cycloheximide chx Search Results


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  • 99
    Thermo Fisher chx
    C5a induces loss of <t>HUVEC</t> viability in the presence of <t>CHX.</t> A) MTS assay for cell viability. Cells were treated with C5a (50 ng/ml), CHX (10 μg/ml), or C5a (50 ng/ml) plus CHX (10 μg/ml) for 18 h. *P
    Chx, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 421 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore cycloheximide chx
    SGK2 regulates c-Myc/β-catenin gene expression. (A). WB results showed the protein levels of β-catenin and c-Myc were significantly decreased after down-regulating SGK2 expression in UMUC3 and J82 cells. (B). WB results showed the protein levels of β-catenin and c-Myc were significantly increased after overexpressing SGK2 in T24 cell. (C) . Immunoprecipitation (IP) assay indicated SGK2 could directly interact with β-catenin protein in T24 cell. (D). <t>MG132</t> (10 µM) abrogated shSGK2 induced inhibition of β-catenin protein expression in UMUC3 cell. (E). A time-dependent decrease in β-catenin protein expression in T24-vector and T24-oeSGK2 cell lines exposed to <t>CHX</t> (10 µg/mL). However, the decrease speed was marked slower in T24-oeSGK2 than T24-vector group. (F). The expression of c-Myc and CTNNB1 gene (encoding β-catenin protein) were significantly associated with tumor stage of bladder cancer ( P
    Cycloheximide Chx, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3341 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Novoprotein cycloheximide chx
    Mitotic slippage is induced in <t>nocodazole-treated</t> cells after inhibition of transcription or translation. Characterization of NIH3T3 cells: control cells, nocodazole (noc)-treated cells (arrested in mitosis), noc-cells incubated with cycloheximide <t>(CHX;</t> 35.5 µM) or actinomycin D (ActD; 8 µM) for 4 h. A) Phase-contrast microscopy; magnification: 200×. B) Immunofluorescence microscopy; arrowheads indicate mitotic cells and arrows indicate cells that underwent mitotic slippage; DAPI stains the DNA (blue) and anti-α-tubulin antibody stains the microtubules (green). Scale bar = 25 µm. C) Mitotic and multinucleation indexes of noc-treated cells with or without CHX or ActD for 4 h. Data presented as mean ± S.D. from three independent experiments; ** p
    Cycloheximide Chx, supplied by Novoprotein, used in various techniques. Bioz Stars score: 93/100, based on 574 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Novoprotein cyclohexamide
    LANA stabilizes Par3 in KSHV positive cells. (A) HEK-293-BAC-KSHV cells were transfected with sh-LANA and sh-Control. Endogenous Par3 expression was measured and presented in graph. GAPDH was used as a protein loading control. (B, C) Stabilization of Par3 was examined with <t>cyclohexamide</t> in HEK-293 and BJAB cells. Left panels and right panels were used as vector control and LANA expression, in a time dependent manner. (D) The proteosome inhibitor MG132 was used to determine if Par3 stability was linked to the proteosome degradation pathway. GFP and GAPDH were used as transfection and endogenous protein loading controls. (E) BC-3-shControl and BC-3-shLANA cells were treated with cyclohexamide and observed Par3 endogenous on hours dependent manner. (F) BC-3-shControl and BC-3shLANA cells were treated with MG132 and followed immunoprecipitation of Par3. Endogenous ubiquitin and Par3 were detected using specific antibodies in both the cell lines. GAPDH was monitored as an internal control for loading in the input section.
    Cyclohexamide, supplied by Novoprotein, used in various techniques. Bioz Stars score: 92/100, based on 111 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Beyotime cycloheximide chx
    HBXIP-enhanced acetylation of HOXB13 stabilizes HOXB13 in the facilitation of TAM resistance. a Immunoblotting analysis of HOXB13 in MCF-7 cells treated with the indicated concentrations of leupeptin for 36 h (lower panel). The upper panel is the quantification of the intensity relative to β-actin. b Immunoblotting analysis of HOXB13 in BT474 cells cultured with serum-supplemented or serum-free media for the indicated time courses (lower panel). The upper panel is the quantification of the intensity relative to β-actin. c Immunoblotting analysis of HOXB13 in MCF-7 cells cultured with serum-supplemented or serum-free media for 48 h along with DMSO or <t>TSA</t> (1 μM) (lower panel). Before that, the cells were transiently transfected with pCMV or pCMV-HBXIP (1.5 μg). The protein level of HBXIP was determined by the anti-Flag antibody. The upper panel is the quantification of the intensity relative to β-actin. d Immunoblotting analysis of GFP-HOXB13 in MCF-7 cells time-dependently treated with 100 μg/ml <t>CHX</t> after being transiently transfected with GFP-HOXB13-WT or GFP-HOXB13-K277R (lower panel). The protein level of GFP-HOXB13 was determined by the anti-GFP antibody. The upper panel is the quantification of the intensity relative to β-actin. e Cell viability assay with MCF-7 cells treated with the indicated concentrations of TAM after being transiently transfected with the displayed plasmids. Error bars represent ± SD. * P
    Cycloheximide Chx, supplied by Beyotime, used in various techniques. Bioz Stars score: 92/100, based on 63 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc chx
    ASOs dose-dependently reduce NMD and increase mRNA in vitro. a <t>RT-PCR</t> (left panel) and qPCR (right panel) of a selected PCCA ASO (ASO-29) targeting the exon inclusion event transfected in HEK293 cells at increasing concentrations for 24 h. Exact p -values for bars with asterisks are 0.0008 and 0.0001, respectively. b RT-PCR (left panel) and qPCR (right panel) of a selected SYNGAP1 ASO (ASO-71) targeting the alternative 3′ss transfected in HEK293 cells at increasing concentrations for 24 h. Exact p -values for bars with asterisks are 0.0001 and 0.0003, 6.99e-5 and 4.25e-5, respectively. c RT-PCR (left panel) and qPCR (right panel) of a selected CD274 ASO (ASO-125) targeting the alternative intron transfected in Huh7 cells at increasing concentrations for 24 h. Cell were treated with 50 μg/mL of <t>CHX</t> for 3 h prior to harvesting to visualize and quantify the non-productive mRNA. Exact p -values for bars with asterisks are 6.38e-6 and 9.21e-5, respectively. d RT-PCR (left panel) and qPCR (right panel) of two selected SCN1A ASOs (ASO-135 and ASO-136) targeting the exon inclusion event delivered by free uptake into ReNCell VM cells at increasing concentrations for 72 h. RT-PCR results (bar graphs on the left) show dose-dependent reductions of the non-productive mRNA and qPCR results (bar graphs on the right) show dose-dependent increases of productive mRNA. Exact p-values for bars with asterisks are 1.69e-10, 2.50e-11, and 1.49e-11; 2.91e-8, 2.05e-10, and 2.59e-11; 4.21e-7, 1.10e-8, and 1.35e-10; and 7.26e-5, 4.01e-8, and 9.08e-12, respectively. No-ASO (−), scramble (SC), and mismatch (MM) controls were included in each experiment at the same increasing concentrations as the respective targeting ASOs. All experiments were performed in three biological replicates. Data are presented as mean values ±SD. P -values were calculated using two-sided t -test. Asterisks denote ASOs that are statistically significant ( p
    Chx, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Novoprotein de novo protein synthesis inhibitor cycloheximide chx
    CBP mediates acetylation and stabilization of <t>FBXL19.</t> A ) IP of proteins from lysates of veh (-), TSA-, or TSA+C646-treated FBXL19-V5 –transfected MLE12 cells with an anti-Ack antibody followed by immunoblotting (IB) analysis of the precipitates with an anti-V5 tag antibody. IB analysis of V5-tagged proteins and β-actin in input cell lysates was performed. B ) IP of proteins from lysates of FBXL19-V5 + Ubiquitin-HA –transfected MLE12 cells cotransfected with or without CBP shRNA with an anti-HA antibody followed by IB analysis of the precipitates with anti-V5 tag. IB analysis of CBP, V5-tagged proteins, and β-actin in input cell lysates was performed. C ) IP of proteins from lysates of FBXL19-V5 - or FBXL19 K141R -V5 –transfected cells with or without CBP-HA cotransfection with an anti-V5 tag antibody followed by IB analysis of precipitates with an anti–acetylated lysine antibody. IB analysis of V5-tagged proteins, HA-tagged proteins, and β-actin in input cell lysates was performed. D ) IB analysis of V5-tagged proteins, CBP, and β-actin in lysates of FBXL19-V5–overexpressing MLE12 cells transfected with or without CBP plasmid and then treated for 0–4 h with <t>CHX</t> and analysis of protein levels by densitometry of the results. *** P
    De Novo Protein Synthesis Inhibitor Cycloheximide Chx, supplied by Novoprotein, used in various techniques. Bioz Stars score: 90/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    chx  (Tocris)
    98
    Tocris chx
    CBP mediates acetylation and stabilization of <t>FBXL19.</t> A ) IP of proteins from lysates of veh (-), TSA-, or TSA+C646-treated FBXL19-V5 –transfected MLE12 cells with an anti-Ack antibody followed by immunoblotting (IB) analysis of the precipitates with an anti-V5 tag antibody. IB analysis of V5-tagged proteins and β-actin in input cell lysates was performed. B ) IP of proteins from lysates of FBXL19-V5 + Ubiquitin-HA –transfected MLE12 cells cotransfected with or without CBP shRNA with an anti-HA antibody followed by IB analysis of the precipitates with anti-V5 tag. IB analysis of CBP, V5-tagged proteins, and β-actin in input cell lysates was performed. C ) IP of proteins from lysates of FBXL19-V5 - or FBXL19 K141R -V5 –transfected cells with or without CBP-HA cotransfection with an anti-V5 tag antibody followed by IB analysis of precipitates with an anti–acetylated lysine antibody. IB analysis of V5-tagged proteins, HA-tagged proteins, and β-actin in input cell lysates was performed. D ) IB analysis of V5-tagged proteins, CBP, and β-actin in lysates of FBXL19-V5–overexpressing MLE12 cells transfected with or without CBP plasmid and then treated for 0–4 h with <t>CHX</t> and analysis of protein levels by densitometry of the results. *** P
    Chx, supplied by Tocris, used in various techniques. Bioz Stars score: 98/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Novoprotein protein synthesis inhibitor cycloheximide chx
    CE-ERa Promotes Misfolded Proinsulin Degradation via <t>HRD1-ERAD</t> Stabilization (A) Diagram of misfolded protein degradation via ERAD including misfolded protein retro-translocation to the cytosol via E3 ubiquitin ligase HRD1 and its scaffold adaptor SEL1L followed by HRD1-mediated ubiquitination and proteasomal degradation. (B and C) Male Ins2 +/Akita cells were treated with CE and BZA (B) or PPT (C) for 24 hr, followed by exposure to Tg for 4 hr. Representative image of western blot for HRD1 expression and quantification (n = 4–7 distinct experiments/condition). (D) COS-7 cells were transfected with HA-WT proinsulin or HA-C 96 Y proinsulin along with ERa and FLAG-ubiquitin in the absence or presence of HRD-1. Cells were treated with CE for 24 hr, fol-lowed by exposure to Tg for 4 hr. Co-immuno-precipitations were carried out using anti-HA antibody followed by western blotting using anti-FLAG or anti-HA antibodies (left). Quantification of relative proinsulin ubiquitination (middle) and proinsulin stability (right) (n = 3 distinct experiments/ condition). (E) Male Ins2 +/Akita cells were treated like in (B), followed by qRT-PCR quantification of HRD1 mRNA (n = 8 experiments). (F–I) Male Ins2 +/Akita cells were treated with cycloheximide <t>(CHX)</t> plus Tg and either vehicle (F and G) or CE (H and I) in the presence (G and I) or in the absence (F and H) of MG132 for the indicated time course. Western blots for HRD1 were performed on cell lysates. Relative HRD1 protein levels were determined by densitometry and were normalized to that obtained at time 0 (n = 4–6 distinct experi-ments/condition). (J) Islets from female human donors were treated like in (B). Representative image of western blot for HRD1 expression and bar graph of quantification (n = 3). Data are shown as mean ± SEM. *p
    Protein Synthesis Inhibitor Cycloheximide Chx, supplied by Novoprotein, used in various techniques. Bioz Stars score: 89/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    FUJIFILM cycloheximide chx
    PXK expression accelerates EGFR degradation. COS7 cells cultured on coverslips were transfected with the gene for FLAG-PXK. Cells showing FLAG fluorescence are indicated by asterisks at the nucleus, and the cellular edges are traced by broken lines. (A) Thirty-six hours after transfection, cells were serum starved for 12 h, followed by preincubation with 20 μg/ml <t>CHX</t> and then left unstimulated (none) or stimulated with 100 nM EGF for 5 or 30 min. Cells were then fixed and treated with mouse anti-FLAG antibody and rabbit anti-EGFR antibody, followed by visualization with Alexa Fluor 488-conjugated anti-mouse antibody (green) and Alexa Fluor 594-conjugated anti-rabbit antibody (red), respectively. (B) Cells were preincubated with 10 μM MG-132 or 0.25 μM <t>bafilomycin</t> A1 in the presence of 20 μg/ml CHX for 30 min and then stimulated with 100 nM EGF for 30 min, followed by treatment as described above. Bars, 10 μm.
    Cycloheximide Chx, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 93/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore cycloheximide chx treatment
    PXK expression accelerates EGFR degradation. COS7 cells cultured on coverslips were transfected with the gene for FLAG-PXK. Cells showing FLAG fluorescence are indicated by asterisks at the nucleus, and the cellular edges are traced by broken lines. (A) Thirty-six hours after transfection, cells were serum starved for 12 h, followed by preincubation with 20 μg/ml <t>CHX</t> and then left unstimulated (none) or stimulated with 100 nM EGF for 5 or 30 min. Cells were then fixed and treated with mouse anti-FLAG antibody and rabbit anti-EGFR antibody, followed by visualization with Alexa Fluor 488-conjugated anti-mouse antibody (green) and Alexa Fluor 594-conjugated anti-rabbit antibody (red), respectively. (B) Cells were preincubated with 10 μM MG-132 or 0.25 μM <t>bafilomycin</t> A1 in the presence of 20 μg/ml CHX for 30 min and then stimulated with 100 nM EGF for 30 min, followed by treatment as described above. Bars, 10 μm.
    Cycloheximide Chx Treatment, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cayman Chemical cycloheximide chx
    Characterization of the degradability of BiFP-modc C-terminal fusion proteins . ( A ) Schematic representation of destabilized BiFP constructs with zero to three copies of MODC PEST domain at the C-terminus of BiFP. The fusion constructs were generated and cloned in the pSEH retroviral vector, yielding pSEH-BiFP, pSEH-BiFP-modc, pSEH-BiFP-2modc, and pSEH-BiFP-3modc, respectively. ( B ) BiFP fusion constructs were used for retrovirus packaging and generating stable lines in HCT116 cells after <t>hygromycin</t> selection. Subconfluent stable lines were treated with <t>CHX</t> (100 μg/ml). Fluorescence signals were recorded at the indicated time points. Representative images are shown. ( C ) Quantitative analysis of the fluorescence signal for the CHX-treated stable lines shown in ( B ). “*” p
    Cycloheximide Chx, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 90/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Selleck Chemicals cycloheximide chx
    HDAC6 mediates the deacetylation of PA at Lys(664). (A) Schematic diagram of PA modification. NLS, nuclear localization signal. (B) 293T cells were transfected with Flag-PA or its acetylation dead mutants as indicated and then treated with <t>tubacin</t> (10 μM) for 17 h. Flag antibody was used to immunoprecipitate the wild type or acetylation dead mutants of Flag-PA, which were then analyzed by immunoblotting with the indicated antibodies. WCL, whole-cell lysates. (C) 293T cells were transfected with the indicated plasmids, followed by tubacin or DMSO treatment for 17 h. The cells were then treated with <t>CHX</t> (10 μg/ml) at the indicated time points. PA and actin were detected by immunoblotting with the indicated antibodies. (D) Calculated relative half-lives of PA, PA-K664R, and PA-K664Q, using the data from panel C. The percent intensity (log 10 ) was plotted versus time.
    Cycloheximide Chx, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 90/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Amresco cycloheximide chx
    TNFα increased basal lipolysis and degraded PLIN1, which requires Cathepsin B a <t>3T3-L1</t> adipocytes were individually treated with 10 ng/ml and 50 ng/ml TNFα for 3 h, then media was removed for glycerol measurement. b 3T3-L1 adipocytes were stimulated with 10 ng/ml TNFα for 3 h with or without 1 h CA074 pre-treatment, then media was removed for glycerol measurement. c–d C57BL/6 mice were fed a HFD or standard chow for 24 weeks, then expression levels of PLIN1 in EAT were determined by qRT-PCR c and western blot d ( n = 6). e 3T3-L1 adipocytes were treated with the proteasome inhibitor MG-132 (10 μ m ) or the lysosomal protease inhibitor leupeptin (Leu; 10 μg/ml) for 24 h, then the content of PLIN1 were assessed by western blot. f PLIN1 stability in response to TNFα (10 ng/ml) was evaluated in a cycloheximide <t>(CHX,</t> 100 μg/ml) chasing experiment. Immunoblot of perilipin after 6, 12, 18, and 24 h treatments with CHX in adipocytes. g Adipocytes were stimulated with TNFα (10 ng/ml) in the presence or absence of CA074 treatment (10 µ m ). Immunoblotting of PLIN1 was perform after treatment with CHX for 6, 12, 18, and 24 h. ** p
    Cycloheximide Chx, supplied by Amresco, used in various techniques. Bioz Stars score: 92/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Nacalai cycloheximide chx
    Intracellular dynamics of M16/O16 chimeric protein in <t>sec12</t> cells. YMO59 cells (A) and YMO60 cells harboring pRS316-M16/O16-GFP (B) were treated with 100 μg/ml <t>CHX</t> for 30 min at 23°C, incubated at 37.5°C for 40 min, and then observed.
    Cycloheximide Chx, supplied by Nacalai, used in various techniques. Bioz Stars score: 90/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher cycloheximide chx
    Intracellular dynamics of M16/O16 chimeric protein in <t>sec12</t> cells. YMO59 cells (A) and YMO60 cells harboring pRS316-M16/O16-GFP (B) were treated with 100 μg/ml <t>CHX</t> for 30 min at 23°C, incubated at 37.5°C for 40 min, and then observed.
    Cycloheximide Chx, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Beyotime chx
    Intracellular dynamics of M16/O16 chimeric protein in <t>sec12</t> cells. YMO59 cells (A) and YMO60 cells harboring pRS316-M16/O16-GFP (B) were treated with 100 μg/ml <t>CHX</t> for 30 min at 23°C, incubated at 37.5°C for 40 min, and then observed.
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    Santa Cruz Biotechnology cycloheximide chx
    Intracellular dynamics of M16/O16 chimeric protein in <t>sec12</t> cells. YMO59 cells (A) and YMO60 cells harboring pRS316-M16/O16-GFP (B) were treated with 100 μg/ml <t>CHX</t> for 30 min at 23°C, incubated at 37.5°C for 40 min, and then observed.
    Cycloheximide Chx, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore cycloheximide solution
    Intracellular dynamics of M16/O16 chimeric protein in <t>sec12</t> cells. YMO59 cells (A) and YMO60 cells harboring pRS316-M16/O16-GFP (B) were treated with 100 μg/ml <t>CHX</t> for 30 min at 23°C, incubated at 37.5°C for 40 min, and then observed.
    Cycloheximide Solution, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    C5a induces loss of HUVEC viability in the presence of CHX. A) MTS assay for cell viability. Cells were treated with C5a (50 ng/ml), CHX (10 μg/ml), or C5a (50 ng/ml) plus CHX (10 μg/ml) for 18 h. *P

    Journal: Inflammation research : official journal of the European Histamine Research Society ... [et al.]

    Article Title: Activation by C5a of endothelial cell caspase 8 and cFLIP

    doi: 10.1007/s00011-008-8156-9

    Figure Lengend Snippet: C5a induces loss of HUVEC viability in the presence of CHX. A) MTS assay for cell viability. Cells were treated with C5a (50 ng/ml), CHX (10 μg/ml), or C5a (50 ng/ml) plus CHX (10 μg/ml) for 18 h. *P

    Article Snippet: HUVEC were stimulated with C5a alone or in the presence of CHX for 0, 4, and 8 h. CHX control involved incubating HUVEC with CHX for 8 h. One microgram total RNA was reversed transcribed with superscript reverse transcriptase (Invitrogen, Carlsbad, CA) using random and poly dT primers for first-strand cDNA synthesis.

    Techniques: MTS Assay

    C5a stimulation of HUVEC initiates apoptotic pathway activity. A) Western blots for protein expression of PARP. Cell lysates were collected after 2, 8, 18 h following stimulation with C5a (50 ng/ml) alone or with C5a (50 ng/ml) and CHX (10 μg/ml).

    Journal: Inflammation research : official journal of the European Histamine Research Society ... [et al.]

    Article Title: Activation by C5a of endothelial cell caspase 8 and cFLIP

    doi: 10.1007/s00011-008-8156-9

    Figure Lengend Snippet: C5a stimulation of HUVEC initiates apoptotic pathway activity. A) Western blots for protein expression of PARP. Cell lysates were collected after 2, 8, 18 h following stimulation with C5a (50 ng/ml) alone or with C5a (50 ng/ml) and CHX (10 μg/ml).

    Article Snippet: HUVEC were stimulated with C5a alone or in the presence of CHX for 0, 4, and 8 h. CHX control involved incubating HUVEC with CHX for 8 h. One microgram total RNA was reversed transcribed with superscript reverse transcriptase (Invitrogen, Carlsbad, CA) using random and poly dT primers for first-strand cDNA synthesis.

    Techniques: Activity Assay, Western Blot, Expressing

    Western blotting of Caspase-8 and cFLIP. A) Cell lysates were collected 18 h following C5a (50 ng/ml) alone or in the presence of CHX (10 μg/ml). Control included non-stimulated (−) and 18 h CHX (10 μg/ml) treatment (+). The activity

    Journal: Inflammation research : official journal of the European Histamine Research Society ... [et al.]

    Article Title: Activation by C5a of endothelial cell caspase 8 and cFLIP

    doi: 10.1007/s00011-008-8156-9

    Figure Lengend Snippet: Western blotting of Caspase-8 and cFLIP. A) Cell lysates were collected 18 h following C5a (50 ng/ml) alone or in the presence of CHX (10 μg/ml). Control included non-stimulated (−) and 18 h CHX (10 μg/ml) treatment (+). The activity

    Article Snippet: HUVEC were stimulated with C5a alone or in the presence of CHX for 0, 4, and 8 h. CHX control involved incubating HUVEC with CHX for 8 h. One microgram total RNA was reversed transcribed with superscript reverse transcriptase (Invitrogen, Carlsbad, CA) using random and poly dT primers for first-strand cDNA synthesis.

    Techniques: Western Blot, Activity Assay

    SGK2 regulates c-Myc/β-catenin gene expression. (A). WB results showed the protein levels of β-catenin and c-Myc were significantly decreased after down-regulating SGK2 expression in UMUC3 and J82 cells. (B). WB results showed the protein levels of β-catenin and c-Myc were significantly increased after overexpressing SGK2 in T24 cell. (C) . Immunoprecipitation (IP) assay indicated SGK2 could directly interact with β-catenin protein in T24 cell. (D). MG132 (10 µM) abrogated shSGK2 induced inhibition of β-catenin protein expression in UMUC3 cell. (E). A time-dependent decrease in β-catenin protein expression in T24-vector and T24-oeSGK2 cell lines exposed to CHX (10 µg/mL). However, the decrease speed was marked slower in T24-oeSGK2 than T24-vector group. (F). The expression of c-Myc and CTNNB1 gene (encoding β-catenin protein) were significantly associated with tumor stage of bladder cancer ( P

    Journal: Journal of Cancer

    Article Title: Glucocorticoid-Inducible Kinase 2 Promotes Bladder Cancer Cell Proliferation, Migration and Invasion by Enhancing β-catenin/c-Myc Signaling Pathway

    doi: 10.7150/jca.25811

    Figure Lengend Snippet: SGK2 regulates c-Myc/β-catenin gene expression. (A). WB results showed the protein levels of β-catenin and c-Myc were significantly decreased after down-regulating SGK2 expression in UMUC3 and J82 cells. (B). WB results showed the protein levels of β-catenin and c-Myc were significantly increased after overexpressing SGK2 in T24 cell. (C) . Immunoprecipitation (IP) assay indicated SGK2 could directly interact with β-catenin protein in T24 cell. (D). MG132 (10 µM) abrogated shSGK2 induced inhibition of β-catenin protein expression in UMUC3 cell. (E). A time-dependent decrease in β-catenin protein expression in T24-vector and T24-oeSGK2 cell lines exposed to CHX (10 µg/mL). However, the decrease speed was marked slower in T24-oeSGK2 than T24-vector group. (F). The expression of c-Myc and CTNNB1 gene (encoding β-catenin protein) were significantly associated with tumor stage of bladder cancer ( P

    Article Snippet: MG132 and CHX (cycloheximide) were obtained from Sigma-Aldrich (MO, USA).

    Techniques: Expressing, Western Blot, Immunoprecipitation, Inhibition, Plasmid Preparation

    Co-inoculation of E. amylovora with CHX reduces apoplastic ROS accumulation. (A,B) Leaves of 5-weeks-old plants were mock-treated (mock) or inoculated with wild-type E. amylovora ( Ea ) and co-inoculated (+) or not (-) with 4 μg/ml cycloheximide. Leaves were stained with DCFH-DA or DAB to detect H 2 O 2 accumulation 18 hpi. (A) Only the top half of the leaf blade was inoculated. Leaves stained with DCFH-DA show intracellular H 2 O 2 accumulation in green. (B) Leaves stained with DAB show H 2 O 2 accumulation in brown. Arrows indicate DAB staining in the apoplast. Bar: 50 μm.

    Journal: Frontiers in Plant Science

    Article Title: DspA/E Contributes to Apoplastic Accumulation of ROS in Non-host A. thaliana

    doi: 10.3389/fpls.2016.00545

    Figure Lengend Snippet: Co-inoculation of E. amylovora with CHX reduces apoplastic ROS accumulation. (A,B) Leaves of 5-weeks-old plants were mock-treated (mock) or inoculated with wild-type E. amylovora ( Ea ) and co-inoculated (+) or not (-) with 4 μg/ml cycloheximide. Leaves were stained with DCFH-DA or DAB to detect H 2 O 2 accumulation 18 hpi. (A) Only the top half of the leaf blade was inoculated. Leaves stained with DCFH-DA show intracellular H 2 O 2 accumulation in green. (B) Leaves stained with DAB show H 2 O 2 accumulation in brown. Arrows indicate DAB staining in the apoplast. Bar: 50 μm.

    Article Snippet: For CHX treatment, E. amylovora was co-inoculated with the translation inhibitor cycloheximide (Sigma) at 4 μg/ml as described in .

    Techniques: Staining

    Overview of Arabidopsis thaliana non-host resistance against E. amylovora . (A) Overview of ROS production and bacterial growth in the different combinations of inoculum and genotype. Depending on the combination, ROS accumulate in the apoplast and/or in the cytoplasm or don’t accumulate. The combinations lead, 24 hpi, to either a rapid decrease in bacterial titers (-), a weak and transient increase (+) or a strong increase in bacterial titer (+++). (B) Model of A. thaliana non-host resistance against E. amylovora . The T3E DspA/E and other elements trigger apoplastic ROS and other defenses that can be repressed by co-inoculation with CHX. CHX: cycloheximide, Dsp: DspA/E, Ea : E. amylovora , ap: apoplast, cy: cytoplasm, RPL: ribosomal protein, dashed lines: defense that reduces bacterial titers, continuous lines: defense repression that increases bacterial titers. Numbers in between brackets indicate the reference of the corresponding work.

    Journal: Frontiers in Plant Science

    Article Title: DspA/E Contributes to Apoplastic Accumulation of ROS in Non-host A. thaliana

    doi: 10.3389/fpls.2016.00545

    Figure Lengend Snippet: Overview of Arabidopsis thaliana non-host resistance against E. amylovora . (A) Overview of ROS production and bacterial growth in the different combinations of inoculum and genotype. Depending on the combination, ROS accumulate in the apoplast and/or in the cytoplasm or don’t accumulate. The combinations lead, 24 hpi, to either a rapid decrease in bacterial titers (-), a weak and transient increase (+) or a strong increase in bacterial titer (+++). (B) Model of A. thaliana non-host resistance against E. amylovora . The T3E DspA/E and other elements trigger apoplastic ROS and other defenses that can be repressed by co-inoculation with CHX. CHX: cycloheximide, Dsp: DspA/E, Ea : E. amylovora , ap: apoplast, cy: cytoplasm, RPL: ribosomal protein, dashed lines: defense that reduces bacterial titers, continuous lines: defense repression that increases bacterial titers. Numbers in between brackets indicate the reference of the corresponding work.

    Article Snippet: For CHX treatment, E. amylovora was co-inoculated with the translation inhibitor cycloheximide (Sigma) at 4 μg/ml as described in .

    Techniques:

    Mitotic slippage is induced in nocodazole-treated cells after inhibition of transcription or translation. Characterization of NIH3T3 cells: control cells, nocodazole (noc)-treated cells (arrested in mitosis), noc-cells incubated with cycloheximide (CHX; 35.5 µM) or actinomycin D (ActD; 8 µM) for 4 h. A) Phase-contrast microscopy; magnification: 200×. B) Immunofluorescence microscopy; arrowheads indicate mitotic cells and arrows indicate cells that underwent mitotic slippage; DAPI stains the DNA (blue) and anti-α-tubulin antibody stains the microtubules (green). Scale bar = 25 µm. C) Mitotic and multinucleation indexes of noc-treated cells with or without CHX or ActD for 4 h. Data presented as mean ± S.D. from three independent experiments; ** p

    Journal: PLoS ONE

    Article Title: Sustained Spindle-Assembly Checkpoint Response Requires De Novo Transcription and Translation of Cyclin B1

    doi: 10.1371/journal.pone.0013037

    Figure Lengend Snippet: Mitotic slippage is induced in nocodazole-treated cells after inhibition of transcription or translation. Characterization of NIH3T3 cells: control cells, nocodazole (noc)-treated cells (arrested in mitosis), noc-cells incubated with cycloheximide (CHX; 35.5 µM) or actinomycin D (ActD; 8 µM) for 4 h. A) Phase-contrast microscopy; magnification: 200×. B) Immunofluorescence microscopy; arrowheads indicate mitotic cells and arrows indicate cells that underwent mitotic slippage; DAPI stains the DNA (blue) and anti-α-tubulin antibody stains the microtubules (green). Scale bar = 25 µm. C) Mitotic and multinucleation indexes of noc-treated cells with or without CHX or ActD for 4 h. Data presented as mean ± S.D. from three independent experiments; ** p

    Article Snippet: NIH3T3 cells were incubated for 14 h with nocodazole and further treated for 4 h with either 35.5 µM of cycloheximide (CHX), a de novo protein synthesis inhibitor , or 8 µM of actinomycin D (ActD), an inhibitor of transcription .

    Techniques: Inhibition, Incubation, Microscopy, Immunofluorescence

    Overexpression of Cyclin B1 protein rescues the mitotic phenotype. HEK293 cells were transfected with the wild-type and the non-degradable forms of Cyclin B1 (CyclinB1-wt and CyclinB1-R42A, respectively); pCMS-EGFP was used as transfection control. A, B and C) Analysis of Cyclin B1 endogenous (indicated with one asterisk) and exogenous (indicated with double asterisk) protein levels by western blotting in cells transfected with pCMS-EGFP (A), CyclinB1-wt (B) and CyclinB1-R42A (C). After transfection, HEK293 cells were incubated with nocodazole (noc) for 14 h and treated with actinomycin D (ActD; 8 µM) or cycloheximide (CHX; 35.5 µM) for 6 h; β-actin was used as loading control. All lanes presented in C are from the same experiment. D) Mitotic index of nocodazole (Noc) treated cells with or without CHX or ActD for 6 h. Data presented as mean ± S.D. from three independent experiments; *** p

    Journal: PLoS ONE

    Article Title: Sustained Spindle-Assembly Checkpoint Response Requires De Novo Transcription and Translation of Cyclin B1

    doi: 10.1371/journal.pone.0013037

    Figure Lengend Snippet: Overexpression of Cyclin B1 protein rescues the mitotic phenotype. HEK293 cells were transfected with the wild-type and the non-degradable forms of Cyclin B1 (CyclinB1-wt and CyclinB1-R42A, respectively); pCMS-EGFP was used as transfection control. A, B and C) Analysis of Cyclin B1 endogenous (indicated with one asterisk) and exogenous (indicated with double asterisk) protein levels by western blotting in cells transfected with pCMS-EGFP (A), CyclinB1-wt (B) and CyclinB1-R42A (C). After transfection, HEK293 cells were incubated with nocodazole (noc) for 14 h and treated with actinomycin D (ActD; 8 µM) or cycloheximide (CHX; 35.5 µM) for 6 h; β-actin was used as loading control. All lanes presented in C are from the same experiment. D) Mitotic index of nocodazole (Noc) treated cells with or without CHX or ActD for 6 h. Data presented as mean ± S.D. from three independent experiments; *** p

    Article Snippet: NIH3T3 cells were incubated for 14 h with nocodazole and further treated for 4 h with either 35.5 µM of cycloheximide (CHX), a de novo protein synthesis inhibitor , or 8 µM of actinomycin D (ActD), an inhibitor of transcription .

    Techniques: Over Expression, Transfection, Western Blot, Incubation

    Cyclin B1 and Securin are degraded during mitotic slippage induced by inhibition of transcription and translation. A) Analysis of Cyclin B1, Securin and CDK1 levels by western blotting. NIH3T3 cells were incubated with nocodazole (noc) for 14 h and treated with actinomycin D (ActD; 8 µM) or cycloheximide (CHX; 35.5 µM) with or without the inhibitor of the proteasome MG132 (25 µM), for 4 h; β-actin was used as loading control. B) Quantification of Cyclin B1 and Securin proteins in noc-, noc-ActD-, noc-ActD-MG, noc-CHX and noc-CHX-MG-cells. Normalization of Cyclin B1 and Securin was calculated on the ratio of these proteins per β-actin protein present in each condition. Data presented as mean ± S.D. from three independent experiments. C and D) Immunofluorescence microscopy: asynchronous population (control) and noc-cells treated with ActD for 4 h; DAPI stains the DNA (blue) and Cyclin B1 (C) and Securin (D) are stained in green. Arrowheads point mitotic cells and arrows point cell that undergone mitotic slippage. The adapted cells shown are representative of both ActD and CHX induced mitotic slippage. Scale bar = 25 µm.

    Journal: PLoS ONE

    Article Title: Sustained Spindle-Assembly Checkpoint Response Requires De Novo Transcription and Translation of Cyclin B1

    doi: 10.1371/journal.pone.0013037

    Figure Lengend Snippet: Cyclin B1 and Securin are degraded during mitotic slippage induced by inhibition of transcription and translation. A) Analysis of Cyclin B1, Securin and CDK1 levels by western blotting. NIH3T3 cells were incubated with nocodazole (noc) for 14 h and treated with actinomycin D (ActD; 8 µM) or cycloheximide (CHX; 35.5 µM) with or without the inhibitor of the proteasome MG132 (25 µM), for 4 h; β-actin was used as loading control. B) Quantification of Cyclin B1 and Securin proteins in noc-, noc-ActD-, noc-ActD-MG, noc-CHX and noc-CHX-MG-cells. Normalization of Cyclin B1 and Securin was calculated on the ratio of these proteins per β-actin protein present in each condition. Data presented as mean ± S.D. from three independent experiments. C and D) Immunofluorescence microscopy: asynchronous population (control) and noc-cells treated with ActD for 4 h; DAPI stains the DNA (blue) and Cyclin B1 (C) and Securin (D) are stained in green. Arrowheads point mitotic cells and arrows point cell that undergone mitotic slippage. The adapted cells shown are representative of both ActD and CHX induced mitotic slippage. Scale bar = 25 µm.

    Article Snippet: NIH3T3 cells were incubated for 14 h with nocodazole and further treated for 4 h with either 35.5 µM of cycloheximide (CHX), a de novo protein synthesis inhibitor , or 8 µM of actinomycin D (ActD), an inhibitor of transcription .

    Techniques: Inhibition, Western Blot, Incubation, Immunofluorescence, Microscopy, Staining

    Inhibition of transcription or translation induces mitotic slippage in nocodazole treated HEK293 cells. Characterization of HEK293 cells: control cells, nocodazole (noc)-treated cells (arrested in mitosis), noc-cells incubated with cycloheximide (CHX; 35.5 µM) or actinomycin D (ActD; 8 µM) for 6 h. A) Phase-contrast microscopy; magnification: 200×. B) Mitotic index of noc-treated cells with or without CHX or ActD for 6 h. Data presented as mean ± S.D. from three independent experiments; ** p

    Journal: PLoS ONE

    Article Title: Sustained Spindle-Assembly Checkpoint Response Requires De Novo Transcription and Translation of Cyclin B1

    doi: 10.1371/journal.pone.0013037

    Figure Lengend Snippet: Inhibition of transcription or translation induces mitotic slippage in nocodazole treated HEK293 cells. Characterization of HEK293 cells: control cells, nocodazole (noc)-treated cells (arrested in mitosis), noc-cells incubated with cycloheximide (CHX; 35.5 µM) or actinomycin D (ActD; 8 µM) for 6 h. A) Phase-contrast microscopy; magnification: 200×. B) Mitotic index of noc-treated cells with or without CHX or ActD for 6 h. Data presented as mean ± S.D. from three independent experiments; ** p

    Article Snippet: NIH3T3 cells were incubated for 14 h with nocodazole and further treated for 4 h with either 35.5 µM of cycloheximide (CHX), a de novo protein synthesis inhibitor , or 8 µM of actinomycin D (ActD), an inhibitor of transcription .

    Techniques: Inhibition, Incubation, Microscopy

    LANA stabilizes Par3 in KSHV positive cells. (A) HEK-293-BAC-KSHV cells were transfected with sh-LANA and sh-Control. Endogenous Par3 expression was measured and presented in graph. GAPDH was used as a protein loading control. (B, C) Stabilization of Par3 was examined with cyclohexamide in HEK-293 and BJAB cells. Left panels and right panels were used as vector control and LANA expression, in a time dependent manner. (D) The proteosome inhibitor MG132 was used to determine if Par3 stability was linked to the proteosome degradation pathway. GFP and GAPDH were used as transfection and endogenous protein loading controls. (E) BC-3-shControl and BC-3-shLANA cells were treated with cyclohexamide and observed Par3 endogenous on hours dependent manner. (F) BC-3-shControl and BC-3shLANA cells were treated with MG132 and followed immunoprecipitation of Par3. Endogenous ubiquitin and Par3 were detected using specific antibodies in both the cell lines. GAPDH was monitored as an internal control for loading in the input section.

    Journal: PLoS Pathogens

    Article Title: KSHV-Mediated Regulation of Par3 and SNAIL Contributes to B-Cell Proliferation

    doi: 10.1371/journal.ppat.1005801

    Figure Lengend Snippet: LANA stabilizes Par3 in KSHV positive cells. (A) HEK-293-BAC-KSHV cells were transfected with sh-LANA and sh-Control. Endogenous Par3 expression was measured and presented in graph. GAPDH was used as a protein loading control. (B, C) Stabilization of Par3 was examined with cyclohexamide in HEK-293 and BJAB cells. Left panels and right panels were used as vector control and LANA expression, in a time dependent manner. (D) The proteosome inhibitor MG132 was used to determine if Par3 stability was linked to the proteosome degradation pathway. GFP and GAPDH were used as transfection and endogenous protein loading controls. (E) BC-3-shControl and BC-3-shLANA cells were treated with cyclohexamide and observed Par3 endogenous on hours dependent manner. (F) BC-3-shControl and BC-3shLANA cells were treated with MG132 and followed immunoprecipitation of Par3. Endogenous ubiquitin and Par3 were detected using specific antibodies in both the cell lines. GAPDH was monitored as an internal control for loading in the input section.

    Article Snippet: We further performed cyclohexamide experiments in BC-3-shControl and BC-3-shLANA cell lines to block de novo protein synthesis.

    Techniques: BAC Assay, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation

    CCND1 mutations interfere with T286 phosphorylation and protein ubiquitination ( A , B ) UPN-1 or Z138 cells expressing WT, Y44D CCND1-HA, or an empty vector control were treated with 10 μM of cyclohexamide (CHX) for 16 hours and released to normal growth medium for indicated times. Cell lysates (10 μg per lane) were prepared for immunoblot analysis with indicated antibodies. Arrow, CCND1-HA protein. Arrowhead, endogenous CCND1. Numbers below the blots are relative densitometric values of corresponding bands after normalization to β-ACTIN loading controls. Graphical representation of selected densitometric values was shown in the bar graphs on the right. ( C ) Z-138 cells expressing an empty vector (EV), WT, or mutant CCND1-HA were immunoprecipitated with HA antibody and immunoblotted with indicated antibodies. Lysates before immunoprecipitation were used as input samples. ( D , E ) Analysis of mutant CCND1 interaction with GSK3B. (D) JEKO-1 cells expressing an empty vector (EV), WT, or mutant CCND1-HA were immunoprecipitated with HA antibody and immunoblotted with indicated antibodies. (E) JEKO-1 cell lines described in (D) were immunoprecipitated with GSK3B antibody and immunoblotted with indicated antibodies. Arrow, CCND1-HA protein. Arrowhead, endogenous CCND1. ( F ) Y44D CCND1 mutation affects ubiquitin (Ub)-dependent degradation. UPN-1 cells that stably expressed an empty vector (EV) control, WT or Y44D CCND1-HA were treated with 5 nM of the proteasome inhibitor PS-341 for 2 h. Treated cells were lysed in 2% SDS and heated at 95°C for 5 min. Lysates were subsequently immunoprecipitated with HA antibodies and Ub conjugates on CCND1 were examined by immunoblot analysis with anti-Ub antibody.

    Journal: Oncotarget

    Article Title: CCND1 mutations increase protein stability and promote ibrutinib resistance in mantle cell lymphoma

    doi: 10.18632/oncotarget.12434

    Figure Lengend Snippet: CCND1 mutations interfere with T286 phosphorylation and protein ubiquitination ( A , B ) UPN-1 or Z138 cells expressing WT, Y44D CCND1-HA, or an empty vector control were treated with 10 μM of cyclohexamide (CHX) for 16 hours and released to normal growth medium for indicated times. Cell lysates (10 μg per lane) were prepared for immunoblot analysis with indicated antibodies. Arrow, CCND1-HA protein. Arrowhead, endogenous CCND1. Numbers below the blots are relative densitometric values of corresponding bands after normalization to β-ACTIN loading controls. Graphical representation of selected densitometric values was shown in the bar graphs on the right. ( C ) Z-138 cells expressing an empty vector (EV), WT, or mutant CCND1-HA were immunoprecipitated with HA antibody and immunoblotted with indicated antibodies. Lysates before immunoprecipitation were used as input samples. ( D , E ) Analysis of mutant CCND1 interaction with GSK3B. (D) JEKO-1 cells expressing an empty vector (EV), WT, or mutant CCND1-HA were immunoprecipitated with HA antibody and immunoblotted with indicated antibodies. (E) JEKO-1 cell lines described in (D) were immunoprecipitated with GSK3B antibody and immunoblotted with indicated antibodies. Arrow, CCND1-HA protein. Arrowhead, endogenous CCND1. ( F ) Y44D CCND1 mutation affects ubiquitin (Ub)-dependent degradation. UPN-1 cells that stably expressed an empty vector (EV) control, WT or Y44D CCND1-HA were treated with 5 nM of the proteasome inhibitor PS-341 for 2 h. Treated cells were lysed in 2% SDS and heated at 95°C for 5 min. Lysates were subsequently immunoprecipitated with HA antibodies and Ub conjugates on CCND1 were examined by immunoblot analysis with anti-Ub antibody.

    Article Snippet: We treated WT or mutant CCND1 -expressing cells with cyclohexamide (CHX) to inhibit de novo protein synthesis, and CCND1 proteolysis was examined by immunoblot analysis.

    Techniques: Expressing, Plasmid Preparation, Mutagenesis, Immunoprecipitation, Stable Transfection

    CCND1 mutations in primary MCL ( A ) Sequencing chromatograms of CCND1 point mutations in MCL samples. WT and mutated CCND1 alleles on residue cysteine-47 (yellow bar) from MCL tumors 10, 11, 12 and 47 are shown. For MCL 47, complementary sequence from the reverse primer is shown. ( B ) CCND1 expression in MCL tumors. Lysates (10 μg per lane) from indicated MCL tumors were immunoblotted with indicated antibodies. Peripheral blood mononuclear cells (PBMC) were used as negative control for CCND1 expression. Bar graphs show relative densitometric values of CCND1 expression from the immunoblot after normalization to GAPDH loading control. Red arrows highlight the CCND1 mutated samples. ( C ) Analysis of CCND1 truncated 3′UTR transcript in MCL tumors. The relative abundance between the full-length and truncated 3′UTR was determined by using proximal and distal primers (see primer locations in the diagram of the CCND1 mRNA transcript). ( D ) CCND1 stability in MCL tumors. Indicated MCL tumors were treated with 10 μM of cyclohexamide (CHX) for indicated times and 10 μg of cell lysates were prepared for immunoblot analysis with indicated antibodies. Bar graphs show relative densitometric values of CCND1 expression from corresponding blots after normalization to GAPDH. ( E ) Sensitivity of MCL tumors to ibrutinib. Indicated MCL tumors were treated with indicated doses of ibrutinib for three days and metabolically active cells were assessed by CellTiter-Glo Luminescent assay (Promega). Shown are fractions of luminescence signals from metabolically active cells normalized to untreated samples. Line graphs show mean values from three independent experiments. Error bars, S.E.M. IC 50 values were calculated by GraphPad Prism 7 software. The table summarizes the ibrutinib response with respect to CCND1 mutation status or protein stability. Correlation of either mutation status or protein stability with ibrutinib sensitivity was analyzed by the two-sided Fisher's exact test.

    Journal: Oncotarget

    Article Title: CCND1 mutations increase protein stability and promote ibrutinib resistance in mantle cell lymphoma

    doi: 10.18632/oncotarget.12434

    Figure Lengend Snippet: CCND1 mutations in primary MCL ( A ) Sequencing chromatograms of CCND1 point mutations in MCL samples. WT and mutated CCND1 alleles on residue cysteine-47 (yellow bar) from MCL tumors 10, 11, 12 and 47 are shown. For MCL 47, complementary sequence from the reverse primer is shown. ( B ) CCND1 expression in MCL tumors. Lysates (10 μg per lane) from indicated MCL tumors were immunoblotted with indicated antibodies. Peripheral blood mononuclear cells (PBMC) were used as negative control for CCND1 expression. Bar graphs show relative densitometric values of CCND1 expression from the immunoblot after normalization to GAPDH loading control. Red arrows highlight the CCND1 mutated samples. ( C ) Analysis of CCND1 truncated 3′UTR transcript in MCL tumors. The relative abundance between the full-length and truncated 3′UTR was determined by using proximal and distal primers (see primer locations in the diagram of the CCND1 mRNA transcript). ( D ) CCND1 stability in MCL tumors. Indicated MCL tumors were treated with 10 μM of cyclohexamide (CHX) for indicated times and 10 μg of cell lysates were prepared for immunoblot analysis with indicated antibodies. Bar graphs show relative densitometric values of CCND1 expression from corresponding blots after normalization to GAPDH. ( E ) Sensitivity of MCL tumors to ibrutinib. Indicated MCL tumors were treated with indicated doses of ibrutinib for three days and metabolically active cells were assessed by CellTiter-Glo Luminescent assay (Promega). Shown are fractions of luminescence signals from metabolically active cells normalized to untreated samples. Line graphs show mean values from three independent experiments. Error bars, S.E.M. IC 50 values were calculated by GraphPad Prism 7 software. The table summarizes the ibrutinib response with respect to CCND1 mutation status or protein stability. Correlation of either mutation status or protein stability with ibrutinib sensitivity was analyzed by the two-sided Fisher's exact test.

    Article Snippet: We treated WT or mutant CCND1 -expressing cells with cyclohexamide (CHX) to inhibit de novo protein synthesis, and CCND1 proteolysis was examined by immunoblot analysis.

    Techniques: Sequencing, Expressing, Negative Control, Metabolic Labelling, Luminescence Assay, Software, Mutagenesis

    HBXIP-enhanced acetylation of HOXB13 stabilizes HOXB13 in the facilitation of TAM resistance. a Immunoblotting analysis of HOXB13 in MCF-7 cells treated with the indicated concentrations of leupeptin for 36 h (lower panel). The upper panel is the quantification of the intensity relative to β-actin. b Immunoblotting analysis of HOXB13 in BT474 cells cultured with serum-supplemented or serum-free media for the indicated time courses (lower panel). The upper panel is the quantification of the intensity relative to β-actin. c Immunoblotting analysis of HOXB13 in MCF-7 cells cultured with serum-supplemented or serum-free media for 48 h along with DMSO or TSA (1 μM) (lower panel). Before that, the cells were transiently transfected with pCMV or pCMV-HBXIP (1.5 μg). The protein level of HBXIP was determined by the anti-Flag antibody. The upper panel is the quantification of the intensity relative to β-actin. d Immunoblotting analysis of GFP-HOXB13 in MCF-7 cells time-dependently treated with 100 μg/ml CHX after being transiently transfected with GFP-HOXB13-WT or GFP-HOXB13-K277R (lower panel). The protein level of GFP-HOXB13 was determined by the anti-GFP antibody. The upper panel is the quantification of the intensity relative to β-actin. e Cell viability assay with MCF-7 cells treated with the indicated concentrations of TAM after being transiently transfected with the displayed plasmids. Error bars represent ± SD. * P

    Journal: Journal of Hematology & Oncology

    Article Title: Oncoprotein HBXIP enhances HOXB13 acetylation and co-activates HOXB13 to confer tamoxifen resistance in breast cancer

    doi: 10.1186/s13045-018-0577-5

    Figure Lengend Snippet: HBXIP-enhanced acetylation of HOXB13 stabilizes HOXB13 in the facilitation of TAM resistance. a Immunoblotting analysis of HOXB13 in MCF-7 cells treated with the indicated concentrations of leupeptin for 36 h (lower panel). The upper panel is the quantification of the intensity relative to β-actin. b Immunoblotting analysis of HOXB13 in BT474 cells cultured with serum-supplemented or serum-free media for the indicated time courses (lower panel). The upper panel is the quantification of the intensity relative to β-actin. c Immunoblotting analysis of HOXB13 in MCF-7 cells cultured with serum-supplemented or serum-free media for 48 h along with DMSO or TSA (1 μM) (lower panel). Before that, the cells were transiently transfected with pCMV or pCMV-HBXIP (1.5 μg). The protein level of HBXIP was determined by the anti-Flag antibody. The upper panel is the quantification of the intensity relative to β-actin. d Immunoblotting analysis of GFP-HOXB13 in MCF-7 cells time-dependently treated with 100 μg/ml CHX after being transiently transfected with GFP-HOXB13-WT or GFP-HOXB13-K277R (lower panel). The protein level of GFP-HOXB13 was determined by the anti-GFP antibody. The upper panel is the quantification of the intensity relative to β-actin. e Cell viability assay with MCF-7 cells treated with the indicated concentrations of TAM after being transiently transfected with the displayed plasmids. Error bars represent ± SD. * P

    Article Snippet: Trichostatin A (TSA) and cycloheximide (CHX) were separately purchased from Beyotime Biotechnology (China) and MedChem Express (USA).

    Techniques: Cell Culture, Transfection, Viability Assay

    HBXIP enhances acetylation of HOXB13 at K277 site via acetylase p300. a Immunoblotting analysis of HOXB13 in MCF-7 cells time-dependently treated with 100 μg/ml cycloheximide (CHX) after being transiently transfected with the indicated plasmids. b Immunoblotting analysis of exogenous Flag-HOXB13 in HEK293T cells. The cells were transiently transfected with pCMV-HOXB13 accompanied by pcDNA or pcDNA-HBXIP. The protein level of Flag-HOXB13 was examined by the anti-Flag antibody. c Acetylation level of HOXB13 was detected by a Co-IP assay using GFP-beads and an immunoblotting analysis performed with the anti-acetyl-lysine antibody in HEK293T cells. The cells were transiently transfected with the indicated plasmids. The protein levels of HBXIP and HOXB13 were examined by anti-Flag or anti-HOXB13 antibodies, respectively. The left and right panels are identical results that differ in exposure time. d , e Immunoblotting analysis of HOXB13 in MCF-7 cells and HEK293T cells treated with 100 μg/ml CHX along with different concentrations of trichostatin A (TSA) for 18 h ( d ) or 1 μM TSA ( e ) for the indicated time points. f Acetylation level of HOXB13 was detected by a Co-IP assay using GFP-beads and an immunoblotting analysis performed with the anti-acetyl-lysine antibody in HEK293T cells. The cells were separately transiently transfected with GFP-HOXB13-WT, GFP-HOXB13-K270R, GFP-HOXB13-K277R, or GFP-HOXB13-DM along with pCMV or pCMV-HBXIP. The protein levels of HBXIP and HOXB13 were examined by anti-Flag or anti-GFP antibodies, respectively. g Acetylation level of HOXB13 was detected by a Co-IP assay using GFP-beads and an immunoblotting analysis with the anti-acetyl-lysine antibody in HEK293T cells. The cells were transiently transfected with GFP-HOXB13-WT along with the indicated plasmids or siRNAs. The protein levels of HBXIP and HOXB13 were separately examined by anti-Flag or anti-GFP antibodies. All experiments were repeated at least three times

    Journal: Journal of Hematology & Oncology

    Article Title: Oncoprotein HBXIP enhances HOXB13 acetylation and co-activates HOXB13 to confer tamoxifen resistance in breast cancer

    doi: 10.1186/s13045-018-0577-5

    Figure Lengend Snippet: HBXIP enhances acetylation of HOXB13 at K277 site via acetylase p300. a Immunoblotting analysis of HOXB13 in MCF-7 cells time-dependently treated with 100 μg/ml cycloheximide (CHX) after being transiently transfected with the indicated plasmids. b Immunoblotting analysis of exogenous Flag-HOXB13 in HEK293T cells. The cells were transiently transfected with pCMV-HOXB13 accompanied by pcDNA or pcDNA-HBXIP. The protein level of Flag-HOXB13 was examined by the anti-Flag antibody. c Acetylation level of HOXB13 was detected by a Co-IP assay using GFP-beads and an immunoblotting analysis performed with the anti-acetyl-lysine antibody in HEK293T cells. The cells were transiently transfected with the indicated plasmids. The protein levels of HBXIP and HOXB13 were examined by anti-Flag or anti-HOXB13 antibodies, respectively. The left and right panels are identical results that differ in exposure time. d , e Immunoblotting analysis of HOXB13 in MCF-7 cells and HEK293T cells treated with 100 μg/ml CHX along with different concentrations of trichostatin A (TSA) for 18 h ( d ) or 1 μM TSA ( e ) for the indicated time points. f Acetylation level of HOXB13 was detected by a Co-IP assay using GFP-beads and an immunoblotting analysis performed with the anti-acetyl-lysine antibody in HEK293T cells. The cells were separately transiently transfected with GFP-HOXB13-WT, GFP-HOXB13-K270R, GFP-HOXB13-K277R, or GFP-HOXB13-DM along with pCMV or pCMV-HBXIP. The protein levels of HBXIP and HOXB13 were examined by anti-Flag or anti-GFP antibodies, respectively. g Acetylation level of HOXB13 was detected by a Co-IP assay using GFP-beads and an immunoblotting analysis with the anti-acetyl-lysine antibody in HEK293T cells. The cells were transiently transfected with GFP-HOXB13-WT along with the indicated plasmids or siRNAs. The protein levels of HBXIP and HOXB13 were separately examined by anti-Flag or anti-GFP antibodies. All experiments were repeated at least three times

    Article Snippet: Trichostatin A (TSA) and cycloheximide (CHX) were separately purchased from Beyotime Biotechnology (China) and MedChem Express (USA).

    Techniques: Transfection, Co-Immunoprecipitation Assay

    ASOs dose-dependently reduce NMD and increase mRNA in vitro. a RT-PCR (left panel) and qPCR (right panel) of a selected PCCA ASO (ASO-29) targeting the exon inclusion event transfected in HEK293 cells at increasing concentrations for 24 h. Exact p -values for bars with asterisks are 0.0008 and 0.0001, respectively. b RT-PCR (left panel) and qPCR (right panel) of a selected SYNGAP1 ASO (ASO-71) targeting the alternative 3′ss transfected in HEK293 cells at increasing concentrations for 24 h. Exact p -values for bars with asterisks are 0.0001 and 0.0003, 6.99e-5 and 4.25e-5, respectively. c RT-PCR (left panel) and qPCR (right panel) of a selected CD274 ASO (ASO-125) targeting the alternative intron transfected in Huh7 cells at increasing concentrations for 24 h. Cell were treated with 50 μg/mL of CHX for 3 h prior to harvesting to visualize and quantify the non-productive mRNA. Exact p -values for bars with asterisks are 6.38e-6 and 9.21e-5, respectively. d RT-PCR (left panel) and qPCR (right panel) of two selected SCN1A ASOs (ASO-135 and ASO-136) targeting the exon inclusion event delivered by free uptake into ReNCell VM cells at increasing concentrations for 72 h. RT-PCR results (bar graphs on the left) show dose-dependent reductions of the non-productive mRNA and qPCR results (bar graphs on the right) show dose-dependent increases of productive mRNA. Exact p-values for bars with asterisks are 1.69e-10, 2.50e-11, and 1.49e-11; 2.91e-8, 2.05e-10, and 2.59e-11; 4.21e-7, 1.10e-8, and 1.35e-10; and 7.26e-5, 4.01e-8, and 9.08e-12, respectively. No-ASO (−), scramble (SC), and mismatch (MM) controls were included in each experiment at the same increasing concentrations as the respective targeting ASOs. All experiments were performed in three biological replicates. Data are presented as mean values ±SD. P -values were calculated using two-sided t -test. Asterisks denote ASOs that are statistically significant ( p

    Journal: Nature Communications

    Article Title: Antisense oligonucleotide modulation of non-productive alternative splicing upregulates gene expression

    doi: 10.1038/s41467-020-17093-9

    Figure Lengend Snippet: ASOs dose-dependently reduce NMD and increase mRNA in vitro. a RT-PCR (left panel) and qPCR (right panel) of a selected PCCA ASO (ASO-29) targeting the exon inclusion event transfected in HEK293 cells at increasing concentrations for 24 h. Exact p -values for bars with asterisks are 0.0008 and 0.0001, respectively. b RT-PCR (left panel) and qPCR (right panel) of a selected SYNGAP1 ASO (ASO-71) targeting the alternative 3′ss transfected in HEK293 cells at increasing concentrations for 24 h. Exact p -values for bars with asterisks are 0.0001 and 0.0003, 6.99e-5 and 4.25e-5, respectively. c RT-PCR (left panel) and qPCR (right panel) of a selected CD274 ASO (ASO-125) targeting the alternative intron transfected in Huh7 cells at increasing concentrations for 24 h. Cell were treated with 50 μg/mL of CHX for 3 h prior to harvesting to visualize and quantify the non-productive mRNA. Exact p -values for bars with asterisks are 6.38e-6 and 9.21e-5, respectively. d RT-PCR (left panel) and qPCR (right panel) of two selected SCN1A ASOs (ASO-135 and ASO-136) targeting the exon inclusion event delivered by free uptake into ReNCell VM cells at increasing concentrations for 72 h. RT-PCR results (bar graphs on the left) show dose-dependent reductions of the non-productive mRNA and qPCR results (bar graphs on the right) show dose-dependent increases of productive mRNA. Exact p-values for bars with asterisks are 1.69e-10, 2.50e-11, and 1.49e-11; 2.91e-8, 2.05e-10, and 2.59e-11; 4.21e-7, 1.10e-8, and 1.35e-10; and 7.26e-5, 4.01e-8, and 9.08e-12, respectively. No-ASO (−), scramble (SC), and mismatch (MM) controls were included in each experiment at the same increasing concentrations as the respective targeting ASOs. All experiments were performed in three biological replicates. Data are presented as mean values ±SD. P -values were calculated using two-sided t -test. Asterisks denote ASOs that are statistically significant ( p

    Article Snippet: For RT-PCR analysis, cells were treated, as indicated, with 50 μg/mL of CHX (Cell Signaling Technology) in DMSO for 3 h 21 h post transfection.

    Techniques: In Vitro, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Allele-specific Oligonucleotide, Transfection

    CBP mediates acetylation and stabilization of FBXL19. A ) IP of proteins from lysates of veh (-), TSA-, or TSA+C646-treated FBXL19-V5 –transfected MLE12 cells with an anti-Ack antibody followed by immunoblotting (IB) analysis of the precipitates with an anti-V5 tag antibody. IB analysis of V5-tagged proteins and β-actin in input cell lysates was performed. B ) IP of proteins from lysates of FBXL19-V5 + Ubiquitin-HA –transfected MLE12 cells cotransfected with or without CBP shRNA with an anti-HA antibody followed by IB analysis of the precipitates with anti-V5 tag. IB analysis of CBP, V5-tagged proteins, and β-actin in input cell lysates was performed. C ) IP of proteins from lysates of FBXL19-V5 - or FBXL19 K141R -V5 –transfected cells with or without CBP-HA cotransfection with an anti-V5 tag antibody followed by IB analysis of precipitates with an anti–acetylated lysine antibody. IB analysis of V5-tagged proteins, HA-tagged proteins, and β-actin in input cell lysates was performed. D ) IB analysis of V5-tagged proteins, CBP, and β-actin in lysates of FBXL19-V5–overexpressing MLE12 cells transfected with or without CBP plasmid and then treated for 0–4 h with CHX and analysis of protein levels by densitometry of the results. *** P

    Journal: The FASEB Journal

    Article Title: Histone acetyltransferase CBP promotes function of SCF FBXL19 ubiquitin E3 ligase by acetylation and stabilization of its F-box protein subunit

    doi: 10.1096/fj.201701069R

    Figure Lengend Snippet: CBP mediates acetylation and stabilization of FBXL19. A ) IP of proteins from lysates of veh (-), TSA-, or TSA+C646-treated FBXL19-V5 –transfected MLE12 cells with an anti-Ack antibody followed by immunoblotting (IB) analysis of the precipitates with an anti-V5 tag antibody. IB analysis of V5-tagged proteins and β-actin in input cell lysates was performed. B ) IP of proteins from lysates of FBXL19-V5 + Ubiquitin-HA –transfected MLE12 cells cotransfected with or without CBP shRNA with an anti-HA antibody followed by IB analysis of the precipitates with anti-V5 tag. IB analysis of CBP, V5-tagged proteins, and β-actin in input cell lysates was performed. C ) IP of proteins from lysates of FBXL19-V5 - or FBXL19 K141R -V5 –transfected cells with or without CBP-HA cotransfection with an anti-V5 tag antibody followed by IB analysis of precipitates with an anti–acetylated lysine antibody. IB analysis of V5-tagged proteins, HA-tagged proteins, and β-actin in input cell lysates was performed. D ) IB analysis of V5-tagged proteins, CBP, and β-actin in lysates of FBXL19-V5–overexpressing MLE12 cells transfected with or without CBP plasmid and then treated for 0–4 h with CHX and analysis of protein levels by densitometry of the results. *** P

    Article Snippet: To examine whether FBXL19 is stable, MLE12 cells were treated with the de novo protein synthesis inhibitor cycloheximide (CHX) (20 µg/ml) for 0–6 h. CHX incubation reduced the amounts of pre-existing FBXL19 in a time-dependent manner , showing that the half-life of endogenous FBXL19 is ∼3 h. To determine which pathway is involved in FBXL19 degradation, MLE12 cells were exposed to a proteasomal inhibitor (MG-132, 20 µM) or to a lysosomal inhibitor (leupeptin, 100 µM) prior to CHX treatment.

    Techniques: Transfection, shRNA, Cotransfection, Plasmid Preparation

    FBXL19 and CBP inversely regulate Cdc42 levels. A ) Immunoblot (IB) analysis of Cdc42 and β-actin in lysates of MLE12 cells treated with CHX together with MG-132 or leupeptin. Intensities of Cdc42 were determined by densitometry of the results. *** P

    Journal: The FASEB Journal

    Article Title: Histone acetyltransferase CBP promotes function of SCF FBXL19 ubiquitin E3 ligase by acetylation and stabilization of its F-box protein subunit

    doi: 10.1096/fj.201701069R

    Figure Lengend Snippet: FBXL19 and CBP inversely regulate Cdc42 levels. A ) Immunoblot (IB) analysis of Cdc42 and β-actin in lysates of MLE12 cells treated with CHX together with MG-132 or leupeptin. Intensities of Cdc42 were determined by densitometry of the results. *** P

    Article Snippet: To examine whether FBXL19 is stable, MLE12 cells were treated with the de novo protein synthesis inhibitor cycloheximide (CHX) (20 µg/ml) for 0–6 h. CHX incubation reduced the amounts of pre-existing FBXL19 in a time-dependent manner , showing that the half-life of endogenous FBXL19 is ∼3 h. To determine which pathway is involved in FBXL19 degradation, MLE12 cells were exposed to a proteasomal inhibitor (MG-132, 20 µM) or to a lysosomal inhibitor (leupeptin, 100 µM) prior to CHX treatment.

    Techniques:

    FBXL19 is degraded in the ubiquitin-proteasome system. A ) Immunoblot analysis of FBXL19 and β-actin (loading control throughout) in lysates of MLE12 cells treated with CHX together with DMSO, MG-132, or leupeptin. Intensities of FBXL19 were determined by densitometry of the results. *** P

    Journal: The FASEB Journal

    Article Title: Histone acetyltransferase CBP promotes function of SCF FBXL19 ubiquitin E3 ligase by acetylation and stabilization of its F-box protein subunit

    doi: 10.1096/fj.201701069R

    Figure Lengend Snippet: FBXL19 is degraded in the ubiquitin-proteasome system. A ) Immunoblot analysis of FBXL19 and β-actin (loading control throughout) in lysates of MLE12 cells treated with CHX together with DMSO, MG-132, or leupeptin. Intensities of FBXL19 were determined by densitometry of the results. *** P

    Article Snippet: To examine whether FBXL19 is stable, MLE12 cells were treated with the de novo protein synthesis inhibitor cycloheximide (CHX) (20 µg/ml) for 0–6 h. CHX incubation reduced the amounts of pre-existing FBXL19 in a time-dependent manner , showing that the half-life of endogenous FBXL19 is ∼3 h. To determine which pathway is involved in FBXL19 degradation, MLE12 cells were exposed to a proteasomal inhibitor (MG-132, 20 µM) or to a lysosomal inhibitor (leupeptin, 100 µM) prior to CHX treatment.

    Techniques:

    CE-ERa Promotes Misfolded Proinsulin Degradation via HRD1-ERAD Stabilization (A) Diagram of misfolded protein degradation via ERAD including misfolded protein retro-translocation to the cytosol via E3 ubiquitin ligase HRD1 and its scaffold adaptor SEL1L followed by HRD1-mediated ubiquitination and proteasomal degradation. (B and C) Male Ins2 +/Akita cells were treated with CE and BZA (B) or PPT (C) for 24 hr, followed by exposure to Tg for 4 hr. Representative image of western blot for HRD1 expression and quantification (n = 4–7 distinct experiments/condition). (D) COS-7 cells were transfected with HA-WT proinsulin or HA-C 96 Y proinsulin along with ERa and FLAG-ubiquitin in the absence or presence of HRD-1. Cells were treated with CE for 24 hr, fol-lowed by exposure to Tg for 4 hr. Co-immuno-precipitations were carried out using anti-HA antibody followed by western blotting using anti-FLAG or anti-HA antibodies (left). Quantification of relative proinsulin ubiquitination (middle) and proinsulin stability (right) (n = 3 distinct experiments/ condition). (E) Male Ins2 +/Akita cells were treated like in (B), followed by qRT-PCR quantification of HRD1 mRNA (n = 8 experiments). (F–I) Male Ins2 +/Akita cells were treated with cycloheximide (CHX) plus Tg and either vehicle (F and G) or CE (H and I) in the presence (G and I) or in the absence (F and H) of MG132 for the indicated time course. Western blots for HRD1 were performed on cell lysates. Relative HRD1 protein levels were determined by densitometry and were normalized to that obtained at time 0 (n = 4–6 distinct experi-ments/condition). (J) Islets from female human donors were treated like in (B). Representative image of western blot for HRD1 expression and bar graph of quantification (n = 3). Data are shown as mean ± SEM. *p

    Journal: Cell reports

    Article Title: Estrogens Promote Misfolded Proinsulin Degradation to Protect Insulin Production and Delay Diabetes

    doi: 10.1016/j.celrep.2018.06.019

    Figure Lengend Snippet: CE-ERa Promotes Misfolded Proinsulin Degradation via HRD1-ERAD Stabilization (A) Diagram of misfolded protein degradation via ERAD including misfolded protein retro-translocation to the cytosol via E3 ubiquitin ligase HRD1 and its scaffold adaptor SEL1L followed by HRD1-mediated ubiquitination and proteasomal degradation. (B and C) Male Ins2 +/Akita cells were treated with CE and BZA (B) or PPT (C) for 24 hr, followed by exposure to Tg for 4 hr. Representative image of western blot for HRD1 expression and quantification (n = 4–7 distinct experiments/condition). (D) COS-7 cells were transfected with HA-WT proinsulin or HA-C 96 Y proinsulin along with ERa and FLAG-ubiquitin in the absence or presence of HRD-1. Cells were treated with CE for 24 hr, fol-lowed by exposure to Tg for 4 hr. Co-immuno-precipitations were carried out using anti-HA antibody followed by western blotting using anti-FLAG or anti-HA antibodies (left). Quantification of relative proinsulin ubiquitination (middle) and proinsulin stability (right) (n = 3 distinct experiments/ condition). (E) Male Ins2 +/Akita cells were treated like in (B), followed by qRT-PCR quantification of HRD1 mRNA (n = 8 experiments). (F–I) Male Ins2 +/Akita cells were treated with cycloheximide (CHX) plus Tg and either vehicle (F and G) or CE (H and I) in the presence (G and I) or in the absence (F and H) of MG132 for the indicated time course. Western blots for HRD1 were performed on cell lysates. Relative HRD1 protein levels were determined by densitometry and were normalized to that obtained at time 0 (n = 4–6 distinct experi-ments/condition). (J) Islets from female human donors were treated like in (B). Representative image of western blot for HRD1 expression and bar graph of quantification (n = 3). Data are shown as mean ± SEM. *p

    Article Snippet: To determine to what extent CE treatment stabilizes HRD1 protein, we studied HRD1 protein turnover in the presence of the protein synthesis inhibitor cycloheximide (CHX) to block de novo protein synthesis.

    Techniques: Translocation Assay, Western Blot, Expressing, Transfection, Quantitative RT-PCR

    CE-ERa Prevents HRD1 Degradation and ER Stress via SEL1L Stabilization (A and B) Male Ins2 +/Akita cells were treated with CE and BZA (A) or PPT (B) for 24 hr, followed by exposure to Tg for 4 hr. Representative image of western blot for SEL1L expression and bar graph of quantification (n = 4–8 distinct experiments/condition). (C and D) Male Ins2 +/Akita cells were transfected with mouse SEL1L-specific siRNA or with a control siRNA and were treated like in (A). Protein expression for SEL1L, HRD1 (C), and CHOP (D) were analyzed by western blotting and quantification (n = 4–6 experiments). (E) Male Ins2 +/Akita cells were treated like in (A) and qRT-PCR quantification of relative SEL1L mRNA was performed and represented as expression relative to vehicle+Tg. n = 8–9 distinct experiments/condition. (F–I) Male Ins2 +/Akita cells were treated with cycloheximide (CHX) plus Tg and vehicle (F and G) or CE (H and I) in the presence (G and I) or absence (F and H) of MG132 for the indicated time course. SEL1L protein was quantified by western blot and relative SEL1L protein levels were determined by densitometry and normalized to that obtained at time 0 (n = 4–6 distinct experiments/condition). (J) Islets from female human donors were treated like in (A). Representative image of western blot for SEL1L protein expression and bar graph of quantification (n = 3 experiments). Data are shown as mean ± SEM. *p

    Journal: Cell reports

    Article Title: Estrogens Promote Misfolded Proinsulin Degradation to Protect Insulin Production and Delay Diabetes

    doi: 10.1016/j.celrep.2018.06.019

    Figure Lengend Snippet: CE-ERa Prevents HRD1 Degradation and ER Stress via SEL1L Stabilization (A and B) Male Ins2 +/Akita cells were treated with CE and BZA (A) or PPT (B) for 24 hr, followed by exposure to Tg for 4 hr. Representative image of western blot for SEL1L expression and bar graph of quantification (n = 4–8 distinct experiments/condition). (C and D) Male Ins2 +/Akita cells were transfected with mouse SEL1L-specific siRNA or with a control siRNA and were treated like in (A). Protein expression for SEL1L, HRD1 (C), and CHOP (D) were analyzed by western blotting and quantification (n = 4–6 experiments). (E) Male Ins2 +/Akita cells were treated like in (A) and qRT-PCR quantification of relative SEL1L mRNA was performed and represented as expression relative to vehicle+Tg. n = 8–9 distinct experiments/condition. (F–I) Male Ins2 +/Akita cells were treated with cycloheximide (CHX) plus Tg and vehicle (F and G) or CE (H and I) in the presence (G and I) or absence (F and H) of MG132 for the indicated time course. SEL1L protein was quantified by western blot and relative SEL1L protein levels were determined by densitometry and normalized to that obtained at time 0 (n = 4–6 distinct experiments/condition). (J) Islets from female human donors were treated like in (A). Representative image of western blot for SEL1L protein expression and bar graph of quantification (n = 3 experiments). Data are shown as mean ± SEM. *p

    Article Snippet: To determine to what extent CE treatment stabilizes HRD1 protein, we studied HRD1 protein turnover in the presence of the protein synthesis inhibitor cycloheximide (CHX) to block de novo protein synthesis.

    Techniques: Western Blot, Expressing, Transfection, Quantitative RT-PCR

    CE-ERa Stabilizes ERAD in β Cells by Inhibiting UBC6e (A) Male Ins2 +/Akita cells were treated with the indicated compounds for 24 hr followed by exposure to Tg for 4 hr. qRT-PCR quantification of relative UBC6e mRNA levels is shown (n = 8 experiments/condition). (B) Female and male WT mouse islets were treated with BZA for 24 hr followed by exposure to Tg for 4 hr, and UBC6e mRNA level were quantified by qRT-PCR (n = 9–15 distinct experiments/condition). (C) Islets from female human donors were treated like in (A). Western blot for UBC6e protein expression and bar graph of quantification (n = 3 experiments). (D) Male Ins2 +/Akita cells were transfected with mouse UBC6e or control expression plasmids and treated like in (A). Western blot for UBC6e, SEL1L, and HRD1 protein expression (upper panel) and bar graph of quantification (lower panel) are shown (n = 4–10 distinct experiments/condition). (E) Male Ins2 +/Akita cells were transfected with mouse UBC6e or control plasmids and treated like in (A). Western blot for BiP protein (upper panel) and bar graph ofqRT-PCR quantification of spliced-XBP1 and ATF4 mRNAs (lower panel) (n = 6 distinct experiments/condition). (F) Cultured islets from female WT, NOER, MOER, and EAAE mice were treated like in (A), followed by qRT-PCR quantification of relative UBC6e mRNA levels (n = 6 distinct experiments/condition). (G) Dispersed islets from female WT, MOER, and NOER mice were treated like in (A). Images of IF staining for CHOP, insulin, and nuclei and quantification ofβ cells with CHOP+ nuclei relative to vehicle (n = 4–6 experiments). (H) Male Ins2 +/Akita cells were treated with the indicated compounds for 24 hr, followed by exposure to Tg for 4 hr. Protein expression (upper panel) and mRNA level (lower panel) of UBC6e (n = 6–10 distinct experiments/condition). (I) Male Ins 2+/Akita cells were treated with CHX plus Tg and either vehicle or EDC in the presence or in the absence of MG132 for the indicated time course. UBC6e protein was measured by western blot and relative UBC6e protein levels were determined by densitometry and were normalized to that obtained at time 0 (n = 3–6 experiments).

    Journal: Cell reports

    Article Title: Estrogens Promote Misfolded Proinsulin Degradation to Protect Insulin Production and Delay Diabetes

    doi: 10.1016/j.celrep.2018.06.019

    Figure Lengend Snippet: CE-ERa Stabilizes ERAD in β Cells by Inhibiting UBC6e (A) Male Ins2 +/Akita cells were treated with the indicated compounds for 24 hr followed by exposure to Tg for 4 hr. qRT-PCR quantification of relative UBC6e mRNA levels is shown (n = 8 experiments/condition). (B) Female and male WT mouse islets were treated with BZA for 24 hr followed by exposure to Tg for 4 hr, and UBC6e mRNA level were quantified by qRT-PCR (n = 9–15 distinct experiments/condition). (C) Islets from female human donors were treated like in (A). Western blot for UBC6e protein expression and bar graph of quantification (n = 3 experiments). (D) Male Ins2 +/Akita cells were transfected with mouse UBC6e or control expression plasmids and treated like in (A). Western blot for UBC6e, SEL1L, and HRD1 protein expression (upper panel) and bar graph of quantification (lower panel) are shown (n = 4–10 distinct experiments/condition). (E) Male Ins2 +/Akita cells were transfected with mouse UBC6e or control plasmids and treated like in (A). Western blot for BiP protein (upper panel) and bar graph ofqRT-PCR quantification of spliced-XBP1 and ATF4 mRNAs (lower panel) (n = 6 distinct experiments/condition). (F) Cultured islets from female WT, NOER, MOER, and EAAE mice were treated like in (A), followed by qRT-PCR quantification of relative UBC6e mRNA levels (n = 6 distinct experiments/condition). (G) Dispersed islets from female WT, MOER, and NOER mice were treated like in (A). Images of IF staining for CHOP, insulin, and nuclei and quantification ofβ cells with CHOP+ nuclei relative to vehicle (n = 4–6 experiments). (H) Male Ins2 +/Akita cells were treated with the indicated compounds for 24 hr, followed by exposure to Tg for 4 hr. Protein expression (upper panel) and mRNA level (lower panel) of UBC6e (n = 6–10 distinct experiments/condition). (I) Male Ins 2+/Akita cells were treated with CHX plus Tg and either vehicle or EDC in the presence or in the absence of MG132 for the indicated time course. UBC6e protein was measured by western blot and relative UBC6e protein levels were determined by densitometry and were normalized to that obtained at time 0 (n = 3–6 experiments).

    Article Snippet: To determine to what extent CE treatment stabilizes HRD1 protein, we studied HRD1 protein turnover in the presence of the protein synthesis inhibitor cycloheximide (CHX) to block de novo protein synthesis.

    Techniques: Quantitative RT-PCR, Western Blot, Expressing, Transfection, Polymerase Chain Reaction, Cell Culture, Mouse Assay, Staining

    PXK expression accelerates EGFR degradation. COS7 cells cultured on coverslips were transfected with the gene for FLAG-PXK. Cells showing FLAG fluorescence are indicated by asterisks at the nucleus, and the cellular edges are traced by broken lines. (A) Thirty-six hours after transfection, cells were serum starved for 12 h, followed by preincubation with 20 μg/ml CHX and then left unstimulated (none) or stimulated with 100 nM EGF for 5 or 30 min. Cells were then fixed and treated with mouse anti-FLAG antibody and rabbit anti-EGFR antibody, followed by visualization with Alexa Fluor 488-conjugated anti-mouse antibody (green) and Alexa Fluor 594-conjugated anti-rabbit antibody (red), respectively. (B) Cells were preincubated with 10 μM MG-132 or 0.25 μM bafilomycin A1 in the presence of 20 μg/ml CHX for 30 min and then stimulated with 100 nM EGF for 30 min, followed by treatment as described above. Bars, 10 μm.

    Journal: Molecular and Cellular Biology

    Article Title: Characterization of PXK as a Protein Involved in Epidermal Growth Factor Receptor Trafficking ▿

    doi: 10.1128/MCB.01105-09

    Figure Lengend Snippet: PXK expression accelerates EGFR degradation. COS7 cells cultured on coverslips were transfected with the gene for FLAG-PXK. Cells showing FLAG fluorescence are indicated by asterisks at the nucleus, and the cellular edges are traced by broken lines. (A) Thirty-six hours after transfection, cells were serum starved for 12 h, followed by preincubation with 20 μg/ml CHX and then left unstimulated (none) or stimulated with 100 nM EGF for 5 or 30 min. Cells were then fixed and treated with mouse anti-FLAG antibody and rabbit anti-EGFR antibody, followed by visualization with Alexa Fluor 488-conjugated anti-mouse antibody (green) and Alexa Fluor 594-conjugated anti-rabbit antibody (red), respectively. (B) Cells were preincubated with 10 μM MG-132 or 0.25 μM bafilomycin A1 in the presence of 20 μg/ml CHX for 30 min and then stimulated with 100 nM EGF for 30 min, followed by treatment as described above. Bars, 10 μm.

    Article Snippet: Bafilomycin A1 and cycloheximide (CHX) were from Wako Pure Chemical Industries (Osaka, Japan).

    Techniques: Expressing, Cell Culture, Transfection, Fluorescence

    Characterization of the degradability of BiFP-modc C-terminal fusion proteins . ( A ) Schematic representation of destabilized BiFP constructs with zero to three copies of MODC PEST domain at the C-terminus of BiFP. The fusion constructs were generated and cloned in the pSEH retroviral vector, yielding pSEH-BiFP, pSEH-BiFP-modc, pSEH-BiFP-2modc, and pSEH-BiFP-3modc, respectively. ( B ) BiFP fusion constructs were used for retrovirus packaging and generating stable lines in HCT116 cells after hygromycin selection. Subconfluent stable lines were treated with CHX (100 μg/ml). Fluorescence signals were recorded at the indicated time points. Representative images are shown. ( C ) Quantitative analysis of the fluorescence signal for the CHX-treated stable lines shown in ( B ). “*” p

    Journal: Genes & Diseases

    Article Title: The development of a sensitive fluorescent protein-based transcript reporter for high throughput screening of negative modulators of lncRNAs

    doi: 10.1016/j.gendis.2018.02.001

    Figure Lengend Snippet: Characterization of the degradability of BiFP-modc C-terminal fusion proteins . ( A ) Schematic representation of destabilized BiFP constructs with zero to three copies of MODC PEST domain at the C-terminus of BiFP. The fusion constructs were generated and cloned in the pSEH retroviral vector, yielding pSEH-BiFP, pSEH-BiFP-modc, pSEH-BiFP-2modc, and pSEH-BiFP-3modc, respectively. ( B ) BiFP fusion constructs were used for retrovirus packaging and generating stable lines in HCT116 cells after hygromycin selection. Subconfluent stable lines were treated with CHX (100 μg/ml). Fluorescence signals were recorded at the indicated time points. Representative images are shown. ( C ) Quantitative analysis of the fluorescence signal for the CHX-treated stable lines shown in ( B ). “*” p

    Article Snippet: 293pTP and RAPA cells derived from HEK-293 cells as previously characterized., All cell lines were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gemini Bio-Products, West Sacramento, CA), containing 100 U/ml penicillin and 100 μg/ml streptomycin, at 37 °C in 5% CO2 as described., , Cycloheximide (CHX) and hygromycin B were purchased from Cayman Chemical (Ann Arbor, MI).

    Techniques: Construct, Generated, Clone Assay, Plasmid Preparation, Selection, Fluorescence

    Fusion proteins of eGFP and Gaussia luciferase (GLuc) with multiple copies of the MODC PEST domain exhibit varied instabilities . ( A ) Schematic representation of the construction of eGFP-3modc C-terminal fusion and its control eGFP vectors. Both constructs were generated on the basis of our homemade pSEH retroviral vector, yielding pSEH-eGFP and pSEH-eGFP-3modc. LTR, long terminal repeat for MSCV retrovirus; Hygro, hygromycin B resistance gene; hEFH, a hybrid promoter consisting of human EF1α promoter and HIV enhancer; eGFP, enhanced green fluorescent protein; stop, stop codon; 3modc, three copies of the PEST-containing degradation domain of mouse ornithine decarboxylase. ( B ) The fluorescence signal of the eGFP-3modc fusion after cycloheximide (CHX) treatment. The retroviral vectors were used to make stable cell lines of HCT116 cells after hygromycin selection. The stable lines were seeded at subconfluency and treated with cycloheximide (100 μg/ml). Fluorescence signals were recorded at the indicated time points. Representative images are shown. ( C ) Quantitative analysis of the fluorescence intensity in the samples prepared in (B). “*” p

    Journal: Genes & Diseases

    Article Title: The development of a sensitive fluorescent protein-based transcript reporter for high throughput screening of negative modulators of lncRNAs

    doi: 10.1016/j.gendis.2018.02.001

    Figure Lengend Snippet: Fusion proteins of eGFP and Gaussia luciferase (GLuc) with multiple copies of the MODC PEST domain exhibit varied instabilities . ( A ) Schematic representation of the construction of eGFP-3modc C-terminal fusion and its control eGFP vectors. Both constructs were generated on the basis of our homemade pSEH retroviral vector, yielding pSEH-eGFP and pSEH-eGFP-3modc. LTR, long terminal repeat for MSCV retrovirus; Hygro, hygromycin B resistance gene; hEFH, a hybrid promoter consisting of human EF1α promoter and HIV enhancer; eGFP, enhanced green fluorescent protein; stop, stop codon; 3modc, three copies of the PEST-containing degradation domain of mouse ornithine decarboxylase. ( B ) The fluorescence signal of the eGFP-3modc fusion after cycloheximide (CHX) treatment. The retroviral vectors were used to make stable cell lines of HCT116 cells after hygromycin selection. The stable lines were seeded at subconfluency and treated with cycloheximide (100 μg/ml). Fluorescence signals were recorded at the indicated time points. Representative images are shown. ( C ) Quantitative analysis of the fluorescence intensity in the samples prepared in (B). “*” p

    Article Snippet: 293pTP and RAPA cells derived from HEK-293 cells as previously characterized., All cell lines were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gemini Bio-Products, West Sacramento, CA), containing 100 U/ml penicillin and 100 μg/ml streptomycin, at 37 °C in 5% CO2 as described., , Cycloheximide (CHX) and hygromycin B were purchased from Cayman Chemical (Ann Arbor, MI).

    Techniques: Luciferase, Construct, Generated, Plasmid Preparation, Fluorescence, Stable Transfection, Selection

    HDAC6 mediates the deacetylation of PA at Lys(664). (A) Schematic diagram of PA modification. NLS, nuclear localization signal. (B) 293T cells were transfected with Flag-PA or its acetylation dead mutants as indicated and then treated with tubacin (10 μM) for 17 h. Flag antibody was used to immunoprecipitate the wild type or acetylation dead mutants of Flag-PA, which were then analyzed by immunoblotting with the indicated antibodies. WCL, whole-cell lysates. (C) 293T cells were transfected with the indicated plasmids, followed by tubacin or DMSO treatment for 17 h. The cells were then treated with CHX (10 μg/ml) at the indicated time points. PA and actin were detected by immunoblotting with the indicated antibodies. (D) Calculated relative half-lives of PA, PA-K664R, and PA-K664Q, using the data from panel C. The percent intensity (log 10 ) was plotted versus time.

    Journal: Journal of Virology

    Article Title: HDAC6 Restricts Influenza A Virus by Deacetylation of the RNA Polymerase PA Subunit

    doi: 10.1128/JVI.01896-18

    Figure Lengend Snippet: HDAC6 mediates the deacetylation of PA at Lys(664). (A) Schematic diagram of PA modification. NLS, nuclear localization signal. (B) 293T cells were transfected with Flag-PA or its acetylation dead mutants as indicated and then treated with tubacin (10 μM) for 17 h. Flag antibody was used to immunoprecipitate the wild type or acetylation dead mutants of Flag-PA, which were then analyzed by immunoblotting with the indicated antibodies. WCL, whole-cell lysates. (C) 293T cells were transfected with the indicated plasmids, followed by tubacin or DMSO treatment for 17 h. The cells were then treated with CHX (10 μg/ml) at the indicated time points. PA and actin were detected by immunoblotting with the indicated antibodies. (D) Calculated relative half-lives of PA, PA-K664R, and PA-K664Q, using the data from panel C. The percent intensity (log 10 ) was plotted versus time.

    Article Snippet: Tubacin, TSA, and cycloheximide (CHX) were purchased from Selleck.

    Techniques: Modification, Transfection

    TNFα increased basal lipolysis and degraded PLIN1, which requires Cathepsin B a 3T3-L1 adipocytes were individually treated with 10 ng/ml and 50 ng/ml TNFα for 3 h, then media was removed for glycerol measurement. b 3T3-L1 adipocytes were stimulated with 10 ng/ml TNFα for 3 h with or without 1 h CA074 pre-treatment, then media was removed for glycerol measurement. c–d C57BL/6 mice were fed a HFD or standard chow for 24 weeks, then expression levels of PLIN1 in EAT were determined by qRT-PCR c and western blot d ( n = 6). e 3T3-L1 adipocytes were treated with the proteasome inhibitor MG-132 (10 μ m ) or the lysosomal protease inhibitor leupeptin (Leu; 10 μg/ml) for 24 h, then the content of PLIN1 were assessed by western blot. f PLIN1 stability in response to TNFα (10 ng/ml) was evaluated in a cycloheximide (CHX, 100 μg/ml) chasing experiment. Immunoblot of perilipin after 6, 12, 18, and 24 h treatments with CHX in adipocytes. g Adipocytes were stimulated with TNFα (10 ng/ml) in the presence or absence of CA074 treatment (10 µ m ). Immunoblotting of PLIN1 was perform after treatment with CHX for 6, 12, 18, and 24 h. ** p

    Journal: Cell Death & Disease

    Article Title: Obesity-associated inflammation triggers an autophagy–lysosomal response in adipocytes and causes degradation of perilipin 1

    doi: 10.1038/s41419-019-1393-8

    Figure Lengend Snippet: TNFα increased basal lipolysis and degraded PLIN1, which requires Cathepsin B a 3T3-L1 adipocytes were individually treated with 10 ng/ml and 50 ng/ml TNFα for 3 h, then media was removed for glycerol measurement. b 3T3-L1 adipocytes were stimulated with 10 ng/ml TNFα for 3 h with or without 1 h CA074 pre-treatment, then media was removed for glycerol measurement. c–d C57BL/6 mice were fed a HFD or standard chow for 24 weeks, then expression levels of PLIN1 in EAT were determined by qRT-PCR c and western blot d ( n = 6). e 3T3-L1 adipocytes were treated with the proteasome inhibitor MG-132 (10 μ m ) or the lysosomal protease inhibitor leupeptin (Leu; 10 μg/ml) for 24 h, then the content of PLIN1 were assessed by western blot. f PLIN1 stability in response to TNFα (10 ng/ml) was evaluated in a cycloheximide (CHX, 100 μg/ml) chasing experiment. Immunoblot of perilipin after 6, 12, 18, and 24 h treatments with CHX in adipocytes. g Adipocytes were stimulated with TNFα (10 ng/ml) in the presence or absence of CA074 treatment (10 µ m ). Immunoblotting of PLIN1 was perform after treatment with CHX for 6, 12, 18, and 24 h. ** p

    Article Snippet: To determine the stability of PLIN1 in 3T3-L1 adipocytes, on day 8 after induction, cells were treated with cycloheximide (CHX) (Amresco, 94271) and harvested at different time points after the addition of CHX.

    Techniques: Mouse Assay, Expressing, Quantitative RT-PCR, Western Blot, Protease Inhibitor

    Intracellular dynamics of M16/O16 chimeric protein in sec12 cells. YMO59 cells (A) and YMO60 cells harboring pRS316-M16/O16-GFP (B) were treated with 100 μg/ml CHX for 30 min at 23°C, incubated at 37.5°C for 40 min, and then observed.

    Journal:

    Article Title: The Cytoplasmic Region of ?-1,6-Mannosyltransferase Mnn9p Is Crucial for Retrograde Transport from the Golgi Apparatus to the Endoplasmic Reticulum in Saccharomyces cerevisiae ▿ ▿ †

    doi: 10.1128/EC.00333-07

    Figure Lengend Snippet: Intracellular dynamics of M16/O16 chimeric protein in sec12 cells. YMO59 cells (A) and YMO60 cells harboring pRS316-M16/O16-GFP (B) were treated with 100 μg/ml CHX for 30 min at 23°C, incubated at 37.5°C for 40 min, and then observed.

    Article Snippet: The sec12-4 mutant cells were grown to exponential phase at 23°C, 100 μg/ml cycloheximide (CHX) (Nacalai Tesque, Kyoto, Japan) was added, and the culture was incubated at 23°C for a further 30 min. Then, the cells were incubated at 37.5°C for 40 min.

    Techniques: Incubation

    Intracellular dynamics of mutant mnn9(R9/11A) protein in sec12 cells. (A) YMO69 cells were incubated at 23°C in SC−Ura medium and treated with 100 μg/ml CHX for 30 min. mnn9(R9/11A)-mRFP was observed 0 or 40 min after being shifted

    Journal:

    Article Title: The Cytoplasmic Region of ?-1,6-Mannosyltransferase Mnn9p Is Crucial for Retrograde Transport from the Golgi Apparatus to the Endoplasmic Reticulum in Saccharomyces cerevisiae ▿ ▿ †

    doi: 10.1128/EC.00333-07

    Figure Lengend Snippet: Intracellular dynamics of mutant mnn9(R9/11A) protein in sec12 cells. (A) YMO69 cells were incubated at 23°C in SC−Ura medium and treated with 100 μg/ml CHX for 30 min. mnn9(R9/11A)-mRFP was observed 0 or 40 min after being shifted

    Article Snippet: The sec12-4 mutant cells were grown to exponential phase at 23°C, 100 μg/ml cycloheximide (CHX) (Nacalai Tesque, Kyoto, Japan) was added, and the culture was incubated at 23°C for a further 30 min. Then, the cells were incubated at 37.5°C for 40 min.

    Techniques: Mutagenesis, Incubation