic representation of rSYNV-GFP and rSYNV-GFP mutants with Δsc4, ΔM or ΔG deletions. Note that the SYNV gene order is shown in the antigenome sense. (B) Cell-to-cell movement of rSYNV-GFP and deletion derivatives. N . benthamiana leaves were agroinfiltrated with the rSYNV-GFP plasmid or the indicated mutant rSYNV-GFP plasmids along with supporting plasmids for expression of the N, P, L core proteins and the VSRs. Leaves were photographed with a fluorescence microscope at 8 and 14 dpi. Scale bar, 100 μm. (C) Complementation of rSYNV-eGFP-Δsc4 cell-to-cell movement by the sc4 protein expressed in trans from the MR-sc4-RFP minireplicon. Agrobacterium strains harboring the rSYNV-eGFP-Δsc4 plasmid, the MR-sc4-RFP plasmid, along with the supporting plasmids indicated in the panel B legend, were mixed and infiltrated into N . benthamiana leaves. Fluorescence images for GFP, RFP and the overlaid images are shown at 8 and 14 dpi. Scale bar, 100 μm.

    Article Snippet: Proteins separated by SDS-PAGE were either stained with Coomassie blue or transferred to nitrocellulose membranes and probed with polyclonal antiserum specific to the disrupted SYNV virions [ ], the SYNV G [ ], or monoclonal antibodies against GFP and RFP (Abcam, Cambridge, UK).

    Techniques: Plasmid Preparation, Mutagenesis, Expressing, Fluorescence, Microscopy

    Improvement of SYNV minireplicon (MR) expression by optimizing the core protein ratios. (A) Schematic representation of the SYNV MR-GFP-RFP containing GFP and RFP reporter genes substituted for the N and P ORFs. The SYNV MR-GFP-RFP antigenomic RNA was transcribed from a CaMV double 35S promoter (2X35S), and flanked by a Hammerhead ribozyme (HH Rz) and HDV ribozyme (HDV Rz) sequence to produce exact 5′- and 3′- ends. le: leader; tr: trailer; LB: left border sequence; RB: right border sequence; Nos: nopaline synthase terminator; N/P GJ: N/P gene junction. (B) Visualization of plaques expressing GFP reporter protein in infiltrated plants. Equal volumes of 0.8 OD Agrobacterium cultures harboring the SYNV MR-GFP-RFP, pGD-NPL and the three VSRs plasmids were mixed and infiltrated into N . benthamiana leaves. Additional volumes of bacterial cultures containing the pGD-N (upper panels), pGD-P (middle panels) or pGD-L plasmids (bottom panels) at 0.2, 0.4 or 0.8 OD as indicated on the top of panels, were also included in the mixture to test their effects on reporter expression. Infiltrated leaves were photographed at 9 dpi with a fluorescence microscope under the GFP channel. Scale bar, 200 μm. (c) Detection of GFP and RFP protein levels in agroinfiltrated leaves by Western blotting using GFP- and RFP-specific antibodies. The Coomassie blue-stained Rubisco large subunit (Rub L) serves as a total protein loading control.

    Journal: PLoS Pathogens

    Article Title: Rescue of a Plant Negative-Strand RNA Virus from Cloned cDNA: Insights into Enveloped Plant Virus Movement and Morphogenesis

    doi: 10.1371/journal.ppat.1005223

    Figure Lengend Snippet: Improvement of SYNV minireplicon (MR) expression by optimizing the core protein ratios. (A) Schematic representation of the SYNV MR-GFP-RFP containing GFP and RFP reporter genes substituted for the N and P ORFs. The SYNV MR-GFP-RFP antigenomic RNA was transcribed from a CaMV double 35S promoter (2X35S), and flanked by a Hammerhead ribozyme (HH Rz) and HDV ribozyme (HDV Rz) sequence to produce exact 5′- and 3′- ends. le: leader; tr: trailer; LB: left border sequence; RB: right border sequence; Nos: nopaline synthase terminator; N/P GJ: N/P gene junction. (B) Visualization of plaques expressing GFP reporter protein in infiltrated plants. Equal volumes of 0.8 OD Agrobacterium cultures harboring the SYNV MR-GFP-RFP, pGD-NPL and the three VSRs plasmids were mixed and infiltrated into N . benthamiana leaves. Additional volumes of bacterial cultures containing the pGD-N (upper panels), pGD-P (middle panels) or pGD-L plasmids (bottom panels) at 0.2, 0.4 or 0.8 OD as indicated on the top of panels, were also included in the mixture to test their effects on reporter expression. Infiltrated leaves were photographed at 9 dpi with a fluorescence microscope under the GFP channel. Scale bar, 200 μm. (c) Detection of GFP and RFP protein levels in agroinfiltrated leaves by Western blotting using GFP- and RFP-specific antibodies. The Coomassie blue-stained Rubisco large subunit (Rub L) serves as a total protein loading control.

    Article Snippet: Proteins separated by SDS-PAGE were either stained with Coomassie blue or transferred to nitrocellulose membranes and probed with polyclonal antiserum specific to the disrupted SYNV virions [ ], the SYNV G [ ], or monoclonal antibodies against GFP and RFP (Abcam, Cambridge, UK).

    Techniques: Expressing, Sequencing, Fluorescence, Microscopy, Western Blot, Staining

    Protein sequence of TRIM27 and its expression were determined by RT-PCR and western blotting. A. The human TRIM27 cDNA encodes a protein of 513 amino acids; B. TRIM27 mRNAs are highly expressed in testis and ovary tissues. C. Western blotting shows TRIM27 expressed in both the testes and ovaries.

    Journal: International Journal of Clinical and Experimental Pathology

    Article Title: Role of tripartite motif protein 27 as a gametogenesis-related protein in human germ cells

    doi:

    Figure Lengend Snippet: Protein sequence of TRIM27 and its expression were determined by RT-PCR and western blotting. A. The human TRIM27 cDNA encodes a protein of 513 amino acids; B. TRIM27 mRNAs are highly expressed in testis and ovary tissues. C. Western blotting shows TRIM27 expressed in both the testes and ovaries.

    Article Snippet: Slides were washed in 0.4% Photoflo (Kodak), dried at room temperature, and stained using anti-SYCP3 (1:200, Abcam), anti-γH2AX (1:200, Upstate), and anti-Trim27 (1:200, NOVUS).

    Techniques: Sequencing, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

    Co-localization of TRIM27 and γ-H2AX protein was detected in the testis and chromosome sections. A: TRIM27 (green) and nucleus (blue) signals at low magnification. TRIM27 protein (green) was localized in primary spermatocytes undergoing meiotic prophase during the first cycle of spermatogenesis. B: TRIM27 (green) and nucleus (blue) signals at high magnification. TRIM27 (green) was clearly present in the cytoplasm of round spermatids and nuclear (blue) in the primary spermatocytes. Interestingly, TRIM27 (green) and γ-H2AX (red) were co-localized in the sex body of primary spermatocytes. C: TRIM27 (green) and SYCP3 (red) co-localized in the primary spermatocytes of chromosome sections, suggesting TRIM27 is a meiosis-associated protein. Legend as in Scale bars = 10 μm.

    Journal: International Journal of Clinical and Experimental Pathology

    Article Title: Role of tripartite motif protein 27 as a gametogenesis-related protein in human germ cells

    doi:

    Figure Lengend Snippet: Co-localization of TRIM27 and γ-H2AX protein was detected in the testis and chromosome sections. A: TRIM27 (green) and nucleus (blue) signals at low magnification. TRIM27 protein (green) was localized in primary spermatocytes undergoing meiotic prophase during the first cycle of spermatogenesis. B: TRIM27 (green) and nucleus (blue) signals at high magnification. TRIM27 (green) was clearly present in the cytoplasm of round spermatids and nuclear (blue) in the primary spermatocytes. Interestingly, TRIM27 (green) and γ-H2AX (red) were co-localized in the sex body of primary spermatocytes. C: TRIM27 (green) and SYCP3 (red) co-localized in the primary spermatocytes of chromosome sections, suggesting TRIM27 is a meiosis-associated protein. Legend as in Scale bars = 10 μm.

    Article Snippet: Slides were washed in 0.4% Photoflo (Kodak), dried at room temperature, and stained using anti-SYCP3 (1:200, Abcam), anti-γH2AX (1:200, Upstate), and anti-Trim27 (1:200, NOVUS).

    Techniques:

    TRIM27 expression determined of human testis sections by immunostaining. TRIM27 (green signals) is clearly present in both the nucleus (A) and cytoplasm (B) of testis cells, and was localized in the sex body of primary spermatocytes (B), and also evident in the cytoplasm Sertoli cells. Legend as in Scale bars = 10 μm.

    Journal: International Journal of Clinical and Experimental Pathology

    Article Title: Role of tripartite motif protein 27 as a gametogenesis-related protein in human germ cells

    doi:

    Figure Lengend Snippet: TRIM27 expression determined of human testis sections by immunostaining. TRIM27 (green signals) is clearly present in both the nucleus (A) and cytoplasm (B) of testis cells, and was localized in the sex body of primary spermatocytes (B), and also evident in the cytoplasm Sertoli cells. Legend as in Scale bars = 10 μm.

    Article Snippet: Slides were washed in 0.4% Photoflo (Kodak), dried at room temperature, and stained using anti-SYCP3 (1:200, Abcam), anti-γH2AX (1:200, Upstate), and anti-Trim27 (1:200, NOVUS).

    Techniques: Expressing, Immunostaining

    Localization of TRIM27 protein was detected in developing female gonads. A: TRIM27 was clearly present in the adult ovaries section. B: TRIM27 was also evident in the nucleus of follicle cells, and is diffusely dispersed in the cytoplasm of granule cells. Legend as in Scale bars = 10 μm.

    Journal: International Journal of Clinical and Experimental Pathology

    Article Title: Role of tripartite motif protein 27 as a gametogenesis-related protein in human germ cells

    doi:

    Figure Lengend Snippet: Localization of TRIM27 protein was detected in developing female gonads. A: TRIM27 was clearly present in the adult ovaries section. B: TRIM27 was also evident in the nucleus of follicle cells, and is diffusely dispersed in the cytoplasm of granule cells. Legend as in Scale bars = 10 μm.

    Article Snippet: Slides were washed in 0.4% Photoflo (Kodak), dried at room temperature, and stained using anti-SYCP3 (1:200, Abcam), anti-γH2AX (1:200, Upstate), and anti-Trim27 (1:200, NOVUS).

    Techniques:

    TOMM-40 promotes insulin secretion. ( A ) Adults, carrying an integrated daf-28::gfp transgene ( svIs69) , were analyzed for coelomocyte GFP content. Absent or severely reduced GFP content (a maximum of two faintly fluorescing coelomocytes) was scored as secretion defective. ( B ) Neuronal expression of GFP from the transcriptional reporter transgene P daf-28 ::gfp in tomm-40(peRNAi) and emv(peRNAi) treated animals, imaged with fluorescence optics. ( C ) Fluorescence optics of peRNAi treated animals of the arIs37 strain, carrying a P myo-3 ::ssgfp transgene that expresses ssGFP in body wall muscle, from where it is secreted into the pseudocoelom. Coelomocyte sequestration of GFP indicates functional coelomocyte endocytosis. rme-1(b1045) is a previously characterized endocytosis defective mutant [39] . Arrows indicate GFP labeled coelomocytes. ( D ) Fluorescence micrographs of emv, tomm-20 and tomm-22(RNAi) -treated animals carrying the P hsp-6 ::gfp reporter. ( E–F ) Relative pixel intensity plots of coelomocyte GFP contents in peRNAi or RNAi treated adults carrying ( E ) an Anf::gfp transgene or ( F ) an ins-22::venus transgene. The strongest fluorescing coelomocyte in the posterior most pair was scored. The mean value of the pixel intensity in emv was set to 1 in each experiment. Error bars represent +/− mean standard deviations from three independent experiments. ( G ) Micrographs of RNAi treated sibling animals, carrying either the integrated daf-16::gfp transgene only, or both the integrated daf-16::gfp transgene and an extra chromosomal P daf-28 ::daf-28 transgene for DAF-28 overexpression. Presence of the P daf-28 ::daf-28 transgene is indicated by a coelomocyte RFP co-injection marker (red arrows). White arrowheads indicate nuclear DAF-16::GFP localization in a tomm-40(RNAi) animal. White arrows indicate the absence of DAF-16::GFP in nuclei of intestinal cells in a tomm-40(RNAi); daf-28(++) animal. Scale bars are 25 µm.

    Journal: PLoS ONE

    Article Title: Mitochondrial Function Is Required for Secretion of DAF-28/Insulin in C. elegans

    doi: 10.1371/journal.pone.0014507

    Figure Lengend Snippet: TOMM-40 promotes insulin secretion. ( A ) Adults, carrying an integrated daf-28::gfp transgene ( svIs69) , were analyzed for coelomocyte GFP content. Absent or severely reduced GFP content (a maximum of two faintly fluorescing coelomocytes) was scored as secretion defective. ( B ) Neuronal expression of GFP from the transcriptional reporter transgene P daf-28 ::gfp in tomm-40(peRNAi) and emv(peRNAi) treated animals, imaged with fluorescence optics. ( C ) Fluorescence optics of peRNAi treated animals of the arIs37 strain, carrying a P myo-3 ::ssgfp transgene that expresses ssGFP in body wall muscle, from where it is secreted into the pseudocoelom. Coelomocyte sequestration of GFP indicates functional coelomocyte endocytosis. rme-1(b1045) is a previously characterized endocytosis defective mutant [39] . Arrows indicate GFP labeled coelomocytes. ( D ) Fluorescence micrographs of emv, tomm-20 and tomm-22(RNAi) -treated animals carrying the P hsp-6 ::gfp reporter. ( E–F ) Relative pixel intensity plots of coelomocyte GFP contents in peRNAi or RNAi treated adults carrying ( E ) an Anf::gfp transgene or ( F ) an ins-22::venus transgene. The strongest fluorescing coelomocyte in the posterior most pair was scored. The mean value of the pixel intensity in emv was set to 1 in each experiment. Error bars represent +/− mean standard deviations from three independent experiments. ( G ) Micrographs of RNAi treated sibling animals, carrying either the integrated daf-16::gfp transgene only, or both the integrated daf-16::gfp transgene and an extra chromosomal P daf-28 ::daf-28 transgene for DAF-28 overexpression. Presence of the P daf-28 ::daf-28 transgene is indicated by a coelomocyte RFP co-injection marker (red arrows). White arrowheads indicate nuclear DAF-16::GFP localization in a tomm-40(RNAi) animal. White arrows indicate the absence of DAF-16::GFP in nuclei of intestinal cells in a tomm-40(RNAi); daf-28(++) animal. Scale bars are 25 µm.

    Article Snippet: The pVB288GK plasmid, which contains wild type daf-28 under 3.4 kb of its own promoter, was co-injected with a Punc-122 ::RFP (coelomocyte::RFP) marker (Addgene plasmid 8938).

    Techniques: Expressing, Fluorescence, Functional Assay, Mutagenesis, Labeling, Over Expression, Injection, Marker

    CDS overexpression inhibits LD expansion. HeLa cells were transfected with eCFP-N1 and eCFP-CDS1 or mCherry-N1 and CDS2-mCherry for 48 h, followed by the treatment with oleate for 16 h. A, D: The protein lysates from untreated cells were subjected to Western blots against CDS1 (A), RFP (D), and GAPDH. B, E: LDs were stained with Nile Red (B) or BODIPY 493/503 (E) in oleate-treated cells and visualized using a confocal microscope. Bar = 10 μm. Arrows indicate the CDS-overexpressing cells. C, F: LD size distribution in HeLa cells was determined. Data were collected by measuring the diameter of at least 500 LDs using the ImageJ software. G: Cellular TAG level was determined by TLC following neutral lipid extraction and quantified using ImageJ software. Data are expressed as mean ± SD (n = 4). ** P

    Journal: Journal of Lipid Research

    Article Title: CDP-diacylglycerol synthases regulate the growth of lipid droplets and adipocyte development [S]

    doi: 10.1194/jlr.M060574

    Figure Lengend Snippet: CDS overexpression inhibits LD expansion. HeLa cells were transfected with eCFP-N1 and eCFP-CDS1 or mCherry-N1 and CDS2-mCherry for 48 h, followed by the treatment with oleate for 16 h. A, D: The protein lysates from untreated cells were subjected to Western blots against CDS1 (A), RFP (D), and GAPDH. B, E: LDs were stained with Nile Red (B) or BODIPY 493/503 (E) in oleate-treated cells and visualized using a confocal microscope. Bar = 10 μm. Arrows indicate the CDS-overexpressing cells. C, F: LD size distribution in HeLa cells was determined. Data were collected by measuring the diameter of at least 500 LDs using the ImageJ software. G: Cellular TAG level was determined by TLC following neutral lipid extraction and quantified using ImageJ software. Data are expressed as mean ± SD (n = 4). ** P

    Article Snippet: Immunoblots were performed with the following primary antibodies: anti-CDS1 (#ab88121), anti-RFP (#ab65856), anti-phospho-IRE1α (#ab124945) (Abcam); anti-total-IRE1α (#3294), anti-phospho-eIF2α (#3597), anti-total-eIF2α (#2103), anti-CHOP (#5554), anti-Calnexin (#2433) (Cell Signaling Technology); anti-β-actin (#P2103) (Sigma); and anti-CGI-58 (#12201-1-AP) (ProteinTech).

    Techniques: Over Expression, Transfection, Western Blot, Staining, Microscopy, Software, Thin Layer Chromatography

    Function of Hsp90 on PADI3 regulated CKS1 expression. RFP stable expressing HCT116 cells as the negative control, PADI3 stable expressing HCT116 cells as the positive control, Hsp90 overexpression plasmid was transfected into the PADI3 stable expressing HCT116 cells to study the function of Hsp90 on PADI3 regulated CKS1 expression, GFP overexpression plasmid was transfected into the PADI3 stable expressing HCT116 cells as the negative control group. a Western blot was used to measure the expression level of CKS1, CDK1 and p27kip1 after Hsp90 transfected into PADI3 stable expressing HCT116 cells; b qRT-PCR was used to verify the results of a . c CCK8 analysis was used to study the function of Hsp90 in the regulating of cell proliferation in PADI3 stable expressing HCT116 cells. GAPDH was selected as the internal control, *indicates p

    Journal: Cancer Cell International

    Article Title: PADI3 plays an antitumor role via the Hsp90/CKS1 pathway in colon cancer

    doi: 10.1186/s12935-019-0999-3

    Figure Lengend Snippet: Function of Hsp90 on PADI3 regulated CKS1 expression. RFP stable expressing HCT116 cells as the negative control, PADI3 stable expressing HCT116 cells as the positive control, Hsp90 overexpression plasmid was transfected into the PADI3 stable expressing HCT116 cells to study the function of Hsp90 on PADI3 regulated CKS1 expression, GFP overexpression plasmid was transfected into the PADI3 stable expressing HCT116 cells as the negative control group. a Western blot was used to measure the expression level of CKS1, CDK1 and p27kip1 after Hsp90 transfected into PADI3 stable expressing HCT116 cells; b qRT-PCR was used to verify the results of a . c CCK8 analysis was used to study the function of Hsp90 in the regulating of cell proliferation in PADI3 stable expressing HCT116 cells. GAPDH was selected as the internal control, *indicates p

    Article Snippet: Antibodies information The following primary antibodies used in this study were commercially obtained: PADI3 (Abcam, ab172959), GAPDH (Abcam, ab181603), His-tag (CST, 12698S), Flag (CST, 14793S), red fluorescent protein (RFP) (Abcam, ab28664), CKS1 (ThermoFisher, 36-6800), CDK1 (ThermoFisher, 33-1800), Hsp90 (CST, 4874S), and p27Kip1 (CST, 3686S).

    Techniques: Expressing, Negative Control, Positive Control, Over Expression, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR

    Function of CKS1 in HCT116 and SW620 cells. pCDNA3.1-CKS1-RFP plasmids were transfected to HCT116 and SW620 cells to study the function of CKS1 in colon cancer cells, pCDNA3.1-RFP plasmids transfected cells were used as controls; a CCK-8 assay was used to measure the proliferation ratio of HCT116 cells post plasmid was transfected for 12 h, 24 h and 48 h, respectively; b CCK-8 assay was used to measure the proliferation ratio of SW620 cells post plasmid was transfected for 12 h, 24 h and 48 h, respectively; c the colony formation ability of HCT116 cells was measured and statistically analyzed using a colony formation assay following 10 days of culture; d the colony formation ability of SW620 cells was measured and statistically analyzed using a colony formation assay following 14 days of culture. *Indicates p

    Journal: Cancer Cell International

    Article Title: PADI3 plays an antitumor role via the Hsp90/CKS1 pathway in colon cancer

    doi: 10.1186/s12935-019-0999-3

    Figure Lengend Snippet: Function of CKS1 in HCT116 and SW620 cells. pCDNA3.1-CKS1-RFP plasmids were transfected to HCT116 and SW620 cells to study the function of CKS1 in colon cancer cells, pCDNA3.1-RFP plasmids transfected cells were used as controls; a CCK-8 assay was used to measure the proliferation ratio of HCT116 cells post plasmid was transfected for 12 h, 24 h and 48 h, respectively; b CCK-8 assay was used to measure the proliferation ratio of SW620 cells post plasmid was transfected for 12 h, 24 h and 48 h, respectively; c the colony formation ability of HCT116 cells was measured and statistically analyzed using a colony formation assay following 10 days of culture; d the colony formation ability of SW620 cells was measured and statistically analyzed using a colony formation assay following 14 days of culture. *Indicates p

    Article Snippet: Antibodies information The following primary antibodies used in this study were commercially obtained: PADI3 (Abcam, ab172959), GAPDH (Abcam, ab181603), His-tag (CST, 12698S), Flag (CST, 14793S), red fluorescent protein (RFP) (Abcam, ab28664), CKS1 (ThermoFisher, 36-6800), CDK1 (ThermoFisher, 33-1800), Hsp90 (CST, 4874S), and p27Kip1 (CST, 3686S).

    Techniques: Transfection, CCK-8 Assay, Plasmid Preparation, Colony Assay

    Both 17-AAG and PADI3 can suppress CKS1 expression in HCT116 cells. a Expression profile of PADI3, CKS1, Hsp90, CDK1 and p27 kip1 were detected using western blot in colon cancer tissues and their corresponding adjacent tissues at the translational level, GAPDH was used to normalize the relative expression of them; b statistical analysis of western blot results in A; c 5 μM 17-AAG was used to treat HCT116 cells for 24 h, and western blot was used to measure the expression level of CKS1, CDK1 and p27 kip1 ; d Statistic analysis of c ; e Overexpression of PADI3 was performed in HCT116 cells, and western blot was used to detect the expression level of Hsp90, CKS1, CDK1 and p27 kip1 , overexpression of RFP as the control group; f statistical analysis of western blot results in e . GAPDH was selected as the internal control, *indicates p

    Journal: Cancer Cell International

    Article Title: PADI3 plays an antitumor role via the Hsp90/CKS1 pathway in colon cancer

    doi: 10.1186/s12935-019-0999-3

    Figure Lengend Snippet: Both 17-AAG and PADI3 can suppress CKS1 expression in HCT116 cells. a Expression profile of PADI3, CKS1, Hsp90, CDK1 and p27 kip1 were detected using western blot in colon cancer tissues and their corresponding adjacent tissues at the translational level, GAPDH was used to normalize the relative expression of them; b statistical analysis of western blot results in A; c 5 μM 17-AAG was used to treat HCT116 cells for 24 h, and western blot was used to measure the expression level of CKS1, CDK1 and p27 kip1 ; d Statistic analysis of c ; e Overexpression of PADI3 was performed in HCT116 cells, and western blot was used to detect the expression level of Hsp90, CKS1, CDK1 and p27 kip1 , overexpression of RFP as the control group; f statistical analysis of western blot results in e . GAPDH was selected as the internal control, *indicates p

    Article Snippet: Antibodies information The following primary antibodies used in this study were commercially obtained: PADI3 (Abcam, ab172959), GAPDH (Abcam, ab181603), His-tag (CST, 12698S), Flag (CST, 14793S), red fluorescent protein (RFP) (Abcam, ab28664), CKS1 (ThermoFisher, 36-6800), CDK1 (ThermoFisher, 33-1800), Hsp90 (CST, 4874S), and p27Kip1 (CST, 3686S).

    Techniques: Expressing, Western Blot, Over Expression

    Modulation of the inflammatory reaction at 3 days after LFPI in CCR2 and CX3CR1 transgenic mice. Control Ccr2 RFP /+ ; Cx3cr1 GFP /+ ( a ), Ccr2 RFP / RFP ( b ), and Cx3cr1 GFP / GFP ( c ) mice were subjected to surgery (craniotomy; Sham mice) or mild injury, and brains were collected 3 days later. Serial sections were stained for GFP ( a , b (Mild), and c ) or Iba1 ( b (Surgery)) to visualize microglia or RFP to visualize infiltrating bone marrow-derived monocytes (BMDM). Inflammatory cells are mostly restricted to the site of injury. Scale bar on images with whole slices, 1 cm. The brightness on the images was adjusted to allow an easier distinction of the brain sections from the background; all sections in a series were adjusted to the same brightness level. Magnified images show a close up view of microglial and monocyte distribution around the cavity size. Note the reduced number of RFP + cells in Ccr2 RFP / RFP mice. Scale bar for magnified images, 100 μm

    Journal: Journal of Neuroinflammation

    Article Title: Ccr2 deletion dissociates cavity size and tau pathology after mild traumatic brain injury

    doi: 10.1186/s12974-015-0443-0

    Figure Lengend Snippet: Modulation of the inflammatory reaction at 3 days after LFPI in CCR2 and CX3CR1 transgenic mice. Control Ccr2 RFP /+ ; Cx3cr1 GFP /+ ( a ), Ccr2 RFP / RFP ( b ), and Cx3cr1 GFP / GFP ( c ) mice were subjected to surgery (craniotomy; Sham mice) or mild injury, and brains were collected 3 days later. Serial sections were stained for GFP ( a , b (Mild), and c ) or Iba1 ( b (Surgery)) to visualize microglia or RFP to visualize infiltrating bone marrow-derived monocytes (BMDM). Inflammatory cells are mostly restricted to the site of injury. Scale bar on images with whole slices, 1 cm. The brightness on the images was adjusted to allow an easier distinction of the brain sections from the background; all sections in a series were adjusted to the same brightness level. Magnified images show a close up view of microglial and monocyte distribution around the cavity size. Note the reduced number of RFP + cells in Ccr2 RFP / RFP mice. Scale bar for magnified images, 100 μm

    Article Snippet: To visualize the inflammatory reaction after TBI, serial sections spaced 150 μm apart and spanning ~4 mm thickness around the injury cavity were blocked in 10 % normal goat serum (NGS) and stained overnight at 4 °C with mouse anti-GFP (UCDavis/NIH NeuroMab Facility #75-132, 1:8000 dilution), or rabbit anti-Iba1 (Wako #019-19741, 1:1000) antibodies to identify microglia, and rabbit or rat anti-RFP (Abcam #ab62341, 1:1000 and Chromotek #5 F8 α-Red, 1:1000, respectively) antibodies to identify infiltrating monocytes.

    Techniques: Transgenic Assay, Mouse Assay, Staining, Derivative Assay

    Quantification of cavity volume and inflammatory reaction after TBI. Serial sections were stained for GFP (or Iba1) to visualize microglia or RFP to visualize infiltrating bone marrow-derived monocytes (BMDM). Whole slices, lesion cavity, and area of the slices occupied by monocytes (RFP + cells) or microglia (increased GFP or Iba1 immunoreactivity) were manually outlined to calculate their area; all sections were summed up to calculate volumes. Pathology was quantified as a percentage of the analyzed brain tissue (4 mm total thickness) that contained the lesion cavity ( a ), infiltrating monocytes ( b ), or increased microglial staining ( c ). Statistics: two-way ANOVA and Tukey’s post hoc test. Comparisons between groups are shown with horizontal lines ; vertical line in figure legend indicates main effect of genotype. * p

    Journal: Journal of Neuroinflammation

    Article Title: Ccr2 deletion dissociates cavity size and tau pathology after mild traumatic brain injury

    doi: 10.1186/s12974-015-0443-0

    Figure Lengend Snippet: Quantification of cavity volume and inflammatory reaction after TBI. Serial sections were stained for GFP (or Iba1) to visualize microglia or RFP to visualize infiltrating bone marrow-derived monocytes (BMDM). Whole slices, lesion cavity, and area of the slices occupied by monocytes (RFP + cells) or microglia (increased GFP or Iba1 immunoreactivity) were manually outlined to calculate their area; all sections were summed up to calculate volumes. Pathology was quantified as a percentage of the analyzed brain tissue (4 mm total thickness) that contained the lesion cavity ( a ), infiltrating monocytes ( b ), or increased microglial staining ( c ). Statistics: two-way ANOVA and Tukey’s post hoc test. Comparisons between groups are shown with horizontal lines ; vertical line in figure legend indicates main effect of genotype. * p

    Article Snippet: To visualize the inflammatory reaction after TBI, serial sections spaced 150 μm apart and spanning ~4 mm thickness around the injury cavity were blocked in 10 % normal goat serum (NGS) and stained overnight at 4 °C with mouse anti-GFP (UCDavis/NIH NeuroMab Facility #75-132, 1:8000 dilution), or rabbit anti-Iba1 (Wako #019-19741, 1:1000) antibodies to identify microglia, and rabbit or rat anti-RFP (Abcam #ab62341, 1:1000 and Chromotek #5 F8 α-Red, 1:1000, respectively) antibodies to identify infiltrating monocytes.

    Techniques: Staining, Derivative Assay

    Co-expression of mutant hSOD1 fused to RFP with mutant hSOD1 fused to YFP. In a matrix approach, all possible combinations for the 6 fusion constructs of mutant SOD1 fused to RFP or YFP were examined. In all cases, inclusions contained both proteins in saponin-resistant aggregates. At least three independent transfection experiments were performed and between 200 and 1,000 individual cells were analyzed in compiling these data.

    Journal: PLoS ONE

    Article Title: An Analysis of Interactions between Fluorescently-Tagged Mutant and Wild-Type SOD1 in Intracellular Inclusions

    doi: 10.1371/journal.pone.0083981

    Figure Lengend Snippet: Co-expression of mutant hSOD1 fused to RFP with mutant hSOD1 fused to YFP. In a matrix approach, all possible combinations for the 6 fusion constructs of mutant SOD1 fused to RFP or YFP were examined. In all cases, inclusions contained both proteins in saponin-resistant aggregates. At least three independent transfection experiments were performed and between 200 and 1,000 individual cells were analyzed in compiling these data.

    Article Snippet: A similar approach was used to create SOD1 fusion proteins with RFP [Turbo RFP cDNA obtained from the pTRIPZ empty vector available at Open Biosystems (Huntsville, AL, USA)] by replacing the YFP tag with the RFP tag.

    Techniques: Expressing, Mutagenesis, Construct, Transfection

    Co-expression of mutant hSOD1:RFP with WT-hSOD1mon:YFP or WT-hSOD1:YFP. CHO cells were transiently transfected with expression vectors for the SOD1 constructs shown. After 24:RFP produces inclusions that only weakly bind WT-hSOD1mon:YFP or WT-hSOD1:YFP. At least three independent transfection experiments were performed and between 200 and 1,000 individual cells were analyzed in compiling these data.

    Journal: PLoS ONE

    Article Title: An Analysis of Interactions between Fluorescently-Tagged Mutant and Wild-Type SOD1 in Intracellular Inclusions

    doi: 10.1371/journal.pone.0083981

    Figure Lengend Snippet: Co-expression of mutant hSOD1:RFP with WT-hSOD1mon:YFP or WT-hSOD1:YFP. CHO cells were transiently transfected with expression vectors for the SOD1 constructs shown. After 24:RFP produces inclusions that only weakly bind WT-hSOD1mon:YFP or WT-hSOD1:YFP. At least three independent transfection experiments were performed and between 200 and 1,000 individual cells were analyzed in compiling these data.

    Article Snippet: A similar approach was used to create SOD1 fusion proteins with RFP [Turbo RFP cDNA obtained from the pTRIPZ empty vector available at Open Biosystems (Huntsville, AL, USA)] by replacing the YFP tag with the RFP tag.

    Techniques: Expressing, Mutagenesis, Transfection, Construct

    Co-expression of WT-hSOD1mon:RFP with WT-hSOD1 and WT-hSOD1mon fused to YFP. CHO cells were transiently transfected with expression vectors for the SOD1 constructs shown. After 24-expression of WT-hSOD1:RFP with either WT-hSOD1:YFP or WT-hSOD1mon:YFP does not produce inclusions; all proteins remain soluble in saponin. B, WT-hSOD1:RFP co-expressed with WT-hSOD1mon:YFP demonstrates a lack of tight binding between these proteins. At least three independent transfection experiments were performed and between 200 and 1,000 individual cells were analyzed in compiling these data.

    Journal: PLoS ONE

    Article Title: An Analysis of Interactions between Fluorescently-Tagged Mutant and Wild-Type SOD1 in Intracellular Inclusions

    doi: 10.1371/journal.pone.0083981

    Figure Lengend Snippet: Co-expression of WT-hSOD1mon:RFP with WT-hSOD1 and WT-hSOD1mon fused to YFP. CHO cells were transiently transfected with expression vectors for the SOD1 constructs shown. After 24-expression of WT-hSOD1:RFP with either WT-hSOD1:YFP or WT-hSOD1mon:YFP does not produce inclusions; all proteins remain soluble in saponin. B, WT-hSOD1:RFP co-expressed with WT-hSOD1mon:YFP demonstrates a lack of tight binding between these proteins. At least three independent transfection experiments were performed and between 200 and 1,000 individual cells were analyzed in compiling these data.

    Article Snippet: A similar approach was used to create SOD1 fusion proteins with RFP [Turbo RFP cDNA obtained from the pTRIPZ empty vector available at Open Biosystems (Huntsville, AL, USA)] by replacing the YFP tag with the RFP tag.

    Techniques: Expressing, Transfection, Construct, Binding Assay

    Mutant SOD1 fused to RFP or YFP form similar types of inclusions that resist release by saponin. CHO cells were transiently transfected with expression vectors for A4V-hSOD1:RFP or A4V-hSOD1:YFP. After 24 hours the cells were treated, or not, with saponin, fixed in paraformaldehyde, and imaged. Inclusions formed by mutant hSOD1 fused to either RFP or YFP remained cell-associated after saponin treatment. The images shown are representative of 3 independent transfection experiments, analyzing between 200 and 1,000 individual cells. Similar observations were made with cells expressing G37R or G85R hSOD1 fused to either RFP or YFP (see Figures S1and S2).

    Journal: PLoS ONE

    Article Title: An Analysis of Interactions between Fluorescently-Tagged Mutant and Wild-Type SOD1 in Intracellular Inclusions

    doi: 10.1371/journal.pone.0083981

    Figure Lengend Snippet: Mutant SOD1 fused to RFP or YFP form similar types of inclusions that resist release by saponin. CHO cells were transiently transfected with expression vectors for A4V-hSOD1:RFP or A4V-hSOD1:YFP. After 24 hours the cells were treated, or not, with saponin, fixed in paraformaldehyde, and imaged. Inclusions formed by mutant hSOD1 fused to either RFP or YFP remained cell-associated after saponin treatment. The images shown are representative of 3 independent transfection experiments, analyzing between 200 and 1,000 individual cells. Similar observations were made with cells expressing G37R or G85R hSOD1 fused to either RFP or YFP (see Figures S1and S2).

    Article Snippet: A similar approach was used to create SOD1 fusion proteins with RFP [Turbo RFP cDNA obtained from the pTRIPZ empty vector available at Open Biosystems (Huntsville, AL, USA)] by replacing the YFP tag with the RFP tag.

    Techniques: Mutagenesis, Transfection, Expressing

    Inclusions formed by WT-hSOD1:RFP are not released by saponin. CHO cells were transiently transfected with expression vectors for the two SOD1 constructs shown. After 24-hSOD1:YFP is fully releasable by saponin treatment whereas WT-hSOD1:RFP remained cell-associated. The images shown are representative of 3 independent transfection experiments, analyzing between 200 and 1,000 individual cells.

    Journal: PLoS ONE

    Article Title: An Analysis of Interactions between Fluorescently-Tagged Mutant and Wild-Type SOD1 in Intracellular Inclusions

    doi: 10.1371/journal.pone.0083981

    Figure Lengend Snippet: Inclusions formed by WT-hSOD1:RFP are not released by saponin. CHO cells were transiently transfected with expression vectors for the two SOD1 constructs shown. After 24-hSOD1:YFP is fully releasable by saponin treatment whereas WT-hSOD1:RFP remained cell-associated. The images shown are representative of 3 independent transfection experiments, analyzing between 200 and 1,000 individual cells.

    Article Snippet: A similar approach was used to create SOD1 fusion proteins with RFP [Turbo RFP cDNA obtained from the pTRIPZ empty vector available at Open Biosystems (Huntsville, AL, USA)] by replacing the YFP tag with the RFP tag.

    Techniques: Transfection, Expressing, Construct

    Co-expression of WT-hSOD1:RFP with WT and mutant SOD1 fused to YFP. CHO cells were transiently transfected with expression vectors for the SOD1 constructs shown. After 24-hSOD1:RFP forms well defined round inclusions that are not released by saponin. Co-expressed WT-hSOD1:YFP appears to be closely associated with these inclusions, but after saponin this protein is released whereas the WT-hSOD1:RFP remains cell associated. B, Mutant SOD1:YFP appears to be more tightly bound to the surface of inclusions formed by WT-hSOD1:RFP. At least three independent transfection experiments were performed and between 200 and 1,000 individual cells were analyzed in compiling these data.

    Journal: PLoS ONE

    Article Title: An Analysis of Interactions between Fluorescently-Tagged Mutant and Wild-Type SOD1 in Intracellular Inclusions

    doi: 10.1371/journal.pone.0083981

    Figure Lengend Snippet: Co-expression of WT-hSOD1:RFP with WT and mutant SOD1 fused to YFP. CHO cells were transiently transfected with expression vectors for the SOD1 constructs shown. After 24-hSOD1:RFP forms well defined round inclusions that are not released by saponin. Co-expressed WT-hSOD1:YFP appears to be closely associated with these inclusions, but after saponin this protein is released whereas the WT-hSOD1:RFP remains cell associated. B, Mutant SOD1:YFP appears to be more tightly bound to the surface of inclusions formed by WT-hSOD1:RFP. At least three independent transfection experiments were performed and between 200 and 1,000 individual cells were analyzed in compiling these data.

    Article Snippet: A similar approach was used to create SOD1 fusion proteins with RFP [Turbo RFP cDNA obtained from the pTRIPZ empty vector available at Open Biosystems (Huntsville, AL, USA)] by replacing the YFP tag with the RFP tag.

    Techniques: Expressing, Mutagenesis, Transfection, Construct

    Mutant SOD1 fused to either RFP or YFP forms inclusions with similar morphologies. CHO cells were transiently transfected with vectors to expression WT and ALS-associated variants (A4V, G37R, G85R). After 24 hours, the cells were fixed in paraformaldehyde and imaged. The exposure times are noted on the images. WT-hSOD1:RFP produces round, well defined inclusions. WT-hSOD1:YFP diffusely fills the cytosol (rounded cell in the image shown). Mutant SOD1 fused to either RFP or YFP form variegated perinuclear inclusions. The images shown are representative of 3 independent transfection experiments, analyzing between 200 and 1,000 individual cells.

    Journal: PLoS ONE

    Article Title: An Analysis of Interactions between Fluorescently-Tagged Mutant and Wild-Type SOD1 in Intracellular Inclusions

    doi: 10.1371/journal.pone.0083981

    Figure Lengend Snippet: Mutant SOD1 fused to either RFP or YFP forms inclusions with similar morphologies. CHO cells were transiently transfected with vectors to expression WT and ALS-associated variants (A4V, G37R, G85R). After 24 hours, the cells were fixed in paraformaldehyde and imaged. The exposure times are noted on the images. WT-hSOD1:RFP produces round, well defined inclusions. WT-hSOD1:YFP diffusely fills the cytosol (rounded cell in the image shown). Mutant SOD1 fused to either RFP or YFP form variegated perinuclear inclusions. The images shown are representative of 3 independent transfection experiments, analyzing between 200 and 1,000 individual cells.

    Article Snippet: A similar approach was used to create SOD1 fusion proteins with RFP [Turbo RFP cDNA obtained from the pTRIPZ empty vector available at Open Biosystems (Huntsville, AL, USA)] by replacing the YFP tag with the RFP tag.

    Techniques: Mutagenesis, Transfection, Expressing

    Rh1 and TRPL accumulate within culd 1 photoreceptors. (A,B) The left row shows a schematic diagrams of cross-sectional (A) and longitudinal views (B) of photoreceptor cells from a single ommatidium. The rhabdomere (R) is indicated. The right rows show the whole-mount staining of Rh1 in cross-sectional (A) and longitudinal (B) views. Eyes from ninaE-rh1-gfp flies were dissected and stained for Rh1 (red). GFP fluorescence of Rh1–GFP was directly observed (green). (C–E) Rh1 and TRPL accumulated in large vesicles within the cytoplasm of culd 1 photoreceptor cells. Compound eyes from (C) wild-type (wt) ( ninaE-trpl-gfp ), (D) culd 1 ( cn bw; ninaE-trpl-gfp culd 1 ) and (E) rescue ( cn bw; ninaE-culd-rfp culd 1 ) flies were dissected and immunostained for Rh1. The ninaE-trpl-gfp transgene was present in all genotypes and GFP signals were directly observed. Two-day-old flies with white eyes were placed in the dark for 12 h before being exposed to 2000 Lux white light for 2.5 h. Scale bars: 10 µm.

    Journal: Journal of Cell Science

    Article Title: CULD is required for rhodopsin and TRPL channel endocytic trafficking and survival of photoreceptor cells

    doi: 10.1242/jcs.178764

    Figure Lengend Snippet: Rh1 and TRPL accumulate within culd 1 photoreceptors. (A,B) The left row shows a schematic diagrams of cross-sectional (A) and longitudinal views (B) of photoreceptor cells from a single ommatidium. The rhabdomere (R) is indicated. The right rows show the whole-mount staining of Rh1 in cross-sectional (A) and longitudinal (B) views. Eyes from ninaE-rh1-gfp flies were dissected and stained for Rh1 (red). GFP fluorescence of Rh1–GFP was directly observed (green). (C–E) Rh1 and TRPL accumulated in large vesicles within the cytoplasm of culd 1 photoreceptor cells. Compound eyes from (C) wild-type (wt) ( ninaE-trpl-gfp ), (D) culd 1 ( cn bw; ninaE-trpl-gfp culd 1 ) and (E) rescue ( cn bw; ninaE-culd-rfp culd 1 ) flies were dissected and immunostained for Rh1. The ninaE-trpl-gfp transgene was present in all genotypes and GFP signals were directly observed. Two-day-old flies with white eyes were placed in the dark for 12 h before being exposed to 2000 Lux white light for 2.5 h. Scale bars: 10 µm.

    Article Snippet: The blots were probed with primary antibodies against tubulin (mouse, 1:2000, Developmental Studies Hybridoma Bank), Rh1 (mouse, 1:2000 dilution, Developmental Studies Hybridoma Bank), GFP (rabbit, 1:2000, Torrey Pines Biolabs, Houston, TX), RFP (rabbit, 1:2000, Biovision, Milpitas, CA), TRPL (rabbit, 1:1000, Fisher, Waltham, MA), TRP (rabbit, 1:2000; ), INAD (rat, 1:2000; ), PDH (rabbit, 1:2000; ) and NORPA (rabbit, 1:2000; ).

    Techniques: Staining, Fluorescence

    Light-dependent retinal degeneration in culd 1 flies. Ommatidial morphologies are shown by TEM examinations. Sections were obtained from (A–C) wt ( w 1118 ) or (D–H) culd 1 ( cn bw;culd 1 ) flies, which were maintained under 12-h-light–12-h-dark (L/D) cycles or in the dark for indicated periods of time. (I–L) Suppression of retinal degeneration in culd 1 flies by ninaE P332 . (I) ninaE-culd-rfp,culd 1 , (J) culd 1 , (K) ninaE P332 ,culd 1 , and (L) trpl 302 ; culd 1 flies were raised for 15 days under 12-h-light–12-h-dark cycles. The light intensity is 2000 Lux. Scale bars: 2 µm.

    Journal: Journal of Cell Science

    Article Title: CULD is required for rhodopsin and TRPL channel endocytic trafficking and survival of photoreceptor cells

    doi: 10.1242/jcs.178764

    Figure Lengend Snippet: Light-dependent retinal degeneration in culd 1 flies. Ommatidial morphologies are shown by TEM examinations. Sections were obtained from (A–C) wt ( w 1118 ) or (D–H) culd 1 ( cn bw;culd 1 ) flies, which were maintained under 12-h-light–12-h-dark (L/D) cycles or in the dark for indicated periods of time. (I–L) Suppression of retinal degeneration in culd 1 flies by ninaE P332 . (I) ninaE-culd-rfp,culd 1 , (J) culd 1 , (K) ninaE P332 ,culd 1 , and (L) trpl 302 ; culd 1 flies were raised for 15 days under 12-h-light–12-h-dark cycles. The light intensity is 2000 Lux. Scale bars: 2 µm.

    Article Snippet: The blots were probed with primary antibodies against tubulin (mouse, 1:2000, Developmental Studies Hybridoma Bank), Rh1 (mouse, 1:2000 dilution, Developmental Studies Hybridoma Bank), GFP (rabbit, 1:2000, Torrey Pines Biolabs, Houston, TX), RFP (rabbit, 1:2000, Biovision, Milpitas, CA), TRPL (rabbit, 1:1000, Fisher, Waltham, MA), TRP (rabbit, 1:2000; ), INAD (rat, 1:2000; ), PDH (rabbit, 1:2000; ) and NORPA (rabbit, 1:2000; ).

    Techniques: Transmission Electron Microscopy

    Defective light-dependent turnover of Rh1 and TRPL in culd 1 flies. (A,B) Colocalization between (A) Rab5–RFP (red) and (B) Rab7–RFP with TRPL–GFP (green) in ommatidia. Three-day-old flies with ninaE-trpl-gfp and ninaE-rab5-rfp or ninaE-rab7-rfp transgenes in either wild-type or culd 1 background were placed in the dark for 12 h, before exposed to orange light for 2 h. Scale bar: 10 µm. (C) Quantification of the percentage of TRPL and Rab5 double-positive vesicles among TRPL-positive vesicles (TRPL+Rab5+/TRPL+) and the percentage of TRPL and Rab7 double-positive vesicles among TRPL-positive vesicles (TRPL+Rab7+/TRPL+) are shown for the experiments in A and B. At least nine ommatidia were quantified for each sample as described in Materials and Methods. Error bars indicate s.d. *** P

    Journal: Journal of Cell Science

    Article Title: CULD is required for rhodopsin and TRPL channel endocytic trafficking and survival of photoreceptor cells

    doi: 10.1242/jcs.178764

    Figure Lengend Snippet: Defective light-dependent turnover of Rh1 and TRPL in culd 1 flies. (A,B) Colocalization between (A) Rab5–RFP (red) and (B) Rab7–RFP with TRPL–GFP (green) in ommatidia. Three-day-old flies with ninaE-trpl-gfp and ninaE-rab5-rfp or ninaE-rab7-rfp transgenes in either wild-type or culd 1 background were placed in the dark for 12 h, before exposed to orange light for 2 h. Scale bar: 10 µm. (C) Quantification of the percentage of TRPL and Rab5 double-positive vesicles among TRPL-positive vesicles (TRPL+Rab5+/TRPL+) and the percentage of TRPL and Rab7 double-positive vesicles among TRPL-positive vesicles (TRPL+Rab7+/TRPL+) are shown for the experiments in A and B. At least nine ommatidia were quantified for each sample as described in Materials and Methods. Error bars indicate s.d. *** P

    Article Snippet: The blots were probed with primary antibodies against tubulin (mouse, 1:2000, Developmental Studies Hybridoma Bank), Rh1 (mouse, 1:2000 dilution, Developmental Studies Hybridoma Bank), GFP (rabbit, 1:2000, Torrey Pines Biolabs, Houston, TX), RFP (rabbit, 1:2000, Biovision, Milpitas, CA), TRPL (rabbit, 1:1000, Fisher, Waltham, MA), TRP (rabbit, 1:2000; ), INAD (rat, 1:2000; ), PDH (rabbit, 1:2000; ) and NORPA (rabbit, 1:2000; ).

    Techniques:

    Mutations in culd increase the protein levels of TRPL and Rh1 and decrease light sensitivity. (A) Organization of the culd locus and two culd alleles. The culd 1 allele consists of a PiggyBac insertion in the third intron of culd . The culd 2 allele lacks most of the second and third exons, a deletion induced by gene targeting. (B) Both culd 1 and culd 2 mutations disrupt culd transcription. gpdh served as a loading control. The genotypes are as follows: wild-type (wt), w 1118 ; excised , a precise excision of culd 1 ; rescue , ninaE-culd-rfp, culd 1 . (C) Western blotting revealed that culd loss-of-function increased TRPL levels and the amount of Rh1 aggregations. Protein extracts from half a head were loaded for each lane. Four-day-old flies raised under 12-h-light–12-h-dark cycles were used. (D) ERG recordings from 4-day-old wt ( cn bw ) and culd 1 ( cn bw ; culd 1 ) flies. Flies were dark-adapted for 2 min before exposed to five pulses of white light of increasing intensities. The maximal light intensity is 300 Lux (10 −1 ). (E) Quantification of the ERG amplitudes of wt and culd 1 flies in the intensity of 0.3 Lux (10 −4 in D). Error bars represent s.d. ( n =7). *** P

    Journal: Journal of Cell Science

    Article Title: CULD is required for rhodopsin and TRPL channel endocytic trafficking and survival of photoreceptor cells

    doi: 10.1242/jcs.178764

    Figure Lengend Snippet: Mutations in culd increase the protein levels of TRPL and Rh1 and decrease light sensitivity. (A) Organization of the culd locus and two culd alleles. The culd 1 allele consists of a PiggyBac insertion in the third intron of culd . The culd 2 allele lacks most of the second and third exons, a deletion induced by gene targeting. (B) Both culd 1 and culd 2 mutations disrupt culd transcription. gpdh served as a loading control. The genotypes are as follows: wild-type (wt), w 1118 ; excised , a precise excision of culd 1 ; rescue , ninaE-culd-rfp, culd 1 . (C) Western blotting revealed that culd loss-of-function increased TRPL levels and the amount of Rh1 aggregations. Protein extracts from half a head were loaded for each lane. Four-day-old flies raised under 12-h-light–12-h-dark cycles were used. (D) ERG recordings from 4-day-old wt ( cn bw ) and culd 1 ( cn bw ; culd 1 ) flies. Flies were dark-adapted for 2 min before exposed to five pulses of white light of increasing intensities. The maximal light intensity is 300 Lux (10 −1 ). (E) Quantification of the ERG amplitudes of wt and culd 1 flies in the intensity of 0.3 Lux (10 −4 in D). Error bars represent s.d. ( n =7). *** P

    Article Snippet: The blots were probed with primary antibodies against tubulin (mouse, 1:2000, Developmental Studies Hybridoma Bank), Rh1 (mouse, 1:2000 dilution, Developmental Studies Hybridoma Bank), GFP (rabbit, 1:2000, Torrey Pines Biolabs, Houston, TX), RFP (rabbit, 1:2000, Biovision, Milpitas, CA), TRPL (rabbit, 1:1000, Fisher, Waltham, MA), TRP (rabbit, 1:2000; ), INAD (rat, 1:2000; ), PDH (rabbit, 1:2000; ) and NORPA (rabbit, 1:2000; ).

    Techniques: Western Blot

    CULD locates in TRPL- or Rh1-positive endocytic vesicles in photoreceptor cells. (A) Cross-sectional images of retina dissected from ninaE-culd-HA ( cn bw; ninaE-culd-HA ) flies were stained with anti-HA antibody for detecting CULD (green) and with phalloidin for labeling the rhabdomere (red). (B) Longitudinal images of retina dissected from cn bw; ninaE- culd-rfp/+ flies were labeled with anti-Rh1 antibody for detecting Rh1 (green) and with anti-RFP for detecting CULD (red). (C) Retinas of cn bw; ninaE-culd-rfp/ninaE-trpl-gfp flies were stained with anti-RFP (red for CULD) and anti-GFP (green for TRPL) antibodies. (D) Retinas of cn bw; ninaE-culd-HA/ninaE-rab5-rfp flies were labeled with anti- HA (blue for CULD) and anti-RFP (red for RFP) antibodies. Two-day-old flies with white eyes were placed in the dark for 12 h before exposed to 2000 Lux white light for 2.5 h. Scale bars: 10 µm. (E) Quantification of the colocalization between CULD and the endocytic Rh1, TRPL and Rab5. Number of vesicles positive for CULD and/or Rh1, TRPL or Rab5 were counted in confocal sections as described in Materials and Methods, and divided by the total number of CULD-positive vesicles. Images of retinas from three different flies and at least five 20 µm×20 µm areas of each retina were quantified. Error bars represent s.d.

    Journal: Journal of Cell Science

    Article Title: CULD is required for rhodopsin and TRPL channel endocytic trafficking and survival of photoreceptor cells

    doi: 10.1242/jcs.178764

    Figure Lengend Snippet: CULD locates in TRPL- or Rh1-positive endocytic vesicles in photoreceptor cells. (A) Cross-sectional images of retina dissected from ninaE-culd-HA ( cn bw; ninaE-culd-HA ) flies were stained with anti-HA antibody for detecting CULD (green) and with phalloidin for labeling the rhabdomere (red). (B) Longitudinal images of retina dissected from cn bw; ninaE- culd-rfp/+ flies were labeled with anti-Rh1 antibody for detecting Rh1 (green) and with anti-RFP for detecting CULD (red). (C) Retinas of cn bw; ninaE-culd-rfp/ninaE-trpl-gfp flies were stained with anti-RFP (red for CULD) and anti-GFP (green for TRPL) antibodies. (D) Retinas of cn bw; ninaE-culd-HA/ninaE-rab5-rfp flies were labeled with anti- HA (blue for CULD) and anti-RFP (red for RFP) antibodies. Two-day-old flies with white eyes were placed in the dark for 12 h before exposed to 2000 Lux white light for 2.5 h. Scale bars: 10 µm. (E) Quantification of the colocalization between CULD and the endocytic Rh1, TRPL and Rab5. Number of vesicles positive for CULD and/or Rh1, TRPL or Rab5 were counted in confocal sections as described in Materials and Methods, and divided by the total number of CULD-positive vesicles. Images of retinas from three different flies and at least five 20 µm×20 µm areas of each retina were quantified. Error bars represent s.d.

    Article Snippet: The blots were probed with primary antibodies against tubulin (mouse, 1:2000, Developmental Studies Hybridoma Bank), Rh1 (mouse, 1:2000 dilution, Developmental Studies Hybridoma Bank), GFP (rabbit, 1:2000, Torrey Pines Biolabs, Houston, TX), RFP (rabbit, 1:2000, Biovision, Milpitas, CA), TRPL (rabbit, 1:1000, Fisher, Waltham, MA), TRP (rabbit, 1:2000; ), INAD (rat, 1:2000; ), PDH (rabbit, 1:2000; ) and NORPA (rabbit, 1:2000; ).

    Techniques: Staining, Labeling

    Extrinsic factors are the major determinant of progenitor cell identity in the developing mouse lung. (A) Experimental outline. Epithelial progenitors (tip or stalk) were microdissected from donor E12.5 or E16.5 Tomato + ( Rosa26R mT−mG/+ ) lungs and grafted into the mesenchyme of unlabelled E12.5 hosts. Hosts and grafts were cultured for 8 days without Dx, or with addition of 50 nM Dx at culture day 4 or 5, followed by serial sectioning and staining to determine graft fate. (A′,A″) Grafts integrate into the host lung, grow and form a lumen. (B-G) Sections of grafted lungs with alternate slides stained for: green, LPCAT1 (alveolar fate); red, Tomato (graft); white, PDPN (basal and AT1 cells), or: green, SOX2 (bronchiolar fate); red, RFP (Tomato + graft); white, acetylated tubulin (ACT; cilia) to determine graft fate. Examples of tip grafts with alveolar, bronchiolar and mixed broncho-alveolar fate are shown. Note D/D′ and G/G′ are different sections of the same graft. (H,I) Quantitation of graft fate as a percentage of numbers of grafts analysed. Each type of graft was analysed in at least three independent experiments. Scale bars: 25 μm in A″, 100 μm in B-G′.

    Journal: Development (Cambridge, England)

    Article Title: Lung epithelial tip progenitors integrate glucocorticoid- and STAT3-mediated signals to control progeny fate

    doi: 10.1242/dev.134023

    Figure Lengend Snippet: Extrinsic factors are the major determinant of progenitor cell identity in the developing mouse lung. (A) Experimental outline. Epithelial progenitors (tip or stalk) were microdissected from donor E12.5 or E16.5 Tomato + ( Rosa26R mT−mG/+ ) lungs and grafted into the mesenchyme of unlabelled E12.5 hosts. Hosts and grafts were cultured for 8 days without Dx, or with addition of 50 nM Dx at culture day 4 or 5, followed by serial sectioning and staining to determine graft fate. (A′,A″) Grafts integrate into the host lung, grow and form a lumen. (B-G) Sections of grafted lungs with alternate slides stained for: green, LPCAT1 (alveolar fate); red, Tomato (graft); white, PDPN (basal and AT1 cells), or: green, SOX2 (bronchiolar fate); red, RFP (Tomato + graft); white, acetylated tubulin (ACT; cilia) to determine graft fate. Examples of tip grafts with alveolar, bronchiolar and mixed broncho-alveolar fate are shown. Note D/D′ and G/G′ are different sections of the same graft. (H,I) Quantitation of graft fate as a percentage of numbers of grafts analysed. Each type of graft was analysed in at least three independent experiments. Scale bars: 25 μm in A″, 100 μm in B-G′.

    Article Snippet: Primary antibodies: acetylated tubulin (mouse, 1:3000, Sigma, T7451), CEBPA (rabbit, 1:500, Santa Cruz, sc-61), cleaved caspase 3 (rabbit, 1:100, Abcam, ab2302), E-CAD (rat, 1:1000, Invitrogen, 13-1900; or mouse, 1:1000, BD Biosciences, 610182), GFP (chick, 1:1000, Abcam, AB13970), GR (rabbit, 1:100, Santa Cruz, sc-1004), HOPX (rabbit, 1:50, Santa Cruz, sc-30216, clone FL-73), KI67 (mouse, 1:200, BD, 550609), LAMP3 (rat, 1:100, Dendritics, DDX0192, clone 1006F7.05), LIF (goat, 1:100, R & D Systems, AB-449-NA), LPCAT1 (rabbit, 1:500, Proteintech), PDPN (hamster, 1:1000, DSHB, 8.1.1), RFP (rabbit, 1:250, Rockland, 600-401-379), pro-SFTPC (rabbit, 1:500, Millipore, AB3786), SOX2 (goat, 1:250, Santa Cruz, sc-17320, clone Y-17), SOX9 (goat, 1:200, R & D Systems, AF3075), pSTAT3-Tyr705 (rabbit, 1:200, Cell Signaling, 9145), STAT5a (rabbit, 1:20, Abcam, ab7968).

    Techniques: Cell Culture, Staining, Activated Clotting Time Assay, Quantitation Assay

    IR56d is expressed in two populations of neurons in the labellum. a Immunofluorescence with anti-RFP (magenta), overlaid on bright-field images, on a whole-mount proboscis of a w;UAS-mCD8:RFP;Ir56d-Gal4 animal. The left image corresponds to the maximal projection of the inner face of one labellar palp, and the right image corresponds to the surface of one labellar palp. Scale bar: 25 μm. b Schematic representing the anatomical location of the taste peg neurons (orange) and taste bristle neurons (blue) in the labellum. c Immunofluorescence with anti-GFP (green), anti-IR25a (blue) and anti-RFP (magenta) on a whole-mount proboscis of a w;LexAop-mCD8:GFP-2A-mCD8:GFP/UAS-mCD8:RFP;Ir56d-LexA/Ir76b-Gal4 animal. The images show a close-up of taste peg neurons (arrowheads) to visualise the co-localisation of the three markers. Scale bar: 25 μm. d Immunofluorescence with anti-RFP (magenta) and anti-GFP (green) on a whole-mount proboscis of a w;LexAop-mCD8:GFP-2A-mCD8:GFP/UAS-mCD8:RFP;NompC-LexA/Ir56d-Gal4 animal. The inset in the merged image shows a bright-field view of the imaged tissue (here and in the following panels). Scale bar: 25 μm. e Immunofluorescence with anti-RFP (magenta) and anti-GFP (green) on a whole-mount proboscis of a w;LexAop-mCD8:GFP-2A-mCD8:GFP/E409-Gal4;Ir56d-LexA/UASCD4:tdTomato animal. Scale bar: 25 μm. f Immunofluorescence with anti-RFP (magenta) and anti-GFP (green) on a whole-mount proboscis of a Gr66a-LexA/+; LexAop-rCD2:GFP/UAS-mCD8:RFP;Ir56d-Gal4/(TM6B or TM2) animal. Scale bar: 25 μm. g Immunofluorescence with anti-RFP (magenta) and anti-GFP (green) on a whole-mount proboscis of a w;LexAop-mCD8:GFP-2A-mCD8:GFP/UAS-mCD8:RFP;Gr64f-LexA/Ir56d-Gal4 animal. Scale bar: 25 μm. h Immunofluorescence with anti-RFP (magenta), anti-GFP (green) and nc82 (blue) on a whole-mount brain of a w;LexAop-mCD8:GFP-2A-mCD8:GFP/UAS-mCD8:RFP;Gr64f-LexA/Ir56d-Gal4 animal. Both left and middle panels show the expression of only the Ir56d-Gal4 driver. The left panel shows the maximal projection of the anterior SEZ; the middle panel shows the maximal projection of the most posterior optical slices of the SEZ. The right panel shows the overlay of the Ir56d-Gal4 - and Gr64f-LexA -expressing populations. AMS1 anterior maxillary sensory zone 1, PMS4 posterior maxillary sensory zone 4. Scale bar: 50 μm

    Journal: Nature Communications

    Article Title: An expression atlas of variant ionotropic glutamate receptors identifies a molecular basis of carbonation sensing

    doi: 10.1038/s41467-018-06453-1

    Figure Lengend Snippet: IR56d is expressed in two populations of neurons in the labellum. a Immunofluorescence with anti-RFP (magenta), overlaid on bright-field images, on a whole-mount proboscis of a w;UAS-mCD8:RFP;Ir56d-Gal4 animal. The left image corresponds to the maximal projection of the inner face of one labellar palp, and the right image corresponds to the surface of one labellar palp. Scale bar: 25 μm. b Schematic representing the anatomical location of the taste peg neurons (orange) and taste bristle neurons (blue) in the labellum. c Immunofluorescence with anti-GFP (green), anti-IR25a (blue) and anti-RFP (magenta) on a whole-mount proboscis of a w;LexAop-mCD8:GFP-2A-mCD8:GFP/UAS-mCD8:RFP;Ir56d-LexA/Ir76b-Gal4 animal. The images show a close-up of taste peg neurons (arrowheads) to visualise the co-localisation of the three markers. Scale bar: 25 μm. d Immunofluorescence with anti-RFP (magenta) and anti-GFP (green) on a whole-mount proboscis of a w;LexAop-mCD8:GFP-2A-mCD8:GFP/UAS-mCD8:RFP;NompC-LexA/Ir56d-Gal4 animal. The inset in the merged image shows a bright-field view of the imaged tissue (here and in the following panels). Scale bar: 25 μm. e Immunofluorescence with anti-RFP (magenta) and anti-GFP (green) on a whole-mount proboscis of a w;LexAop-mCD8:GFP-2A-mCD8:GFP/E409-Gal4;Ir56d-LexA/UASCD4:tdTomato animal. Scale bar: 25 μm. f Immunofluorescence with anti-RFP (magenta) and anti-GFP (green) on a whole-mount proboscis of a Gr66a-LexA/+; LexAop-rCD2:GFP/UAS-mCD8:RFP;Ir56d-Gal4/(TM6B or TM2) animal. Scale bar: 25 μm. g Immunofluorescence with anti-RFP (magenta) and anti-GFP (green) on a whole-mount proboscis of a w;LexAop-mCD8:GFP-2A-mCD8:GFP/UAS-mCD8:RFP;Gr64f-LexA/Ir56d-Gal4 animal. Scale bar: 25 μm. h Immunofluorescence with anti-RFP (magenta), anti-GFP (green) and nc82 (blue) on a whole-mount brain of a w;LexAop-mCD8:GFP-2A-mCD8:GFP/UAS-mCD8:RFP;Gr64f-LexA/Ir56d-Gal4 animal. Both left and middle panels show the expression of only the Ir56d-Gal4 driver. The left panel shows the maximal projection of the anterior SEZ; the middle panel shows the maximal projection of the most posterior optical slices of the SEZ. The right panel shows the overlay of the Ir56d-Gal4 - and Gr64f-LexA -expressing populations. AMS1 anterior maxillary sensory zone 1, PMS4 posterior maxillary sensory zone 4. Scale bar: 50 μm

    Article Snippet: Primary antibodies: rabbit anti-IR25a (1:500) , guinea pig anti-IR25a (1:200) , mouse anti-GFP (1:500; Invitrogen), chicken anti-GFP (1:500; Abcam), rabbit anti-RFP (1:500; Abcam) and mouse monoclonal nc82 (1:10; Developmental Studies Hybridoma Bank).

    Techniques: Immunofluorescence, Expressing

    Transduction of BAT after intraiBAT administration of AAV vectors. A : Immunostaining against GFP (brown) in sections of iBAT after intraiBAT administration of 2 × 10 9 vg of AAV8 or AAV9-CAG-GFP. Original magnification ×200 and ×400 ( insets ). B : RFP expression levels in iBAT after administration of 10 10 vg of AAV-CMV-RFP vectors of serotypes 1, 2, 5, 7, 8, or 9 ( n = 3–5 mice/group). AU, arbitrary units. C : Immunostaining against GFP (brown) in sections of iBAT administered with 2 × 10 11 vg of AAV8 or AAV9-mini/UCP1-GFP. Original magnification ×200 and ×400 ( insets ). D : In vivo 2DG uptake by iBAT, eWAT, and heart after intraiBAT administration of 7 × 10 10 vg of AAV8-mini/UCP1-HK2 ( n = 6) or AAV8-mini/UCP1-null vectors ( n = 10). E : VEGF164 and PECAM1 expression levels in iBAT after intraiBAT administration of 2 × 10 11 vg of AAV9-mini/UCP1-VEGF164 or AAV9-mini/UCP1-null vectors ( n = 5). F : Immunostaining against CD105 (brown) in iBAT from the same cohorts. Original magnification ×400 and ×1,000 ( insets ). All analyses were performed 2 weeks after vector administration. Values shown are means ± SEM. * P

    Journal: Diabetes

    Article Title: In Vivo Adeno-Associated Viral Vector–Mediated Genetic Engineering of White and Brown Adipose Tissue in Adult Mice

    doi: 10.2337/db13-0311

    Figure Lengend Snippet: Transduction of BAT after intraiBAT administration of AAV vectors. A : Immunostaining against GFP (brown) in sections of iBAT after intraiBAT administration of 2 × 10 9 vg of AAV8 or AAV9-CAG-GFP. Original magnification ×200 and ×400 ( insets ). B : RFP expression levels in iBAT after administration of 10 10 vg of AAV-CMV-RFP vectors of serotypes 1, 2, 5, 7, 8, or 9 ( n = 3–5 mice/group). AU, arbitrary units. C : Immunostaining against GFP (brown) in sections of iBAT administered with 2 × 10 11 vg of AAV8 or AAV9-mini/UCP1-GFP. Original magnification ×200 and ×400 ( insets ). D : In vivo 2DG uptake by iBAT, eWAT, and heart after intraiBAT administration of 7 × 10 10 vg of AAV8-mini/UCP1-HK2 ( n = 6) or AAV8-mini/UCP1-null vectors ( n = 10). E : VEGF164 and PECAM1 expression levels in iBAT after intraiBAT administration of 2 × 10 11 vg of AAV9-mini/UCP1-VEGF164 or AAV9-mini/UCP1-null vectors ( n = 5). F : Immunostaining against CD105 (brown) in iBAT from the same cohorts. Original magnification ×400 and ×1,000 ( insets ). All analyses were performed 2 weeks after vector administration. Values shown are means ± SEM. * P

    Article Snippet: Sections were incubated overnight at 4°C with a goat anti-GFP antibody (Abcam, Cambridge, MA), with a rabbit anti-RFP antibody (Abcam), with a goat anti-CD105 antibody (R & D Systems Inc., Minneapolis, MN) or with a mouse anti–α-smooth muscle actin (SMA) antibody (Sigma-Aldrich, Saint Louis, MO).

    Techniques: Transduction, Immunostaining, Expressing, Mouse Assay, In Vivo, Plasmid Preparation

    TAPE is localized in endolysosomes. A and B , 293 cells were transfected with EGFP-tagged hTAPE and its deletion mutants, together with an endosomal protein Rab5-DsRed ( A ) or a lysosomal protein Lamp1-RFP ( B ). Transfected cells were then passed into a

    Journal: The Journal of Biological Chemistry

    Article Title: TBK1-associated Protein in Endolysosomes (TAPE) Is an Innate Immune Regulator Modulating the TLR3 and TLR4 Signaling Pathways *

    doi: 10.1074/jbc.M110.164632

    Figure Lengend Snippet: TAPE is localized in endolysosomes. A and B , 293 cells were transfected with EGFP-tagged hTAPE and its deletion mutants, together with an endosomal protein Rab5-DsRed ( A ) or a lysosomal protein Lamp1-RFP ( B ). Transfected cells were then passed into a

    Article Snippet: HEK293 cells in the 6-well plate were transfected with TAPE-EGFP together with Rab5-DsRed (an early endosomal marker) or Lamp1-RFP (a lysosomal marker).

    Techniques: Transfection

    Z-LIG functions as autophagy inhibitor in MCF-7TR5 cells ( A ) Effect of Z-LIG on conversion of LC3II and p62 expression. MCF-7 TR5 cells were treated with Z-LIG with indicated concentrations for 24 h (left panel) or treated with Z-LIG (50 μM) for indicated time points (right panel). Then, the expression of LC3II/I and p62 was analyzed by Western blotting. ( B ) Effect of Z-LIG on RFP-LC3 punctation. Cells were transfected with RFP-LC3 for 6 h, and then MCF-7 TR5 cells were treated with Z-LIG (50 μM) and CQ (20 μM) for 24 h. A punctate distribution of LC3 was observed by confocal microscopy (40×). Scale bars: 10 μm. ( C ) Effect of Z-LIG on p62 mRNA expression. MCF-7 cells were treated with Z-LIG as indicated for 3 or 24 h, then expression of p62 mRNA was determined by RT-PCR. ( D ) Effect of combinatorial Z-LIG and CQ on conversion of LC3II and p62 expression. Cells were treated with CQ (20 μM) or Z-LIG (50 μM) or their combination for 24 h. And then the expression of LC3II/I and p62 was analyzed by Western blotting. The blots were a representative of three independent experiments.

    Journal: Oncotarget

    Article Title: Sensitization of tamoxifen-resistant breast cancer cells by Z-ligustilide through inhibiting autophagy and accumulating DNA damages

    doi: 10.18632/oncotarget.16832

    Figure Lengend Snippet: Z-LIG functions as autophagy inhibitor in MCF-7TR5 cells ( A ) Effect of Z-LIG on conversion of LC3II and p62 expression. MCF-7 TR5 cells were treated with Z-LIG with indicated concentrations for 24 h (left panel) or treated with Z-LIG (50 μM) for indicated time points (right panel). Then, the expression of LC3II/I and p62 was analyzed by Western blotting. ( B ) Effect of Z-LIG on RFP-LC3 punctation. Cells were transfected with RFP-LC3 for 6 h, and then MCF-7 TR5 cells were treated with Z-LIG (50 μM) and CQ (20 μM) for 24 h. A punctate distribution of LC3 was observed by confocal microscopy (40×). Scale bars: 10 μm. ( C ) Effect of Z-LIG on p62 mRNA expression. MCF-7 cells were treated with Z-LIG as indicated for 3 or 24 h, then expression of p62 mRNA was determined by RT-PCR. ( D ) Effect of combinatorial Z-LIG and CQ on conversion of LC3II and p62 expression. Cells were treated with CQ (20 μM) or Z-LIG (50 μM) or their combination for 24 h. And then the expression of LC3II/I and p62 was analyzed by Western blotting. The blots were a representative of three independent experiments.

    Article Snippet: Plasmids transfection The GFP-LC3, RFP-LC3, RFP-LAMP1 and mRFP-LC3 tandem fluorescence-tagged LC3 construct (tfLC3) were provided by Addgene.

    Techniques: Expressing, Western Blot, Transfection, Confocal Microscopy, Reverse Transcription Polymerase Chain Reaction

    Tau-mEOS2 is localized to axons, dendrites and the soma but excluded from the nucleus. Mature DIV18 hippocampal cultures from Tau-mEOS2 mice cotransfected with the spine marker Lifeact-RFP to reveal dendritic spines. (a) Tau-mEOS2 mice show a strong axonal expression of EOS-tagged Tau, with a gradient towards the growth cone of distal axons. (b) Tau-mEOS2 is also present in the somatodendritic domain, ( c ) with very low levels in dendritic spines (zoom-in, white arrow: dendrite; yellow arrow: axon). ( d ) Within the limit of detection, EOS-tagged endogenous Tau cannot be visualized in the nucleus. (e) Time-lapse imaging of photo-converted mEOS2-tagged Tau reveals bidirectional transport of Tau in the axon at about ~0.25 μm/s. Yellow arrows indicate how far photo-converted Tau travelled. Scale bar: 10 μm ( a – d ) and 15 μm ( e ).

    Journal: Scientific Reports

    Article Title: Mobility and subcellular localization of endogenous, gene-edited Tau differs from that of over-expressed human wild-type and P301L mutant Tau

    doi: 10.1038/srep29074

    Figure Lengend Snippet: Tau-mEOS2 is localized to axons, dendrites and the soma but excluded from the nucleus. Mature DIV18 hippocampal cultures from Tau-mEOS2 mice cotransfected with the spine marker Lifeact-RFP to reveal dendritic spines. (a) Tau-mEOS2 mice show a strong axonal expression of EOS-tagged Tau, with a gradient towards the growth cone of distal axons. (b) Tau-mEOS2 is also present in the somatodendritic domain, ( c ) with very low levels in dendritic spines (zoom-in, white arrow: dendrite; yellow arrow: axon). ( d ) Within the limit of detection, EOS-tagged endogenous Tau cannot be visualized in the nucleus. (e) Time-lapse imaging of photo-converted mEOS2-tagged Tau reveals bidirectional transport of Tau in the axon at about ~0.25 μm/s. Yellow arrows indicate how far photo-converted Tau travelled. Scale bar: 10 μm ( a – d ) and 15 μm ( e ).

    Article Snippet: The following constructs were used for transfection: hTau-EGFP (human full-length 2N4R Tau with carboxy-terminally tagged EGFP), hP301L-EGFP (FTD P301L mutant 2N4R Tau with carboxy-terminally tagged EGFP), Lifeact-RFP (pmTagRFP-T-Lifeact-7, Addgene Plasmid #54586), and Lifeact-EGFP (kindly provided by Dr. Fred Meunier).

    Techniques: Mouse Assay, Marker, Expressing, Imaging

    Genetic complementation of Apc Δ/Δ Cdh1 fl/fl adenoma organoid cells with adenovirus either expressing wild-type or EC1 domain mutants of Cdh1 a. Adenovirus transfection of Apc Δ/Δ Cdh1 +/+ and Apc Δ/Δ Cdh1 fl/fl intestinal adenoma organoids with Ad-Cre-GFP, Ad-Cdh1-WT-RFP and Ad-Cdh1-Mut-RFP. Ad-Cre-GFP is expressed in the outer cells of the Apc Δ/Δ Cdh1 +/+ control organoid, and in cells of the disrupted organoid in Apc Δ/Δ Cdh1 fl/fl . Expression of either Ad-Cdh1-WT-RFP or Ad-Cdh1-Mut-RFP results in small contracted organoids in both genotypes. Complementation of Apc Δ/Δ Cdh1 fl/fl Ad-Cre-GFP with Ad-Cdh1-WT-RFP, results in adherent double expressing (yellow) cells in both genotypes. For complementation with Ad-Cdh1-Mut-RFP, double expressing cells (yellow) appear less adherent and separate. b d. Flow cytometry profiles of E-cadherin and β-catenin antibody binding to Apc Δ/Δ Cdh1 +/+ adenoma organoid cells with adenoviral transfection. With Ad-Cre-GFP alone, there was no effect on overall E-cadherin and β-catenin expression, but when combined with either Ad-Cdh1-WT-RFP or Ad-Cdh1-Mut-RFP, results in increased E-cadherin and β-catenin. In Apc Δ/Δ Cdh1 fl/fl cells, loss of E-cadherin and β-catenin occurs following Ad-Cre-GFP transfection, and is rescued by either Ad-Cdh1-WT-RFP or Ad-Cdh1-Mut-RFP. (c) In Apc Δ/Δ Cdh1 +/+ , infection with Ad-Cre-GFP does not result in E-cadherin loss (top row), and Ad-Cdh1-WT-RFP (middle row) or Ad-Cdh1-Mut-RFP (bottom row) expression results in contracted organoids as in a. Note that β-catenin appears cytoplasmic and membrane bound. e. In Apc Δ/Δ Cdh1 fl/fl (top row) Ad-Cre-GFP transfection results in loss of E-cadherin, cytoplasmic and nuclear localisation of β-catenin, and disrupted organoid morphology (large, separate, rounded or elongated) as in a. Complementation of Apc Δ/Δ Cdh1 fl/fl is shown in two example panels. For Ad-Cre-GFP with Ad-Cdh1-WT-RFP (middle row), results in adherent double expressing (yellow) cells, associated with expression of E-cadherin (arrows, left panel) and predominantly membrane bound β-catenin (arrows, right panel). For complementation with Ad-Cdh1-Mut-RFP (yellow cells, bottom row, left panel), this results predominantly in non-adherent cells, with mainly cytoplasmic and nuclear β-catenin expression (white arrow heads, right panel). Scale bar 100μm in (a), otherwise 50μm (c, e).

    Journal: Oncotarget

    Article Title: Epithelial-mesenchymal transition and nuclear β-catenin induced by conditional intestinal disruption of Cdh1 with Apc is E-cadherin EC1 domain dependent

    doi: 10.18632/oncotarget.11513

    Figure Lengend Snippet: Genetic complementation of Apc Δ/Δ Cdh1 fl/fl adenoma organoid cells with adenovirus either expressing wild-type or EC1 domain mutants of Cdh1 a. Adenovirus transfection of Apc Δ/Δ Cdh1 +/+ and Apc Δ/Δ Cdh1 fl/fl intestinal adenoma organoids with Ad-Cre-GFP, Ad-Cdh1-WT-RFP and Ad-Cdh1-Mut-RFP. Ad-Cre-GFP is expressed in the outer cells of the Apc Δ/Δ Cdh1 +/+ control organoid, and in cells of the disrupted organoid in Apc Δ/Δ Cdh1 fl/fl . Expression of either Ad-Cdh1-WT-RFP or Ad-Cdh1-Mut-RFP results in small contracted organoids in both genotypes. Complementation of Apc Δ/Δ Cdh1 fl/fl Ad-Cre-GFP with Ad-Cdh1-WT-RFP, results in adherent double expressing (yellow) cells in both genotypes. For complementation with Ad-Cdh1-Mut-RFP, double expressing cells (yellow) appear less adherent and separate. b d. Flow cytometry profiles of E-cadherin and β-catenin antibody binding to Apc Δ/Δ Cdh1 +/+ adenoma organoid cells with adenoviral transfection. With Ad-Cre-GFP alone, there was no effect on overall E-cadherin and β-catenin expression, but when combined with either Ad-Cdh1-WT-RFP or Ad-Cdh1-Mut-RFP, results in increased E-cadherin and β-catenin. In Apc Δ/Δ Cdh1 fl/fl cells, loss of E-cadherin and β-catenin occurs following Ad-Cre-GFP transfection, and is rescued by either Ad-Cdh1-WT-RFP or Ad-Cdh1-Mut-RFP. (c) In Apc Δ/Δ Cdh1 +/+ , infection with Ad-Cre-GFP does not result in E-cadherin loss (top row), and Ad-Cdh1-WT-RFP (middle row) or Ad-Cdh1-Mut-RFP (bottom row) expression results in contracted organoids as in a. Note that β-catenin appears cytoplasmic and membrane bound. e. In Apc Δ/Δ Cdh1 fl/fl (top row) Ad-Cre-GFP transfection results in loss of E-cadherin, cytoplasmic and nuclear localisation of β-catenin, and disrupted organoid morphology (large, separate, rounded or elongated) as in a. Complementation of Apc Δ/Δ Cdh1 fl/fl is shown in two example panels. For Ad-Cre-GFP with Ad-Cdh1-WT-RFP (middle row), results in adherent double expressing (yellow) cells, associated with expression of E-cadherin (arrows, left panel) and predominantly membrane bound β-catenin (arrows, right panel). For complementation with Ad-Cdh1-Mut-RFP (yellow cells, bottom row, left panel), this results predominantly in non-adherent cells, with mainly cytoplasmic and nuclear β-catenin expression (white arrow heads, right panel). Scale bar 100μm in (a), otherwise 50μm (c, e).

    Article Snippet: Adenovirus expressing murine E-cadherin CMV promoter driven adenovirus expressing either murine wild-type (Cdh1WT ) or an EC1 domain mutated cDNA, were combined with an IRES for the fluorescent reporter RFP (Vector Biolabs).

    Techniques: Expressing, Transfection, Flow Cytometry, Cytometry, Binding Assay, Infection

    Localization of nuclear β-catenin in Apc Δ/Δ Cdh1 fl/fl adenoma organoids following complementation with adenovirus expressing wild-type and EC1 domain mutant of Cdh1 a. Validation of image stream analysis of β-catenin localization in MCF7 and Colo201 cells with cytoplasmic and nuclear β-catenin localization, respectively. Note difference in the % of nuclear β-catenin and similarity dilate between the cell lines, with MCF being cytoplasmic and Colo201 being nuclear. Image stream representative images displaying differential β-catenin localization in the cell lines. b. Image stream representative images displaying differential β-catenin localization in the adenoma organoid cells with respect to genotype. c. Image stream evaluation for nuclear β-catenin utilises either a DAPI mask (left graph, % nuclear β-catenin) or similarity dilate (right graph, direct comparison of DAPI with β-catenin). Shown are the profiles of nuclear vs. cytoplasmic β-catenin localization for non-Ad-Cre-GFP transfected (grey shading) versus Ad-Cre-GFP (green), with either Ad-Cdh1-WT-RFP (red) or Ad-Cdh1-Mut (purple) co- transfected adenoma organoids. d. Quantification (mean and 95% confidence interval with outliers) and statistical analysis of the % nuclear β-catenin and similarity dilate (R12 region, Supplementary Figure S14 ) with respect to genotype and intervention. Note the rescue of the increase of nuclear β-catenin localization in double Ad-Cre-GFP and Ad-Cdh1-WT-RFP transfected Apc Δ/Δ Cdh1 fl/fl cells, and the significant overall increase in nuclear β-catenin localization following Ad-Cdh1-Mut (see also Supplementary Figure S14 ). *p

    Journal: Oncotarget

    Article Title: Epithelial-mesenchymal transition and nuclear β-catenin induced by conditional intestinal disruption of Cdh1 with Apc is E-cadherin EC1 domain dependent

    doi: 10.18632/oncotarget.11513

    Figure Lengend Snippet: Localization of nuclear β-catenin in Apc Δ/Δ Cdh1 fl/fl adenoma organoids following complementation with adenovirus expressing wild-type and EC1 domain mutant of Cdh1 a. Validation of image stream analysis of β-catenin localization in MCF7 and Colo201 cells with cytoplasmic and nuclear β-catenin localization, respectively. Note difference in the % of nuclear β-catenin and similarity dilate between the cell lines, with MCF being cytoplasmic and Colo201 being nuclear. Image stream representative images displaying differential β-catenin localization in the cell lines. b. Image stream representative images displaying differential β-catenin localization in the adenoma organoid cells with respect to genotype. c. Image stream evaluation for nuclear β-catenin utilises either a DAPI mask (left graph, % nuclear β-catenin) or similarity dilate (right graph, direct comparison of DAPI with β-catenin). Shown are the profiles of nuclear vs. cytoplasmic β-catenin localization for non-Ad-Cre-GFP transfected (grey shading) versus Ad-Cre-GFP (green), with either Ad-Cdh1-WT-RFP (red) or Ad-Cdh1-Mut (purple) co- transfected adenoma organoids. d. Quantification (mean and 95% confidence interval with outliers) and statistical analysis of the % nuclear β-catenin and similarity dilate (R12 region, Supplementary Figure S14 ) with respect to genotype and intervention. Note the rescue of the increase of nuclear β-catenin localization in double Ad-Cre-GFP and Ad-Cdh1-WT-RFP transfected Apc Δ/Δ Cdh1 fl/fl cells, and the significant overall increase in nuclear β-catenin localization following Ad-Cdh1-Mut (see also Supplementary Figure S14 ). *p

    Article Snippet: Adenovirus expressing murine E-cadherin CMV promoter driven adenovirus expressing either murine wild-type (Cdh1WT ) or an EC1 domain mutated cDNA, were combined with an IRES for the fluorescent reporter RFP (Vector Biolabs).

    Techniques: Expressing, Mutagenesis, Transfection

    TIRF microscopy of clathrin light chain (YFP-CLC or RFP-CLC) reveals an increased lifetime that is accentuated by transferrin labeling (cargo loading) on the basal surface of fibroblasts in MIIB KO cells. (A) Panels on left show kymographs of YFP-CLC in Wt and MIIB KO cells and also in MIIB KO cells following partial rescue by transient expression of the MIIB heavy chain. The MIIB KO shows greater persistence of spots (longer streaks). Images were taken at 5 s intervals. Panels on right show recordings from regions of interest containing a few spots. In the MIIB KO some spots (circled in red) persist for prolonged periods. Time is in seconds. Note that time 0 represents the beginning of the example sequence not the initiation of recording (the spots appeared during the recording). (B) Quantitative analysis of YFP-CLC lifetime. The average lifetime per cell (±S.E.M) is shown along with the average for the group (thick horizontal black line). At minimum, 50 spots were analyzed in each cell. MIIB KO cells show a significant increase in lifetime (t-test; P=0.02, the number of cells is represented by the points on the graph; from 3–4 cultures). This is an underrepresentation of the difference because 2–10% of the YFP-CLC spots in the MIIB KO cells persisted throughout the entire recording and were not included in the analysis. Rescue by transient expression of the MIIB heavy chain in the MIIB KO cells was done blindly (using an adenovirus vector to infect cells) because the vector did not express a fluorescent marker protein. Typical infection rates were 75–80% and most cells showed a shorter lifetime compared to MIIB KO cells. A few cells had longer lifetimes and were negative for MIIB heavy chain expression as determined by post hoc fixation and immunolabeling. (C) Comparison of lifetimes in transferrin-labeled and unlabeled cells. YFP-CLC lifetimes in the MIIB KO cells increase after transferrin labeling. Cells were labeled with Alexa-546 transferrin (100 μg/ml) and then recorded. The difference was not significant in the Wt cells (t-test, P=0.09, N=8 cells each from 3 cultures) but was significant in the MIIB KO cells (t-test, P=0.02, N=8 each from 3 cultures). (D) On left are examples of kymographs for Wt and MIIB KO cells expressing RFP-CLC. On the right are RFP-CLC spots from cells analyzed for lifetime by TIRF microscopy. Co-expression of either GFP-MIIB or GFP-R709C MIIB with RFP-CLC decreased the signal to noise. (E) Quantitative analysis of lifetimes from kymographs. Expression of the Wt MIIB decreased lifetimes MIIB KO cells (rescue), but expression of the mutated MIIB had no detectable affect on lifetimes. Difference were significant, t-test left to right * P≤0.001, Wt, N=12 cells from 4 cultures, MIIB KO N=11 cells from 4 cultures, ** P≤0.001, GFP-MIIB expression in MIIB KO cells N=8 cells from 3 cultures, ***P≤0.001, GFP-R790C expression in MIIB KO cells, N=8 cells from 3 cultures.

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Nonmuscle Myosin II is a Critical Regulator of Clathrin Mediated Endocytosis

    doi: 10.1111/tra.12152

    Figure Lengend Snippet: TIRF microscopy of clathrin light chain (YFP-CLC or RFP-CLC) reveals an increased lifetime that is accentuated by transferrin labeling (cargo loading) on the basal surface of fibroblasts in MIIB KO cells. (A) Panels on left show kymographs of YFP-CLC in Wt and MIIB KO cells and also in MIIB KO cells following partial rescue by transient expression of the MIIB heavy chain. The MIIB KO shows greater persistence of spots (longer streaks). Images were taken at 5 s intervals. Panels on right show recordings from regions of interest containing a few spots. In the MIIB KO some spots (circled in red) persist for prolonged periods. Time is in seconds. Note that time 0 represents the beginning of the example sequence not the initiation of recording (the spots appeared during the recording). (B) Quantitative analysis of YFP-CLC lifetime. The average lifetime per cell (±S.E.M) is shown along with the average for the group (thick horizontal black line). At minimum, 50 spots were analyzed in each cell. MIIB KO cells show a significant increase in lifetime (t-test; P=0.02, the number of cells is represented by the points on the graph; from 3–4 cultures). This is an underrepresentation of the difference because 2–10% of the YFP-CLC spots in the MIIB KO cells persisted throughout the entire recording and were not included in the analysis. Rescue by transient expression of the MIIB heavy chain in the MIIB KO cells was done blindly (using an adenovirus vector to infect cells) because the vector did not express a fluorescent marker protein. Typical infection rates were 75–80% and most cells showed a shorter lifetime compared to MIIB KO cells. A few cells had longer lifetimes and were negative for MIIB heavy chain expression as determined by post hoc fixation and immunolabeling. (C) Comparison of lifetimes in transferrin-labeled and unlabeled cells. YFP-CLC lifetimes in the MIIB KO cells increase after transferrin labeling. Cells were labeled with Alexa-546 transferrin (100 μg/ml) and then recorded. The difference was not significant in the Wt cells (t-test, P=0.09, N=8 cells each from 3 cultures) but was significant in the MIIB KO cells (t-test, P=0.02, N=8 each from 3 cultures). (D) On left are examples of kymographs for Wt and MIIB KO cells expressing RFP-CLC. On the right are RFP-CLC spots from cells analyzed for lifetime by TIRF microscopy. Co-expression of either GFP-MIIB or GFP-R709C MIIB with RFP-CLC decreased the signal to noise. (E) Quantitative analysis of lifetimes from kymographs. Expression of the Wt MIIB decreased lifetimes MIIB KO cells (rescue), but expression of the mutated MIIB had no detectable affect on lifetimes. Difference were significant, t-test left to right * P≤0.001, Wt, N=12 cells from 4 cultures, MIIB KO N=11 cells from 4 cultures, ** P≤0.001, GFP-MIIB expression in MIIB KO cells N=8 cells from 3 cultures, ***P≤0.001, GFP-R790C expression in MIIB KO cells, N=8 cells from 3 cultures.

    Article Snippet: The cultures were transfected with plasmids expressing YFP-CLC (Addgene plasmid #20921) or RFP-CLC (Addgene plasmid 14435), GFP-MIIB heavy chain or the GFP-R709C mutated MIIB heavy chain ( ) by Lipofectamine 2000.

    Techniques: Microscopy, Labeling, Expressing, Sequencing, T-Test, Plasmid Preparation, Marker, Infection, Immunolabeling

    Trafficking of proteasomes versus synaptobrevin in motor neurons. (A) Top, the set up used to record movies of axonal transport of Syb–GFP (left) and Prosβ5–RFP (right) in axons of motor neurons projecting from the ventral nerve cord (VNC) of the Drosophila third-instar larvae. Bottom, representative kymographs from live-imaging of Syb–GFP (left kymograph) and Prosβ5–RFP (right kymograph) particles in Drosophila third-instar larvae motor neurons ( OK6-Gal4/UAS-syb-GFP , OK6-Gal4,UAS-prosβ5-RFP;UAS-prosβ5-RFP ). Kymographs represent cumulative movement (displacement on the x -axis) over time ( y -axis). Mobile particles appear as diagonal lines moving either in the anterograde (to the right) or retrograde (to the left) direction. (B) Average proportion of Syb–GFP (light gray, n =429 particles) and Prosβ5–RFP (dark gray, n =325 particles) particles moving in anterograde direction, retrograde direction, and that are stationary and reversing. (C,D) Mean run length (C) and mean segmental velocity (D) of Syb–GFP (light gray, n =197 particles) and Prosβ5–RFP (dark gray, n =138 particles) particles moving in the anterograde and retrograde direction as determined from analysis of live imaging; 10–20 animals per genotype were analyzed. (E,F) Normalized frequency distribution of mean anterograde (E) and retrograde (F) segmental velocities of Syb–GFP (light gray) and Prosβ5–RFP (dark gray) during axonal transport as determined from analysis of live imaging. Error bars indicate s.e.m. * P

    Journal: Journal of Cell Science

    Article Title: The Drosophila LC8 homolog cut up specifies the axonal transport of proteasomes

    doi: 10.1242/jcs.207027

    Figure Lengend Snippet: Trafficking of proteasomes versus synaptobrevin in motor neurons. (A) Top, the set up used to record movies of axonal transport of Syb–GFP (left) and Prosβ5–RFP (right) in axons of motor neurons projecting from the ventral nerve cord (VNC) of the Drosophila third-instar larvae. Bottom, representative kymographs from live-imaging of Syb–GFP (left kymograph) and Prosβ5–RFP (right kymograph) particles in Drosophila third-instar larvae motor neurons ( OK6-Gal4/UAS-syb-GFP , OK6-Gal4,UAS-prosβ5-RFP;UAS-prosβ5-RFP ). Kymographs represent cumulative movement (displacement on the x -axis) over time ( y -axis). Mobile particles appear as diagonal lines moving either in the anterograde (to the right) or retrograde (to the left) direction. (B) Average proportion of Syb–GFP (light gray, n =429 particles) and Prosβ5–RFP (dark gray, n =325 particles) particles moving in anterograde direction, retrograde direction, and that are stationary and reversing. (C,D) Mean run length (C) and mean segmental velocity (D) of Syb–GFP (light gray, n =197 particles) and Prosβ5–RFP (dark gray, n =138 particles) particles moving in the anterograde and retrograde direction as determined from analysis of live imaging; 10–20 animals per genotype were analyzed. (E,F) Normalized frequency distribution of mean anterograde (E) and retrograde (F) segmental velocities of Syb–GFP (light gray) and Prosβ5–RFP (dark gray) during axonal transport as determined from analysis of live imaging. Error bars indicate s.e.m. * P

    Article Snippet: Membranes were blocked with 5% milk in 1% Tween 20 in Tris-buffered saline (TBS) pH 7.4, at room temperature for 30 min. Membranes were probed with the rabbit anti-19S 5SA (1:1000; Abcam, Cambridge, MA) and rabbit anti-Drosophila Prosβ5 (dβ5) (1:1000; a gift from the Figueiredo-Pereira laboratory; ), mouse anti-RFP (1:1000; cat. no. ab125244; Abcam, Cambridge, MA) or mouse anti-GFP (1:1000; cat. no. ab1218; Abcam, Cambridge, MA) antibodies followed by secondary horseradish peroxidase (HRP)-labeled goat anti-rabbit-IgG antibody (1:2500; cat. no. sc-2004; Santa Cruz Biotechnology, Dallas, TX) or goat anti-mouse IgG antibody (1:2500; cat. no.:sc-2005; Santa Cruz Biotechnology).

    Techniques: Imaging

    Proteasomes at the NMJ. (A) Top, still image of the NMJ at muscle 4 from live-imaging of Prosβ5–RFP expressed in neurons of third-instar larvae ( C155-Gal4;UAS-prosβ5-RFP;UAS-prosβ5-RFP ). Middle and bottom, immunofluorescence image of the same fixed NMJ co-stained with anti-HRP (nerve terminal membrane) and anti-Futsch (synaptic MTs). Scale bar: 1 μm. Asterisks (*) mark individual boutons. Arrow indicates bouton containing Prosβ5–RFP fluorescence. (B) Representative still images from live-imaging of Prosβ5–RFP particles in muscle 4 NMJ of the Drosophila third-instar larvae demonstrating two individual Prosβ5–RFP puncta moving towards each other (black arrow and white arrowheads). Asterisks (*) mark individual boutons. (C) Mean segmental velocity of moving Prosβ5–RFP particles in axons (black bars, n =218 particles) and synapses (gray bars, n =51 particles) determined from live-imaging analysis. (D) Average proportion of Prosβ5–RFP particles that are stationary (black bars) and reversing (gray bars) in axons ( n =21 animals) and synapses ( n =17 animals) demonstrating a significant increase in the percentage of stationary and reversing Prosβ5-RFP in the synapse. Error bars indicate s.e.m. ** P

    Journal: Journal of Cell Science

    Article Title: The Drosophila LC8 homolog cut up specifies the axonal transport of proteasomes

    doi: 10.1242/jcs.207027

    Figure Lengend Snippet: Proteasomes at the NMJ. (A) Top, still image of the NMJ at muscle 4 from live-imaging of Prosβ5–RFP expressed in neurons of third-instar larvae ( C155-Gal4;UAS-prosβ5-RFP;UAS-prosβ5-RFP ). Middle and bottom, immunofluorescence image of the same fixed NMJ co-stained with anti-HRP (nerve terminal membrane) and anti-Futsch (synaptic MTs). Scale bar: 1 μm. Asterisks (*) mark individual boutons. Arrow indicates bouton containing Prosβ5–RFP fluorescence. (B) Representative still images from live-imaging of Prosβ5–RFP particles in muscle 4 NMJ of the Drosophila third-instar larvae demonstrating two individual Prosβ5–RFP puncta moving towards each other (black arrow and white arrowheads). Asterisks (*) mark individual boutons. (C) Mean segmental velocity of moving Prosβ5–RFP particles in axons (black bars, n =218 particles) and synapses (gray bars, n =51 particles) determined from live-imaging analysis. (D) Average proportion of Prosβ5–RFP particles that are stationary (black bars) and reversing (gray bars) in axons ( n =21 animals) and synapses ( n =17 animals) demonstrating a significant increase in the percentage of stationary and reversing Prosβ5-RFP in the synapse. Error bars indicate s.e.m. ** P

    Article Snippet: Membranes were blocked with 5% milk in 1% Tween 20 in Tris-buffered saline (TBS) pH 7.4, at room temperature for 30 min. Membranes were probed with the rabbit anti-19S 5SA (1:1000; Abcam, Cambridge, MA) and rabbit anti-Drosophila Prosβ5 (dβ5) (1:1000; a gift from the Figueiredo-Pereira laboratory; ), mouse anti-RFP (1:1000; cat. no. ab125244; Abcam, Cambridge, MA) or mouse anti-GFP (1:1000; cat. no. ab1218; Abcam, Cambridge, MA) antibodies followed by secondary horseradish peroxidase (HRP)-labeled goat anti-rabbit-IgG antibody (1:2500; cat. no. sc-2004; Santa Cruz Biotechnology, Dallas, TX) or goat anti-mouse IgG antibody (1:2500; cat. no.:sc-2005; Santa Cruz Biotechnology).

    Techniques: Imaging, Immunofluorescence, Staining, Fluorescence

    Independent trafficking of Syb–GFP and Prosβ5–RFP. (A–D) Mean segmental velocity (A,C) and mean run length (B,D) for the anterograde (A,B) and retrograde (C,D) direction of movement of Syb–GFP and Prosβ5–RFP particles in control [ctrl, black bars, n =186 (Syb-GFP), n =230 particles (Prosβ5-RFP)] and K lc mutants [gray bars, n =76 (Syb-GFP), n =143 particles (Prosβ5-RFP)]; 12–16 animals per genotype were analyzed. Error bars indicate s.e.m. * P

    Journal: Journal of Cell Science

    Article Title: The Drosophila LC8 homolog cut up specifies the axonal transport of proteasomes

    doi: 10.1242/jcs.207027

    Figure Lengend Snippet: Independent trafficking of Syb–GFP and Prosβ5–RFP. (A–D) Mean segmental velocity (A,C) and mean run length (B,D) for the anterograde (A,B) and retrograde (C,D) direction of movement of Syb–GFP and Prosβ5–RFP particles in control [ctrl, black bars, n =186 (Syb-GFP), n =230 particles (Prosβ5-RFP)] and K lc mutants [gray bars, n =76 (Syb-GFP), n =143 particles (Prosβ5-RFP)]; 12–16 animals per genotype were analyzed. Error bars indicate s.e.m. * P

    Article Snippet: Membranes were blocked with 5% milk in 1% Tween 20 in Tris-buffered saline (TBS) pH 7.4, at room temperature for 30 min. Membranes were probed with the rabbit anti-19S 5SA (1:1000; Abcam, Cambridge, MA) and rabbit anti-Drosophila Prosβ5 (dβ5) (1:1000; a gift from the Figueiredo-Pereira laboratory; ), mouse anti-RFP (1:1000; cat. no. ab125244; Abcam, Cambridge, MA) or mouse anti-GFP (1:1000; cat. no. ab1218; Abcam, Cambridge, MA) antibodies followed by secondary horseradish peroxidase (HRP)-labeled goat anti-rabbit-IgG antibody (1:2500; cat. no. sc-2004; Santa Cruz Biotechnology, Dallas, TX) or goat anti-mouse IgG antibody (1:2500; cat. no.:sc-2005; Santa Cruz Biotechnology).

    Techniques:

    DLC RNAi screen of proteasome transport. (A) Un-rooted phylogenetic tree of all CG RNAi lines tested in the screen determined from multiple sequence alignment using CLUSTALW. hs, human proteins. TcText1, DYNLT1. (B) Protein alignment between Drosophila CG6998 (Ctp) and human DLC2 (DYNLL2) and DLC1 (DYNLL1) demonstrating a high level of conservation between human DLC2 and Drosophila . Helical and β regions are indicated. (C) Graph represents mean segmental velocity of Prosβ5–RFP motility in axons of neurons from the indicated CG RNAi lines and wild-type control (black bar) determined from live-imaging analysis. Expression of CG6998 RNAi line resulted in the slowest axonal transport velocity of Prosβ5–RFP (red bar); 60–90 particles from three animals per genotype were analyzed. (D) Representative kymograph from live-imaging of Prosβ5–RFP in axons from a no-RNAi control (top) and with CG6998 RNAi (bottom). Kymograph represents cumulative movement (displacement on the x -axis) over time ( y -axis). Stationary particles appear as vertical lines, whereas motile particles appear as diagonal lines.

    Journal: Journal of Cell Science

    Article Title: The Drosophila LC8 homolog cut up specifies the axonal transport of proteasomes

    doi: 10.1242/jcs.207027

    Figure Lengend Snippet: DLC RNAi screen of proteasome transport. (A) Un-rooted phylogenetic tree of all CG RNAi lines tested in the screen determined from multiple sequence alignment using CLUSTALW. hs, human proteins. TcText1, DYNLT1. (B) Protein alignment between Drosophila CG6998 (Ctp) and human DLC2 (DYNLL2) and DLC1 (DYNLL1) demonstrating a high level of conservation between human DLC2 and Drosophila . Helical and β regions are indicated. (C) Graph represents mean segmental velocity of Prosβ5–RFP motility in axons of neurons from the indicated CG RNAi lines and wild-type control (black bar) determined from live-imaging analysis. Expression of CG6998 RNAi line resulted in the slowest axonal transport velocity of Prosβ5–RFP (red bar); 60–90 particles from three animals per genotype were analyzed. (D) Representative kymograph from live-imaging of Prosβ5–RFP in axons from a no-RNAi control (top) and with CG6998 RNAi (bottom). Kymograph represents cumulative movement (displacement on the x -axis) over time ( y -axis). Stationary particles appear as vertical lines, whereas motile particles appear as diagonal lines.

    Article Snippet: Membranes were blocked with 5% milk in 1% Tween 20 in Tris-buffered saline (TBS) pH 7.4, at room temperature for 30 min. Membranes were probed with the rabbit anti-19S 5SA (1:1000; Abcam, Cambridge, MA) and rabbit anti-Drosophila Prosβ5 (dβ5) (1:1000; a gift from the Figueiredo-Pereira laboratory; ), mouse anti-RFP (1:1000; cat. no. ab125244; Abcam, Cambridge, MA) or mouse anti-GFP (1:1000; cat. no. ab1218; Abcam, Cambridge, MA) antibodies followed by secondary horseradish peroxidase (HRP)-labeled goat anti-rabbit-IgG antibody (1:2500; cat. no. sc-2004; Santa Cruz Biotechnology, Dallas, TX) or goat anti-mouse IgG antibody (1:2500; cat. no.:sc-2005; Santa Cruz Biotechnology).

    Techniques: Sequencing, Imaging, Expressing

    MT-based axonal transport of proteasomes. (A) A schematic of the Drosophila proteasome β5 subunit (Prosβ5) locus including coding sequences (CDS) and untranslated regions (UTR). A full-length Prosβ5 cDNA (LD0717) was fused with RFP at the C-terminus. (B) The setup used to record movies of the axonal transport in third-instar larvae, and representative still images from live-imaging of Prosβ5–RFP particles and tubulin–GFP co-expressed in Drosophila neurons ( C155-Gal4;UAS-prosβ5-RFP;UAS-Tub-GFP ). (C) Representative kymograph of Prosβ5–RFP motility in Drosophila third-instar neurons. Kymograph represents cumulative movement (displacement on the x -axis) over time ( y -axis). Stationary particles appear as vertical lines, whereas motile particles appear as diagonal lines. Two examples of ‘reversing’ behavior are indicated by the arrowheads. (D) Percentage of Prosβ5–RFP particles that are stationary in wild-type (black bars, n =448 particles) and DNGlued mutants (gray bars, n =326 particles). (E,F) Mean run length (E) and mean segmental velocity (F) of moving Prosβ5–RFP particles in wild-type (black bars, n =219 particles) and DNGlued mutants (gray bars, n =227 particles). Wild-type genotype, C155-Gal4;UAS-prosβ5-RFP;UAS-prosβ5-RFP ; DNGlued genotype, C155-Gal4;UAS-prosβ5-RFP;UAS-prosβ5-RFP,UAS-DNGlued . At least 12 larvae were dissected and analyzed for each genotype. Error bars indicate s.e.m. * P

    Journal: Journal of Cell Science

    Article Title: The Drosophila LC8 homolog cut up specifies the axonal transport of proteasomes

    doi: 10.1242/jcs.207027

    Figure Lengend Snippet: MT-based axonal transport of proteasomes. (A) A schematic of the Drosophila proteasome β5 subunit (Prosβ5) locus including coding sequences (CDS) and untranslated regions (UTR). A full-length Prosβ5 cDNA (LD0717) was fused with RFP at the C-terminus. (B) The setup used to record movies of the axonal transport in third-instar larvae, and representative still images from live-imaging of Prosβ5–RFP particles and tubulin–GFP co-expressed in Drosophila neurons ( C155-Gal4;UAS-prosβ5-RFP;UAS-Tub-GFP ). (C) Representative kymograph of Prosβ5–RFP motility in Drosophila third-instar neurons. Kymograph represents cumulative movement (displacement on the x -axis) over time ( y -axis). Stationary particles appear as vertical lines, whereas motile particles appear as diagonal lines. Two examples of ‘reversing’ behavior are indicated by the arrowheads. (D) Percentage of Prosβ5–RFP particles that are stationary in wild-type (black bars, n =448 particles) and DNGlued mutants (gray bars, n =326 particles). (E,F) Mean run length (E) and mean segmental velocity (F) of moving Prosβ5–RFP particles in wild-type (black bars, n =219 particles) and DNGlued mutants (gray bars, n =227 particles). Wild-type genotype, C155-Gal4;UAS-prosβ5-RFP;UAS-prosβ5-RFP ; DNGlued genotype, C155-Gal4;UAS-prosβ5-RFP;UAS-prosβ5-RFP,UAS-DNGlued . At least 12 larvae were dissected and analyzed for each genotype. Error bars indicate s.e.m. * P

    Article Snippet: Membranes were blocked with 5% milk in 1% Tween 20 in Tris-buffered saline (TBS) pH 7.4, at room temperature for 30 min. Membranes were probed with the rabbit anti-19S 5SA (1:1000; Abcam, Cambridge, MA) and rabbit anti-Drosophila Prosβ5 (dβ5) (1:1000; a gift from the Figueiredo-Pereira laboratory; ), mouse anti-RFP (1:1000; cat. no. ab125244; Abcam, Cambridge, MA) or mouse anti-GFP (1:1000; cat. no. ab1218; Abcam, Cambridge, MA) antibodies followed by secondary horseradish peroxidase (HRP)-labeled goat anti-rabbit-IgG antibody (1:2500; cat. no. sc-2004; Santa Cruz Biotechnology, Dallas, TX) or goat anti-mouse IgG antibody (1:2500; cat. no.:sc-2005; Santa Cruz Biotechnology).

    Techniques: Imaging

    ctp is required for axonal transport of proteasomes. (A) A schematic of the Drosophila cup up ( ctp ) gene including coding sequences (CDS), untranslated regions (UTR), and the location of the P-element insertion used for the mutant analysis. (B) Representative kymographs from live-imaging of Prosβ5–RFP in axons of motor neurons from control, ctp/+ and ctp/y third-instar larvae. Kymographs represent cumulative movement (displacement on the x -axis) over time ( y -axis). Stationary particles appear as vertical lines, whereas motile particles appear as diagonal lines (anterograde are to the right, and retrograde are to the left). (C) Average proportion of Prosβ5–RFP particles moving in the anterograde direction, the retrograde direction, or that are stationary and reversing in control (ctrl, black bars, n =271 particles), ctp/+ (dark gray bars, n =401 particles) and ctp/y (light gray bars, n =543 particles). (D,E) Mean segmental retrograde velocity (D) and mean retrograde run length (E) of Prosβ5–RFP puncta in control (ctrl, black bars, n =53), ctp/+ (dark gray bars, n =79) and ctp/y (light gray bars, n =92) determined from an analysis of live imaging; 10–20 animals per genotype were analyzed. (F) Normalized frequency distribution of mean retrograde segmental velocities of Prosβ5–RFP puncta in control (ctrl, black bars) and ctp/y mutant (gray bars) determined from analysis of live imaging. (G,H) Mean segmental anterograde velocity (G) and mean anterograde run length (H) of Prosβ5–RFP puncta in control (ctrl, black bars, n =35 particles), ctp/+ (dark gray bars, n =111 particles) and ctp/y (light gray bars, n =111 particles) determined from analysis of live imaging. (I) Normalized frequency distribution of mean anterograde segmental velocities of Prosβ5–RFP puncta in control (ctrl, black bars) and ctp/y mutant (gray bars) determined from analysis of live imaging. Control genotype, C380-Gal4/y;OK6-Gal4,UAS-prosβ5-RFP/+;UAS-prosβ5-RFP/+ ; ctp/+ genotype, C380-Gal4,ctp/+;OK6-Gal4,UAS-prosβ5-RFP/+;UAS-prosβ5-RFP/+ ; ctp/y genotype, C380-Gal4,ctp/y;OK6-Gal4,UAS-prosβ5-RFP/+;UAS-prosβ5-RFP/+ . Error bars indicate s.e.m. * P

    Journal: Journal of Cell Science

    Article Title: The Drosophila LC8 homolog cut up specifies the axonal transport of proteasomes

    doi: 10.1242/jcs.207027

    Figure Lengend Snippet: ctp is required for axonal transport of proteasomes. (A) A schematic of the Drosophila cup up ( ctp ) gene including coding sequences (CDS), untranslated regions (UTR), and the location of the P-element insertion used for the mutant analysis. (B) Representative kymographs from live-imaging of Prosβ5–RFP in axons of motor neurons from control, ctp/+ and ctp/y third-instar larvae. Kymographs represent cumulative movement (displacement on the x -axis) over time ( y -axis). Stationary particles appear as vertical lines, whereas motile particles appear as diagonal lines (anterograde are to the right, and retrograde are to the left). (C) Average proportion of Prosβ5–RFP particles moving in the anterograde direction, the retrograde direction, or that are stationary and reversing in control (ctrl, black bars, n =271 particles), ctp/+ (dark gray bars, n =401 particles) and ctp/y (light gray bars, n =543 particles). (D,E) Mean segmental retrograde velocity (D) and mean retrograde run length (E) of Prosβ5–RFP puncta in control (ctrl, black bars, n =53), ctp/+ (dark gray bars, n =79) and ctp/y (light gray bars, n =92) determined from an analysis of live imaging; 10–20 animals per genotype were analyzed. (F) Normalized frequency distribution of mean retrograde segmental velocities of Prosβ5–RFP puncta in control (ctrl, black bars) and ctp/y mutant (gray bars) determined from analysis of live imaging. (G,H) Mean segmental anterograde velocity (G) and mean anterograde run length (H) of Prosβ5–RFP puncta in control (ctrl, black bars, n =35 particles), ctp/+ (dark gray bars, n =111 particles) and ctp/y (light gray bars, n =111 particles) determined from analysis of live imaging. (I) Normalized frequency distribution of mean anterograde segmental velocities of Prosβ5–RFP puncta in control (ctrl, black bars) and ctp/y mutant (gray bars) determined from analysis of live imaging. Control genotype, C380-Gal4/y;OK6-Gal4,UAS-prosβ5-RFP/+;UAS-prosβ5-RFP/+ ; ctp/+ genotype, C380-Gal4,ctp/+;OK6-Gal4,UAS-prosβ5-RFP/+;UAS-prosβ5-RFP/+ ; ctp/y genotype, C380-Gal4,ctp/y;OK6-Gal4,UAS-prosβ5-RFP/+;UAS-prosβ5-RFP/+ . Error bars indicate s.e.m. * P

    Article Snippet: Membranes were blocked with 5% milk in 1% Tween 20 in Tris-buffered saline (TBS) pH 7.4, at room temperature for 30 min. Membranes were probed with the rabbit anti-19S 5SA (1:1000; Abcam, Cambridge, MA) and rabbit anti-Drosophila Prosβ5 (dβ5) (1:1000; a gift from the Figueiredo-Pereira laboratory; ), mouse anti-RFP (1:1000; cat. no. ab125244; Abcam, Cambridge, MA) or mouse anti-GFP (1:1000; cat. no. ab1218; Abcam, Cambridge, MA) antibodies followed by secondary horseradish peroxidase (HRP)-labeled goat anti-rabbit-IgG antibody (1:2500; cat. no. sc-2004; Santa Cruz Biotechnology, Dallas, TX) or goat anti-mouse IgG antibody (1:2500; cat. no.:sc-2005; Santa Cruz Biotechnology).

    Techniques: Mutagenesis, Imaging