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  • chx  (Valiant)
    96
    Valiant chx
    Chx, supplied by Valiant, used in various techniques. Bioz Stars score: 96/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/chx/product/Valiant
    Average 96 stars, based on 14 article reviews
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    99
    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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    93
    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 chx
    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 <t>cyclohexamide</t> <t>(CHX)</t> 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.
    Chx, supplied by Novoprotein, used in various techniques. Bioz Stars score: 92/100, based on 875 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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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    90
    Cell Signaling Technology Inc chx
    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 <t>SYNGAP1</t> 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: 90/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology cyclohexamide
    Examination of mRNA and protein stability of mutant and wild type Na + /H + exchangers. A. mRNA levels of wild type and mutant NHE1 proteins 8 hours after treatment with 1.25 μM actinomycin D. mRNA levels were measured by quantitative RT-PCR. Levels are relative to the initial starting mRNA level prior to treatment and were corrected by the level of GAPDH. Results are mean +/- SE of at least 3 experiments. Individual experiments had 8 replicates. B. Protein levels of wild type and mutant NHE1 measured at time 0, (starting time) and up to 8 hours after <t>cyclohexamide</t> (50 μM) addition. Equal amounts of total proteins were loaded in each lane. NHE1 levels were determined by western blotting against the HA tag on the protein. Quantification was via were estimated using Image J 1.35 software. Insets are example western blots showing NHE1 levels (upper panel) in comparison to tubulin levels. Full sized NHE1 protein is indicated with an hour tubulin is indicated with an asterisk. Time points at 0 hr, 2, 4 and 8 are indicated. Results are mean +/- SE of at least 3 experiments.
    Cyclohexamide, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 39 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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    92
    FUJIFILM chx
    <t>RRM2</t> regulates RNA polymerase NS5B protein stability. A , quantification of NS5B mRNA levels relative to those of GAPDH in HuH-7 lenti-NS5B cells treated with RRM2 or control siRNA or mock-treated ( Non ) cells for 72 h. Data are shown as an average value of three independent experiments. Vertical bars , S.D. ( n = 3). B , HuH-7 lenti-NS5B cells were transfected with RRM2 or control siRNA for 48 h and then treated with <t>CHX</t> (100 μg/ml) and puromycin ( Puro ; 50 μg/ml) for 6 h. Cells were harvested and analyzed via Western blotting using anti-NS5B, anti-RRM2, and anti-actin antibodies. C , lenti-NS5B–expressing HuH-7 cells were transfected with the pcDNA6-myc-His-RRM2 vector. An empty vector, pcDNA6-myc-His, was used as a negative control. At 48 h post-transfection, cells were treated with CHX (100 μg/ml) for the indicated times. The cells were harvested and analyzed via Western blotting for NS5B, RRM2, and β-actin as reported in B . Blots shown are representative of three independent experiments ( n = 3).
    Chx, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 92/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    chx  (Tocris)
    98
    Tocris chx
    <t>RRM2</t> regulates RNA polymerase NS5B protein stability. A , quantification of NS5B mRNA levels relative to those of GAPDH in HuH-7 lenti-NS5B cells treated with RRM2 or control siRNA or mock-treated ( Non ) cells for 72 h. Data are shown as an average value of three independent experiments. Vertical bars , S.D. ( n = 3). B , HuH-7 lenti-NS5B cells were transfected with RRM2 or control siRNA for 48 h and then treated with <t>CHX</t> (100 μg/ml) and puromycin ( Puro ; 50 μg/ml) for 6 h. Cells were harvested and analyzed via Western blotting using anti-NS5B, anti-RRM2, and anti-actin antibodies. C , lenti-NS5B–expressing HuH-7 cells were transfected with the pcDNA6-myc-His-RRM2 vector. An empty vector, pcDNA6-myc-His, was used as a negative control. At 48 h post-transfection, cells were treated with CHX (100 μg/ml) for the indicated times. The cells were harvested and analyzed via Western blotting for NS5B, RRM2, and β-actin as reported in B . Blots shown are representative of three independent experiments ( n = 3).
    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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    90
    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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    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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    chx  (Amresco)
    92
    Amresco chx
    <t>EGF</t> stabilizes c-Fos by dissociating c-Fos from KDM2B/FBXL10 (A) EGF induces c-Fos protein accumulation in a time-dependent manner. HEK293 cells stably expressing FLAG-tagged c-Fos were deprived of serum for 8 h, and then treated with 50 μg/L EGF for the indicated length of time. The protein and mRNA levels of FLAG-c-Fos were determined by western blot and qRT-PCR, respectively, and normalized against β-actin. Error bars represent ±SD for triplicate experiments. (B) EGF stabilizes c-Fos. HEK293 cells stably expressing FLAG-tagged c-Fos were pretreated with or without EGF (50 μg/L) for 30 min, followed by <t>CHX</t> treatment (20 mg/L) for the indicated time. The protein levels of c-Fos were determined by western blot and normalized against β-actin. Error bars represent ±SD for triplicate experiments. (C) EGF-induced c-Fos accumulation is blocked by MEK1/2 inhibitor U0126 and an ERK1/2 inhibitor (SCH772984). HEK293 cells stably expressing FLAG-c-Fos were deprived of serum for 8 h, followed by treatment with U0126 (2 μM) or SCH772984 (0.2 or 1 μM) for 5 min. EGF (50 μg/L) was then added for additional 30 min. The protein levels of c-Fos were determined by western blot and normalized against β-actin. (D) EGF-induced c-Fos accumulation is not affected by inhibition of protein synthesis. HEK293 cells stably expressing FLAG-tagged c-Fos were treated with CHX (20 mg/L) for 5 min or 10 min. Solvent or EGF (50 μg/L) was then added for further treatment for 30 min. The protein levels of FLAG-c-Fos were determined by western blot and normalized against β-actin. Data are shown as relative fold change over protein levels in cells without EGF treatment. The arrow represents highly phosphorylated c-Fos (E) Knock down of KDM2B prolongs the high level of c-Fos protein following EGF stimulation. HeLa cells with stable knock down of KDM2B were deprived of serum for 8 h, followed by treatment with EGF (50 μg/L) for the indicated length of time. The protein levels of c-Fos were determined by western blot and normalized against β-actin. Data are shown as relative fold change over cells without EGF treatment (right panel). (F) EGF treatment induces c-Fos S374 phosphorylation and concomitantly reduces the interaction between c-Fos and KDM2B. HEK293 cells were transfected with plasmids expressing indicated proteins and then treated with EGF (50 μg/L) for the indicated length of time. The interactions between c-Fos and KDM2B were determined by Co-IP. (G) The reduction of c-Fos and KDM2B interaction by EGF is blocked by U0126. FLAG-KDM2B and 3HA-c-Fos were co-transfected into HEK293 cells. Cells were pretreated with U0126 (2 μM) for 5 min and then treated with EGF (50 μg/L) for another 30 min. The interactions between c-Fos and KDM2B were determined by Co-IP.
    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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    88
    Merck KGaA cycloheximid
    LAP*/LAP protein levels are stabilised in models of monocytic differentiation. (A) THP-1 monocytic cells were cultivated in the absence (left, 24 h) or presence of differentiating stimuli (PMA: 24 h; VitD3: 72 h). Then, cells were preincubated for 0.5 h with 10 μg/ml <t>CHX</t> before LAP*/LAP protein levels were analysed at the indicated time points (n = 3). To visualize the basically low LAP*/LAP levels in untreated cells, a prolonged exposure time and a more sensitive HRP substrate were applied (5 min Femto Plus vs . 1 min ECL). (B) Densitometric analysis of the experiment shown in A is depicted. The LAP*/LAP levels in THP-1 cells cultivated for 24 h in the absence or presence of PMA were defined as the respective 100% control. (C) THP-1 cells were incubated with PMA for 24 h. Following a 0.5 h treatment of cells with CHX ± RSK-I, the LAP*/LAP protein levels were analysed at the indicated time points (n = 3). (D) MM-6 cells were cultivated ± PMA for 24 h, and following addition of CHX, the LAP*/LAP levels were analysed at the indicated time points (n = 3). (E) Primary human monocytes were differentiated for 3 d (3 d diff.) and then preincubated for 0.5 h with CHX, before LAP*/LAP protein levels were analysed at the indicated time points (n = 3).
    Cycloheximid, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    chx  (Abcam)
    98
    Abcam chx
    LAP*/LAP protein levels are stabilised in models of monocytic differentiation. (A) THP-1 monocytic cells were cultivated in the absence (left, 24 h) or presence of differentiating stimuli (PMA: 24 h; VitD3: 72 h). Then, cells were preincubated for 0.5 h with 10 μg/ml <t>CHX</t> before LAP*/LAP protein levels were analysed at the indicated time points (n = 3). To visualize the basically low LAP*/LAP levels in untreated cells, a prolonged exposure time and a more sensitive HRP substrate were applied (5 min Femto Plus vs . 1 min ECL). (B) Densitometric analysis of the experiment shown in A is depicted. The LAP*/LAP levels in THP-1 cells cultivated for 24 h in the absence or presence of PMA were defined as the respective 100% control. (C) THP-1 cells were incubated with PMA for 24 h. Following a 0.5 h treatment of cells with CHX ± RSK-I, the LAP*/LAP protein levels were analysed at the indicated time points (n = 3). (D) MM-6 cells were cultivated ± PMA for 24 h, and following addition of CHX, the LAP*/LAP levels were analysed at the indicated time points (n = 3). (E) Primary human monocytes were differentiated for 3 d (3 d diff.) and then preincubated for 0.5 h with CHX, before LAP*/LAP protein levels were analysed at the indicated time points (n = 3).
    Chx, supplied by Abcam, used in various techniques. Bioz Stars score: 98/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Beyotime chx
    LAP*/LAP protein levels are stabilised in models of monocytic differentiation. (A) THP-1 monocytic cells were cultivated in the absence (left, 24 h) or presence of differentiating stimuli (PMA: 24 h; VitD3: 72 h). Then, cells were preincubated for 0.5 h with 10 μg/ml <t>CHX</t> before LAP*/LAP protein levels were analysed at the indicated time points (n = 3). To visualize the basically low LAP*/LAP levels in untreated cells, a prolonged exposure time and a more sensitive HRP substrate were applied (5 min Femto Plus vs . 1 min ECL). (B) Densitometric analysis of the experiment shown in A is depicted. The LAP*/LAP levels in THP-1 cells cultivated for 24 h in the absence or presence of PMA were defined as the respective 100% control. (C) THP-1 cells were incubated with PMA for 24 h. Following a 0.5 h treatment of cells with CHX ± RSK-I, the LAP*/LAP protein levels were analysed at the indicated time points (n = 3). (D) MM-6 cells were cultivated ± PMA for 24 h, and following addition of CHX, the LAP*/LAP levels were analysed at the indicated time points (n = 3). (E) Primary human monocytes were differentiated for 3 d (3 d diff.) and then preincubated for 0.5 h with CHX, before LAP*/LAP protein levels were analysed at the indicated time points (n = 3).
    Chx, supplied by Beyotime, used in various techniques. Bioz Stars score: 94/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    chx  (Nacalai)
    92
    Nacalai chx
    LAP*/LAP protein levels are stabilised in models of monocytic differentiation. (A) THP-1 monocytic cells were cultivated in the absence (left, 24 h) or presence of differentiating stimuli (PMA: 24 h; VitD3: 72 h). Then, cells were preincubated for 0.5 h with 10 μg/ml <t>CHX</t> before LAP*/LAP protein levels were analysed at the indicated time points (n = 3). To visualize the basically low LAP*/LAP levels in untreated cells, a prolonged exposure time and a more sensitive HRP substrate were applied (5 min Femto Plus vs . 1 min ECL). (B) Densitometric analysis of the experiment shown in A is depicted. The LAP*/LAP levels in THP-1 cells cultivated for 24 h in the absence or presence of PMA were defined as the respective 100% control. (C) THP-1 cells were incubated with PMA for 24 h. Following a 0.5 h treatment of cells with CHX ± RSK-I, the LAP*/LAP protein levels were analysed at the indicated time points (n = 3). (D) MM-6 cells were cultivated ± PMA for 24 h, and following addition of CHX, the LAP*/LAP levels were analysed at the indicated time points (n = 3). (E) Primary human monocytes were differentiated for 3 d (3 d diff.) and then preincubated for 0.5 h with CHX, before LAP*/LAP protein levels were analysed at the indicated time points (n = 3).
    Chx, supplied by Nacalai, used in various techniques. Bioz Stars score: 92/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    FUJIFILM cycloheximide chx
    H 2 promotes β-catenin degradation in L cells. (a) Cells were treated with control CM (Cont.), Wnt3a CM, 30 mM <t>LiCl,</t> or 2 μM BIO with 10% H 2 or 10% N 2 gas for 24 h. Expression of Ctnnb1 encoding β-catenin was quantified by qRT-PCR ( n = 3). (b) Cells transfected with myc-β-catenin (XE28 XBC plasmid) were exposed to a combination of 10 μg/ml cycloheximide <t>(CHX)</t> and 2 μM BIO with 10% H 2 or 10% N 2 gas for indicated periods of time. Representative Western blots are shown with densitometry of myc-β-catenin/β-actin ( n = 4). Two groups were not statistically different by two-way repeated measures ANOVA. * P
    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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    92
    Enzo Biochem cyclohexamide
    ( A – D ) Examining the effect of proteasome subunit knockdown in different cell lines. 80 shRNAs targeting 20 different proteasome subunits and control hairpins were expressed in HepG2 ( A ), H838 ( B ), T47D ( C ), and H1792 ( D ) cells by viral transduction. Each subunit was targeted by 4 different shRNAs and the cell viability was measured as the relative cell number compared to the average of non-targeting shRNAs 5 days after the initial introduction of the shRNAs. ( E , F ) HepG2 cells with shRNAs targeting PSMC5, PSMD2, and green fluorescent protein (GFP) were grown out. Their relative growth was analyzed in the absence of bortezomib ( E ) and their protein content was analyzed 24 hr after the addition of either 8 or 12 nM of bortezomib ( F ). ( G ) The relative cell number of cells harboring a control shLacZ (black) or each of 5 individual shRNAs targeting shPSMC5 (Cayenne) was analyzed 4 days after addition of the indicated concentrations of bortezomib. ( H ) HepG2 cells stably expressing shRNAs targeting the PSMC5 subunit and a control shRNA (lacZ) were analyzed by Western blot for the indicated proteins 24 hr with or without bortezomib treatment. ( I – N ) The HepG2 cells with shRNAs targeting PSMC5, PSMD2, and GFP (described above) were further exposed to a short panel of stress inducers including bortezomib ( I ), tunicamycin ( J ), rohinitib-RHT ( K ), Hsp90 inhibition ( L ), withaferin A ( M ), and <t>cyclohexamide</t> ( N ) at indicated concentrations and the relative cell number (RFU) was examined after 4 days. The graphs represent the average of at least 4 different measurements and the SEM. DOI: http://dx.doi.org/10.7554/eLife.08467.006
    Cyclohexamide, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    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

    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

    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

    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: Treatment with CHX, cell culture, and transfectionsTo determine the abundance of the non-productive mRNAs, cells (HEK293: PCCA, SYNGAP1; Huh7: CD274; ReNCell VM: SCN1A) were incubated with 50 μg/ml of CHX (Cell Signaling Technology) dissolved in DMSO for 3 h. For PCCA, HEK293 cells were grown in EMEM with 10% FBS and 1 × 105 cells were seeded in 24-well plate and reverse-transfected with 80 nM ASOs for initial screening or 1, 5, and 25 nM of selected ASO using Lipofectamine RNAiMax reagent (Invitrogen) according to manufacturer’s instructions.

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

    Examination of mRNA and protein stability of mutant and wild type Na + /H + exchangers. A. mRNA levels of wild type and mutant NHE1 proteins 8 hours after treatment with 1.25 μM actinomycin D. mRNA levels were measured by quantitative RT-PCR. Levels are relative to the initial starting mRNA level prior to treatment and were corrected by the level of GAPDH. Results are mean +/- SE of at least 3 experiments. Individual experiments had 8 replicates. B. Protein levels of wild type and mutant NHE1 measured at time 0, (starting time) and up to 8 hours after cyclohexamide (50 μM) addition. Equal amounts of total proteins were loaded in each lane. NHE1 levels were determined by western blotting against the HA tag on the protein. Quantification was via were estimated using Image J 1.35 software. Insets are example western blots showing NHE1 levels (upper panel) in comparison to tubulin levels. Full sized NHE1 protein is indicated with an hour tubulin is indicated with an asterisk. Time points at 0 hr, 2, 4 and 8 are indicated. Results are mean +/- SE of at least 3 experiments.

    Journal: PLoS ONE

    Article Title: Stop Codon Polymorphisms in the Human SLC9A1 Gene Disrupt or Compromise Na+/H+ Exchanger Function

    doi: 10.1371/journal.pone.0162902

    Figure Lengend Snippet: Examination of mRNA and protein stability of mutant and wild type Na + /H + exchangers. A. mRNA levels of wild type and mutant NHE1 proteins 8 hours after treatment with 1.25 μM actinomycin D. mRNA levels were measured by quantitative RT-PCR. Levels are relative to the initial starting mRNA level prior to treatment and were corrected by the level of GAPDH. Results are mean +/- SE of at least 3 experiments. Individual experiments had 8 replicates. B. Protein levels of wild type and mutant NHE1 measured at time 0, (starting time) and up to 8 hours after cyclohexamide (50 μM) addition. Equal amounts of total proteins were loaded in each lane. NHE1 levels were determined by western blotting against the HA tag on the protein. Quantification was via were estimated using Image J 1.35 software. Insets are example western blots showing NHE1 levels (upper panel) in comparison to tubulin levels. Full sized NHE1 protein is indicated with an hour tubulin is indicated with an asterisk. Time points at 0 hr, 2, 4 and 8 are indicated. Results are mean +/- SE of at least 3 experiments.

    Article Snippet: Cyclohexamide was Santa Cruz Biotechnology Inc, (Dallas, TX, USA).

    Techniques: Mutagenesis, Quantitative RT-PCR, Western Blot, Software

    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

    RRM2 regulates RNA polymerase NS5B protein stability. A , quantification of NS5B mRNA levels relative to those of GAPDH in HuH-7 lenti-NS5B cells treated with RRM2 or control siRNA or mock-treated ( Non ) cells for 72 h. Data are shown as an average value of three independent experiments. Vertical bars , S.D. ( n = 3). B , HuH-7 lenti-NS5B cells were transfected with RRM2 or control siRNA for 48 h and then treated with CHX (100 μg/ml) and puromycin ( Puro ; 50 μg/ml) for 6 h. Cells were harvested and analyzed via Western blotting using anti-NS5B, anti-RRM2, and anti-actin antibodies. C , lenti-NS5B–expressing HuH-7 cells were transfected with the pcDNA6-myc-His-RRM2 vector. An empty vector, pcDNA6-myc-His, was used as a negative control. At 48 h post-transfection, cells were treated with CHX (100 μg/ml) for the indicated times. The cells were harvested and analyzed via Western blotting for NS5B, RRM2, and β-actin as reported in B . Blots shown are representative of three independent experiments ( n = 3).

    Journal: The Journal of Biological Chemistry

    Article Title: Ribonucleotide reductase M2 promotes RNA replication of hepatitis C virus by protecting NS5B protein from hPLIC1-dependent proteasomal degradation

    doi: 10.1074/jbc.RA118.004397

    Figure Lengend Snippet: RRM2 regulates RNA polymerase NS5B protein stability. A , quantification of NS5B mRNA levels relative to those of GAPDH in HuH-7 lenti-NS5B cells treated with RRM2 or control siRNA or mock-treated ( Non ) cells for 72 h. Data are shown as an average value of three independent experiments. Vertical bars , S.D. ( n = 3). B , HuH-7 lenti-NS5B cells were transfected with RRM2 or control siRNA for 48 h and then treated with CHX (100 μg/ml) and puromycin ( Puro ; 50 μg/ml) for 6 h. Cells were harvested and analyzed via Western blotting using anti-NS5B, anti-RRM2, and anti-actin antibodies. C , lenti-NS5B–expressing HuH-7 cells were transfected with the pcDNA6-myc-His-RRM2 vector. An empty vector, pcDNA6-myc-His, was used as a negative control. At 48 h post-transfection, cells were treated with CHX (100 μg/ml) for the indicated times. The cells were harvested and analyzed via Western blotting for NS5B, RRM2, and β-actin as reported in B . Blots shown are representative of three independent experiments ( n = 3).

    Article Snippet: NS5B stability assays HuH-7 lenti-NS5B cells were transfected with RRM2 or control siRNA for 48 h and then treated with CHX (100 μg/ml; Wako) and puromycin (50 μg/ml; InvivoGen) for 6 h before harvesting.

    Techniques: Transfection, Western Blot, Expressing, Plasmid Preparation, Negative Control

    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

    EGF stabilizes c-Fos by dissociating c-Fos from KDM2B/FBXL10 (A) EGF induces c-Fos protein accumulation in a time-dependent manner. HEK293 cells stably expressing FLAG-tagged c-Fos were deprived of serum for 8 h, and then treated with 50 μg/L EGF for the indicated length of time. The protein and mRNA levels of FLAG-c-Fos were determined by western blot and qRT-PCR, respectively, and normalized against β-actin. Error bars represent ±SD for triplicate experiments. (B) EGF stabilizes c-Fos. HEK293 cells stably expressing FLAG-tagged c-Fos were pretreated with or without EGF (50 μg/L) for 30 min, followed by CHX treatment (20 mg/L) for the indicated time. The protein levels of c-Fos were determined by western blot and normalized against β-actin. Error bars represent ±SD for triplicate experiments. (C) EGF-induced c-Fos accumulation is blocked by MEK1/2 inhibitor U0126 and an ERK1/2 inhibitor (SCH772984). HEK293 cells stably expressing FLAG-c-Fos were deprived of serum for 8 h, followed by treatment with U0126 (2 μM) or SCH772984 (0.2 or 1 μM) for 5 min. EGF (50 μg/L) was then added for additional 30 min. The protein levels of c-Fos were determined by western blot and normalized against β-actin. (D) EGF-induced c-Fos accumulation is not affected by inhibition of protein synthesis. HEK293 cells stably expressing FLAG-tagged c-Fos were treated with CHX (20 mg/L) for 5 min or 10 min. Solvent or EGF (50 μg/L) was then added for further treatment for 30 min. The protein levels of FLAG-c-Fos were determined by western blot and normalized against β-actin. Data are shown as relative fold change over protein levels in cells without EGF treatment. The arrow represents highly phosphorylated c-Fos (E) Knock down of KDM2B prolongs the high level of c-Fos protein following EGF stimulation. HeLa cells with stable knock down of KDM2B were deprived of serum for 8 h, followed by treatment with EGF (50 μg/L) for the indicated length of time. The protein levels of c-Fos were determined by western blot and normalized against β-actin. Data are shown as relative fold change over cells without EGF treatment (right panel). (F) EGF treatment induces c-Fos S374 phosphorylation and concomitantly reduces the interaction between c-Fos and KDM2B. HEK293 cells were transfected with plasmids expressing indicated proteins and then treated with EGF (50 μg/L) for the indicated length of time. The interactions between c-Fos and KDM2B were determined by Co-IP. (G) The reduction of c-Fos and KDM2B interaction by EGF is blocked by U0126. FLAG-KDM2B and 3HA-c-Fos were co-transfected into HEK293 cells. Cells were pretreated with U0126 (2 μM) for 5 min and then treated with EGF (50 μg/L) for another 30 min. The interactions between c-Fos and KDM2B were determined by Co-IP.

    Journal: Oncogene

    Article Title: KDM2B/FBXL10 targets c-Fos for ubiquitylation and degradation in response to mitogenic stimulation

    doi: 10.1038/onc.2015.482

    Figure Lengend Snippet: EGF stabilizes c-Fos by dissociating c-Fos from KDM2B/FBXL10 (A) EGF induces c-Fos protein accumulation in a time-dependent manner. HEK293 cells stably expressing FLAG-tagged c-Fos were deprived of serum for 8 h, and then treated with 50 μg/L EGF for the indicated length of time. The protein and mRNA levels of FLAG-c-Fos were determined by western blot and qRT-PCR, respectively, and normalized against β-actin. Error bars represent ±SD for triplicate experiments. (B) EGF stabilizes c-Fos. HEK293 cells stably expressing FLAG-tagged c-Fos were pretreated with or without EGF (50 μg/L) for 30 min, followed by CHX treatment (20 mg/L) for the indicated time. The protein levels of c-Fos were determined by western blot and normalized against β-actin. Error bars represent ±SD for triplicate experiments. (C) EGF-induced c-Fos accumulation is blocked by MEK1/2 inhibitor U0126 and an ERK1/2 inhibitor (SCH772984). HEK293 cells stably expressing FLAG-c-Fos were deprived of serum for 8 h, followed by treatment with U0126 (2 μM) or SCH772984 (0.2 or 1 μM) for 5 min. EGF (50 μg/L) was then added for additional 30 min. The protein levels of c-Fos were determined by western blot and normalized against β-actin. (D) EGF-induced c-Fos accumulation is not affected by inhibition of protein synthesis. HEK293 cells stably expressing FLAG-tagged c-Fos were treated with CHX (20 mg/L) for 5 min or 10 min. Solvent or EGF (50 μg/L) was then added for further treatment for 30 min. The protein levels of FLAG-c-Fos were determined by western blot and normalized against β-actin. Data are shown as relative fold change over protein levels in cells without EGF treatment. The arrow represents highly phosphorylated c-Fos (E) Knock down of KDM2B prolongs the high level of c-Fos protein following EGF stimulation. HeLa cells with stable knock down of KDM2B were deprived of serum for 8 h, followed by treatment with EGF (50 μg/L) for the indicated length of time. The protein levels of c-Fos were determined by western blot and normalized against β-actin. Data are shown as relative fold change over cells without EGF treatment (right panel). (F) EGF treatment induces c-Fos S374 phosphorylation and concomitantly reduces the interaction between c-Fos and KDM2B. HEK293 cells were transfected with plasmids expressing indicated proteins and then treated with EGF (50 μg/L) for the indicated length of time. The interactions between c-Fos and KDM2B were determined by Co-IP. (G) The reduction of c-Fos and KDM2B interaction by EGF is blocked by U0126. FLAG-KDM2B and 3HA-c-Fos were co-transfected into HEK293 cells. Cells were pretreated with U0126 (2 μM) for 5 min and then treated with EGF (50 μg/L) for another 30 min. The interactions between c-Fos and KDM2B were determined by Co-IP.

    Article Snippet: The target sequences of shKDM2B are below:shKDM2B #1 TGAGCGTGAAAGGTTGTTT shKDM2B #2 GCCTTTACAAGAAGACATT shKDM2B #3 TTCTTCAAACGCTGTGGAA MG132 (C2211, Sigma-Aldrich), CHX (94271, AMRESCO), EGF (AF-100-15, PeproTech), U0126 (S1102, Selleckchem) and SCH772984 (S7101, Selleckchem) were purchased commercially.

    Techniques: Stable Transfection, Expressing, Western Blot, Quantitative RT-PCR, Inhibition, Transfection, Co-Immunoprecipitation Assay

    EGF-mediated phosphorylation at S374 stabilizes c-Fos and promotes cell proliferation (A) Mutation of S374 site to alanine abolishes EGF-induced c-Fos protein accumulation. HEK293 cells stably expressing FLAG-tagged c-Fos were deprived of serum for 8 h, and then treated with EGF (50 μg/L) for 30 min. The protein and mRNA levels of FLAG-c-Fos were determined by western blot and qRT-PCR, respectively, and normalized against β-actin. Data are shown as relative fold change over cells without EGF treatment. Error bars represent ±SD for duplicate experiments. (B) Mutation of S374 site to aspartic acid renders c-Fos resistant to degradation. HEK293 cells stably expressing FLAG-tagged c-Fos WT or S374D mutant were treated with 20mg/L CHX for the indicated length of time. The half-life of c-Fos protein levels were determined by western blot and normalized against β-actin (Bottom). Error bars represent ±SD for triplicate experiments. (C) The reduction of c-Fos and KDM2B interaction by EGF is inhibited by c-Fos S374A mutant. HEK293 cells were co-transfected with plasmids expressing indicated proteins and then treated with or without EGF (50 μg/L) for 30 min. The interactions between c-Fos and KDM2B were determined by Co-IP. (D) KDM2B preferentially interacts with S374 non-phosphorylatable c-Fos. HEK293 cells were co-transfected with plasmids expressing the indicated proteins and then treated with or without EGF (50 μg/L) for 30min. The FLAG-KDM2B was immunoprecipitated and western blot was performed to detect the co-precipitated c-Fos with indicated antibodies. * indicates the heavy chain background around 55 KDa. (E) S374D mutation of c-Fos hinders its binding to KDM2B. HEK293 cells were co-transfected with plasmids expressing indicated proteins, and the interactions between c-Fos and KDM2B were determined by Co-IP. (F) EGF-induced cell proliferation is compromised by S374A mutant of c-Fos. HEK293 cells stably expressing FLAG-c-Fos WT or S374A mutant were cultured in the absence of serum and treated with or without EGF (100 μg/L) for 0, 1, 2 or 3 days, as indicated. EGF was replenished every day. Cell numbers were counted each day. Western blot was performed to show FLAG-c-Fos protein levels on the 3rd day. * denotes the p

    Journal: Oncogene

    Article Title: KDM2B/FBXL10 targets c-Fos for ubiquitylation and degradation in response to mitogenic stimulation

    doi: 10.1038/onc.2015.482

    Figure Lengend Snippet: EGF-mediated phosphorylation at S374 stabilizes c-Fos and promotes cell proliferation (A) Mutation of S374 site to alanine abolishes EGF-induced c-Fos protein accumulation. HEK293 cells stably expressing FLAG-tagged c-Fos were deprived of serum for 8 h, and then treated with EGF (50 μg/L) for 30 min. The protein and mRNA levels of FLAG-c-Fos were determined by western blot and qRT-PCR, respectively, and normalized against β-actin. Data are shown as relative fold change over cells without EGF treatment. Error bars represent ±SD for duplicate experiments. (B) Mutation of S374 site to aspartic acid renders c-Fos resistant to degradation. HEK293 cells stably expressing FLAG-tagged c-Fos WT or S374D mutant were treated with 20mg/L CHX for the indicated length of time. The half-life of c-Fos protein levels were determined by western blot and normalized against β-actin (Bottom). Error bars represent ±SD for triplicate experiments. (C) The reduction of c-Fos and KDM2B interaction by EGF is inhibited by c-Fos S374A mutant. HEK293 cells were co-transfected with plasmids expressing indicated proteins and then treated with or without EGF (50 μg/L) for 30 min. The interactions between c-Fos and KDM2B were determined by Co-IP. (D) KDM2B preferentially interacts with S374 non-phosphorylatable c-Fos. HEK293 cells were co-transfected with plasmids expressing the indicated proteins and then treated with or without EGF (50 μg/L) for 30min. The FLAG-KDM2B was immunoprecipitated and western blot was performed to detect the co-precipitated c-Fos with indicated antibodies. * indicates the heavy chain background around 55 KDa. (E) S374D mutation of c-Fos hinders its binding to KDM2B. HEK293 cells were co-transfected with plasmids expressing indicated proteins, and the interactions between c-Fos and KDM2B were determined by Co-IP. (F) EGF-induced cell proliferation is compromised by S374A mutant of c-Fos. HEK293 cells stably expressing FLAG-c-Fos WT or S374A mutant were cultured in the absence of serum and treated with or without EGF (100 μg/L) for 0, 1, 2 or 3 days, as indicated. EGF was replenished every day. Cell numbers were counted each day. Western blot was performed to show FLAG-c-Fos protein levels on the 3rd day. * denotes the p

    Article Snippet: The target sequences of shKDM2B are below:shKDM2B #1 TGAGCGTGAAAGGTTGTTT shKDM2B #2 GCCTTTACAAGAAGACATT shKDM2B #3 TTCTTCAAACGCTGTGGAA MG132 (C2211, Sigma-Aldrich), CHX (94271, AMRESCO), EGF (AF-100-15, PeproTech), U0126 (S1102, Selleckchem) and SCH772984 (S7101, Selleckchem) were purchased commercially.

    Techniques: Mutagenesis, Stable Transfection, Expressing, Western Blot, Quantitative RT-PCR, Transfection, Co-Immunoprecipitation Assay, Immunoprecipitation, Binding Assay, Cell Culture

    LAP*/LAP protein levels are stabilised in models of monocytic differentiation. (A) THP-1 monocytic cells were cultivated in the absence (left, 24 h) or presence of differentiating stimuli (PMA: 24 h; VitD3: 72 h). Then, cells were preincubated for 0.5 h with 10 μg/ml CHX before LAP*/LAP protein levels were analysed at the indicated time points (n = 3). To visualize the basically low LAP*/LAP levels in untreated cells, a prolonged exposure time and a more sensitive HRP substrate were applied (5 min Femto Plus vs . 1 min ECL). (B) Densitometric analysis of the experiment shown in A is depicted. The LAP*/LAP levels in THP-1 cells cultivated for 24 h in the absence or presence of PMA were defined as the respective 100% control. (C) THP-1 cells were incubated with PMA for 24 h. Following a 0.5 h treatment of cells with CHX ± RSK-I, the LAP*/LAP protein levels were analysed at the indicated time points (n = 3). (D) MM-6 cells were cultivated ± PMA for 24 h, and following addition of CHX, the LAP*/LAP levels were analysed at the indicated time points (n = 3). (E) Primary human monocytes were differentiated for 3 d (3 d diff.) and then preincubated for 0.5 h with CHX, before LAP*/LAP protein levels were analysed at the indicated time points (n = 3).

    Journal: PLoS ONE

    Article Title: C/EBPβ-LAP*/LAP Expression Is Mediated by RSK/eIF4B-Dependent Signalling and Boosted by Increased Protein Stability in Models of Monocytic Differentiation

    doi: 10.1371/journal.pone.0144338

    Figure Lengend Snippet: LAP*/LAP protein levels are stabilised in models of monocytic differentiation. (A) THP-1 monocytic cells were cultivated in the absence (left, 24 h) or presence of differentiating stimuli (PMA: 24 h; VitD3: 72 h). Then, cells were preincubated for 0.5 h with 10 μg/ml CHX before LAP*/LAP protein levels were analysed at the indicated time points (n = 3). To visualize the basically low LAP*/LAP levels in untreated cells, a prolonged exposure time and a more sensitive HRP substrate were applied (5 min Femto Plus vs . 1 min ECL). (B) Densitometric analysis of the experiment shown in A is depicted. The LAP*/LAP levels in THP-1 cells cultivated for 24 h in the absence or presence of PMA were defined as the respective 100% control. (C) THP-1 cells were incubated with PMA for 24 h. Following a 0.5 h treatment of cells with CHX ± RSK-I, the LAP*/LAP protein levels were analysed at the indicated time points (n = 3). (D) MM-6 cells were cultivated ± PMA for 24 h, and following addition of CHX, the LAP*/LAP levels were analysed at the indicated time points (n = 3). (E) Primary human monocytes were differentiated for 3 d (3 d diff.) and then preincubated for 0.5 h with CHX, before LAP*/LAP protein levels were analysed at the indicated time points (n = 3).

    Article Snippet: Determination of protein stability For examining the C/EBPβ protein stability in differentiating monocytic cells, THP-1 or MM-6 cells were treated with PMA for 24 h or with VitD3 for 72h and primary human monocytes were differentiated for 3 d. Following pretreatment of cells with Cycloheximid (CHX; Merck Millipore) for 30 min, LAP*/LAP protein levels were assessed at the indicated time points.

    Techniques: Incubation

    Inhibition of proteasome and calpain activities (A-C) THP-1 cells were treated with CHX ± 50 μM ALLN (A), 50 μM MG-132 (B), or 100 μM Calpain-inhibitor V (calpain-I; C). LAP*/LAP levels were detected at the indicated time points (n = 3 each). (D-E) THP-1 cells were treated with PMA up to 24 or 48 h and the chymotrypsin-like activity of the proteasome (D) as well as the calpain activity (E) were determined in whole cell extracts using protease-specific substrates (mean±SD; n≥3, duplicates). The respective proteolytic activity at 0 h was defined as 100% (dashed line). (F) Primary human monocytes were cultivated up to 3 d. The chymotrypsin-like (CL) and the calpain (C) proteolytic activities were determined at days 0, 1, and 3 (mean±SD; n = 2, representative for n = 4 experiments). The respective proteolytic activity at day 0 was defined as 100% (dashed line). (G) THP-1 cells were incubated for 24 h ± PMA in the absence or presence of RSK-I. The chymotrypsin-like (CL) proteasome and calpain (C) activities were measured as described above (mean±SD; n = 3, duplicates). (H-I) THP-1 cells were incubated with PMA up to 24 h (H) or with VitD3 up to 72 h (I) and the levels of calpastatin were determined at the indicated time points (n = 3).

    Journal: PLoS ONE

    Article Title: C/EBPβ-LAP*/LAP Expression Is Mediated by RSK/eIF4B-Dependent Signalling and Boosted by Increased Protein Stability in Models of Monocytic Differentiation

    doi: 10.1371/journal.pone.0144338

    Figure Lengend Snippet: Inhibition of proteasome and calpain activities (A-C) THP-1 cells were treated with CHX ± 50 μM ALLN (A), 50 μM MG-132 (B), or 100 μM Calpain-inhibitor V (calpain-I; C). LAP*/LAP levels were detected at the indicated time points (n = 3 each). (D-E) THP-1 cells were treated with PMA up to 24 or 48 h and the chymotrypsin-like activity of the proteasome (D) as well as the calpain activity (E) were determined in whole cell extracts using protease-specific substrates (mean±SD; n≥3, duplicates). The respective proteolytic activity at 0 h was defined as 100% (dashed line). (F) Primary human monocytes were cultivated up to 3 d. The chymotrypsin-like (CL) and the calpain (C) proteolytic activities were determined at days 0, 1, and 3 (mean±SD; n = 2, representative for n = 4 experiments). The respective proteolytic activity at day 0 was defined as 100% (dashed line). (G) THP-1 cells were incubated for 24 h ± PMA in the absence or presence of RSK-I. The chymotrypsin-like (CL) proteasome and calpain (C) activities were measured as described above (mean±SD; n = 3, duplicates). (H-I) THP-1 cells were incubated with PMA up to 24 h (H) or with VitD3 up to 72 h (I) and the levels of calpastatin were determined at the indicated time points (n = 3).

    Article Snippet: Determination of protein stability For examining the C/EBPβ protein stability in differentiating monocytic cells, THP-1 or MM-6 cells were treated with PMA for 24 h or with VitD3 for 72h and primary human monocytes were differentiated for 3 d. Following pretreatment of cells with Cycloheximid (CHX; Merck Millipore) for 30 min, LAP*/LAP protein levels were assessed at the indicated time points.

    Techniques: Inhibition, Activity Assay, Incubation

    H 2 promotes β-catenin degradation in L cells. (a) Cells were treated with control CM (Cont.), Wnt3a CM, 30 mM LiCl, or 2 μM BIO with 10% H 2 or 10% N 2 gas for 24 h. Expression of Ctnnb1 encoding β-catenin was quantified by qRT-PCR ( n = 3). (b) Cells transfected with myc-β-catenin (XE28 XBC plasmid) were exposed to a combination of 10 μg/ml cycloheximide (CHX) and 2 μM BIO with 10% H 2 or 10% N 2 gas for indicated periods of time. Representative Western blots are shown with densitometry of myc-β-catenin/β-actin ( n = 4). Two groups were not statistically different by two-way repeated measures ANOVA. * P

    Journal: Scientific Reports

    Article Title: Molecular hydrogen suppresses activated Wnt/β-catenin signaling

    doi: 10.1038/srep31986

    Figure Lengend Snippet: H 2 promotes β-catenin degradation in L cells. (a) Cells were treated with control CM (Cont.), Wnt3a CM, 30 mM LiCl, or 2 μM BIO with 10% H 2 or 10% N 2 gas for 24 h. Expression of Ctnnb1 encoding β-catenin was quantified by qRT-PCR ( n = 3). (b) Cells transfected with myc-β-catenin (XE28 XBC plasmid) were exposed to a combination of 10 μg/ml cycloheximide (CHX) and 2 μM BIO with 10% H 2 or 10% N 2 gas for indicated periods of time. Representative Western blots are shown with densitometry of myc-β-catenin/β-actin ( n = 4). Two groups were not statistically different by two-way repeated measures ANOVA. * P

    Article Snippet: Lithium chloride (LiCl), cycloheximide (CHX), N-benzyoloxycarbonyl (Z)-Leu-Leu-leucinal (MG132), dimethyl sulfoxide (DMSO), and SP600125 were purchased from Wako.

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

    ( A – D ) Examining the effect of proteasome subunit knockdown in different cell lines. 80 shRNAs targeting 20 different proteasome subunits and control hairpins were expressed in HepG2 ( A ), H838 ( B ), T47D ( C ), and H1792 ( D ) cells by viral transduction. Each subunit was targeted by 4 different shRNAs and the cell viability was measured as the relative cell number compared to the average of non-targeting shRNAs 5 days after the initial introduction of the shRNAs. ( E , F ) HepG2 cells with shRNAs targeting PSMC5, PSMD2, and green fluorescent protein (GFP) were grown out. Their relative growth was analyzed in the absence of bortezomib ( E ) and their protein content was analyzed 24 hr after the addition of either 8 or 12 nM of bortezomib ( F ). ( G ) The relative cell number of cells harboring a control shLacZ (black) or each of 5 individual shRNAs targeting shPSMC5 (Cayenne) was analyzed 4 days after addition of the indicated concentrations of bortezomib. ( H ) HepG2 cells stably expressing shRNAs targeting the PSMC5 subunit and a control shRNA (lacZ) were analyzed by Western blot for the indicated proteins 24 hr with or without bortezomib treatment. ( I – N ) The HepG2 cells with shRNAs targeting PSMC5, PSMD2, and GFP (described above) were further exposed to a short panel of stress inducers including bortezomib ( I ), tunicamycin ( J ), rohinitib-RHT ( K ), Hsp90 inhibition ( L ), withaferin A ( M ), and cyclohexamide ( N ) at indicated concentrations and the relative cell number (RFU) was examined after 4 days. The graphs represent the average of at least 4 different measurements and the SEM. DOI: http://dx.doi.org/10.7554/eLife.08467.006

    Journal: eLife

    Article Title: Compromising the 19S proteasome complex protects cells from reduced flux through the proteasome

    doi: 10.7554/eLife.08467

    Figure Lengend Snippet: ( A – D ) Examining the effect of proteasome subunit knockdown in different cell lines. 80 shRNAs targeting 20 different proteasome subunits and control hairpins were expressed in HepG2 ( A ), H838 ( B ), T47D ( C ), and H1792 ( D ) cells by viral transduction. Each subunit was targeted by 4 different shRNAs and the cell viability was measured as the relative cell number compared to the average of non-targeting shRNAs 5 days after the initial introduction of the shRNAs. ( E , F ) HepG2 cells with shRNAs targeting PSMC5, PSMD2, and green fluorescent protein (GFP) were grown out. Their relative growth was analyzed in the absence of bortezomib ( E ) and their protein content was analyzed 24 hr after the addition of either 8 or 12 nM of bortezomib ( F ). ( G ) The relative cell number of cells harboring a control shLacZ (black) or each of 5 individual shRNAs targeting shPSMC5 (Cayenne) was analyzed 4 days after addition of the indicated concentrations of bortezomib. ( H ) HepG2 cells stably expressing shRNAs targeting the PSMC5 subunit and a control shRNA (lacZ) were analyzed by Western blot for the indicated proteins 24 hr with or without bortezomib treatment. ( I – N ) The HepG2 cells with shRNAs targeting PSMC5, PSMD2, and GFP (described above) were further exposed to a short panel of stress inducers including bortezomib ( I ), tunicamycin ( J ), rohinitib-RHT ( K ), Hsp90 inhibition ( L ), withaferin A ( M ), and cyclohexamide ( N ) at indicated concentrations and the relative cell number (RFU) was examined after 4 days. The graphs represent the average of at least 4 different measurements and the SEM. DOI: http://dx.doi.org/10.7554/eLife.08467.006

    Article Snippet: MG132 (EMD Millipore, Billerica, MA, United States), Bortezomib (LC Laboratories # B-1408, Woburn, MA, United States), Cyclohexamide (Enzo life sciences, Farmingdale, NY, United States), withaferin A, tunicamycin (Sigma St. Louis, MO, United States).

    Techniques: Transduction, Stable Transfection, Expressing, shRNA, Western Blot, Inhibition

    ( A – F ) PSMD2 shRNA was induced for 48 hr with 1 μg/ml doxycycline. Cells were then collected, washed, and plated in the absence of doxycycline 24 hr prior to exposure to increasing concentration of bortezomib ( A ), MG132 ( B ), cyclohexamide ( C ), withaferin A ( D ), tunicamycin ( E ), and rotenone ( F ). ( G – I ) PSMD2 KD was induced as described above and then cells were grown in the presence or absence of 15 nM bortezomib for the indicated time points (3, 6, 24 hr). ( G ) Immunoblot analysis of the total proteasome subunits following SDS-PAGE (upper panels) and the 20S proteasome complex levels after native gel electrophoresis. These results were quantified by Imagelab software and plotted ( H ). ( I ) Lysosomal degradation rate was measured in control and PSMD2 knock down (Dox) cells in the presence or absence of 10 nM Bortezomib treatment for 20 hr. DOI: http://dx.doi.org/10.7554/eLife.08467.010

    Journal: eLife

    Article Title: Compromising the 19S proteasome complex protects cells from reduced flux through the proteasome

    doi: 10.7554/eLife.08467

    Figure Lengend Snippet: ( A – F ) PSMD2 shRNA was induced for 48 hr with 1 μg/ml doxycycline. Cells were then collected, washed, and plated in the absence of doxycycline 24 hr prior to exposure to increasing concentration of bortezomib ( A ), MG132 ( B ), cyclohexamide ( C ), withaferin A ( D ), tunicamycin ( E ), and rotenone ( F ). ( G – I ) PSMD2 KD was induced as described above and then cells were grown in the presence or absence of 15 nM bortezomib for the indicated time points (3, 6, 24 hr). ( G ) Immunoblot analysis of the total proteasome subunits following SDS-PAGE (upper panels) and the 20S proteasome complex levels after native gel electrophoresis. These results were quantified by Imagelab software and plotted ( H ). ( I ) Lysosomal degradation rate was measured in control and PSMD2 knock down (Dox) cells in the presence or absence of 10 nM Bortezomib treatment for 20 hr. DOI: http://dx.doi.org/10.7554/eLife.08467.010

    Article Snippet: MG132 (EMD Millipore, Billerica, MA, United States), Bortezomib (LC Laboratories # B-1408, Woburn, MA, United States), Cyclohexamide (Enzo life sciences, Farmingdale, NY, United States), withaferin A, tunicamycin (Sigma St. Louis, MO, United States).

    Techniques: shRNA, Concentration Assay, SDS Page, Nucleic Acid Electrophoresis, Software