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  • 99
    Millipore mg132
    Peli1 binds to and mediates Lys48 ubiquitination of NIK. a Immunoblot analysis of NIK and HSP60 in whole-cell lysates of WT and Peli1 -deficient splenic B cells stimulated with anti-CD40 (αCD40). b Analysis of Lys48 ubiquitination of NIK in WT and KO B cells left unstimulated or stimulated with anti-CD40 (αCD40) for 4 h in the presence of a proteasome inhibitor <t>MG132.</t> IP, immunoprecipitation; IB, immunoblotting. c Ubiquitination of NIK in HEK293 cells transfected with (+) or without (−) indicated expression vectors. d Immunoassays on lysates of WT splenic B cells left unstimulated or stimulated with anti-CD40 (αCD40) for 4 h in the presence of MG132, followed by IP with control IgG or anti-NIK and immunoblot analysis of NIK-associated Peli1. e IP analysis examining the association of NIK with Peli1 in HEK293 cells transfected with (+) or without (−) indicated expression vectors. f Immunoblot analysis of Lys48 ubiquitination of NIK assessed by in vitro ubiquitination assay with a mixture of E1, E2 (UbcH5c), ATP, Flag-NIK, HA-Peli1, or HA-Peli1ΔC after IP with anti-Flag. g Immunoblot analysis of p52, p100, NIK, c-IAP2, Peli1, and HSP60 in total lysis of control and Peli1 -knockdown M12 cells that pretreated with DMSO or smac mimetic BV6 for 4 h, and then stimulated with anti-CD40 (αCD40) (upper panel) or BAFF (lower panel) at the indicated time points. Ctrl, control. h Analysis of Lys48 ubiquitination of NIK in control and Peli1 -knockdown M12 cells that pretreated with DMSO or smac mimetic BV6 for 4 h, then left unstimulated or stimulated with anti-CD40 (αCD40) for 4 h in the presence of MG132. Ctrl, control. Data are shown based on three independent experiments
    Mg132, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 21042 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Selleck Chemicals mg132
    Peli1 binds to and mediates Lys48 ubiquitination of NIK. a Immunoblot analysis of NIK and HSP60 in whole-cell lysates of WT and Peli1 -deficient splenic B cells stimulated with anti-CD40 (αCD40). b Analysis of Lys48 ubiquitination of NIK in WT and KO B cells left unstimulated or stimulated with anti-CD40 (αCD40) for 4 h in the presence of a proteasome inhibitor <t>MG132.</t> IP, immunoprecipitation; IB, immunoblotting. c Ubiquitination of NIK in HEK293 cells transfected with (+) or without (−) indicated expression vectors. d Immunoassays on lysates of WT splenic B cells left unstimulated or stimulated with anti-CD40 (αCD40) for 4 h in the presence of MG132, followed by IP with control IgG or anti-NIK and immunoblot analysis of NIK-associated Peli1. e IP analysis examining the association of NIK with Peli1 in HEK293 cells transfected with (+) or without (−) indicated expression vectors. f Immunoblot analysis of Lys48 ubiquitination of NIK assessed by in vitro ubiquitination assay with a mixture of E1, E2 (UbcH5c), ATP, Flag-NIK, HA-Peli1, or HA-Peli1ΔC after IP with anti-Flag. g Immunoblot analysis of p52, p100, NIK, c-IAP2, Peli1, and HSP60 in total lysis of control and Peli1 -knockdown M12 cells that pretreated with DMSO or smac mimetic BV6 for 4 h, and then stimulated with anti-CD40 (αCD40) (upper panel) or BAFF (lower panel) at the indicated time points. Ctrl, control. h Analysis of Lys48 ubiquitination of NIK in control and Peli1 -knockdown M12 cells that pretreated with DMSO or smac mimetic BV6 for 4 h, then left unstimulated or stimulated with anti-CD40 (αCD40) for 4 h in the presence of MG132. Ctrl, control. Data are shown based on three independent experiments
    Mg132, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 99/100, based on 1124 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Biomol GmbH mg132
    Photographs of immunoblots of electrophoretically separated proteins from lysates of rabbit skin cells infected with HSV-1(F), HSV-1(vCPc0), or HSV-1(vCPc0)R in the presence or absence of <t>MG132.</t> Replicate cultures in 25-cm 2 flasks of rabbit skin cells were either mock infected or exposed to 5 (A) or 10 to 100 (B) PFU of corresponding virus per cell. At 3 h after infection, one set of cultures was replenished with fresh untreated medium (lanes 1, 3, 5, and 7) whereas a second set was replenished with medium containing 10 μM MG132 (lanes 2, 4, 6, and 8). The cells were harvested at 24 h after infection. Proteins were solubilized in disruption buffer and electrophoretically separated in 11% denaturing polyacrylamide gels, transferred to nitrocellulose sheets, and reacted with monoclonal antibodies to ICP0, ICP4, ICP8, ICP27, αTIF, or U S 11 or polyclonal antibody to ICP22.
    Mg132, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 92/100, based on 823 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Cayman Chemical mg132
    Transcriptional and protein consequences of PLK4 mutations a) Schematic of the human PLK4 gene. Coding exons (black), UTRs (white), alternatively spliced region in exon 5 (grey), arrow heads, RT-PCR primer positions. b) PLK4 protein domain structure. Kinase domain (KD)( grey), PEST sequences (1-3) (black), polo-box domains (PBD1-3) (blue/turquoise/green). Middle panel, the c.2811-5G > C mutation creates a new splice acceptor site that leads to retention of 4 bp of intron 15 sequence in the PLK4 mRNA, resulting in premature truncation of the protein at its C-terminus, disrupting the terminal polo-box domain (PB3). Lower panel depicts domain structure for the alternative isoform (ALT) resulting from use of an internal exon 5 splice donor site. c) Sequence electropherograms of the exon 15-16 junction of PLK4 amplified by RT-PCR from patient and control RNA. d) Alternative splicing of an internal exon 5 splice donor site is not detected in other vertebrates by RT-PCR. e) Levels of functional PLK4 are reduced to 25% of normal levels, in patients with the c.1299_1309delTAAAG mutation. Transcript levels plotted from quantitative RT-PCR on RNA extracted from patient primary fibroblast cell lines (n=3 experiments (exp), performed in triplicate; error bars, s.e.m). P value, two-tailed t-test *p≤0.05. Further details and characterization of PLK4 ALT and FL transcript levels, Fig. S5 . f) Immunoblotting demonstrates reduced endogenous PLK4 protein levels in patient fibroblasts. Cell lysates of asynchronous cells. Left two lanes, RNAi of PLK4 in RPE1 cells demonstrating specificity of PLK4 antibody, with two PLK4-specific protein bands visualized. Remaining lanes, patient and control primary fibroblasts, upper panel standard exposure PLK4 immunoblot with middle panel overexposed to demonstrate residual detectable protein in P1 patient. Note also loss of the upper PLK4 band in P6 and P7. Lower panel, loading control, blot probed with anti-Actin antibody. g) PLK4 protein levels at the centrosome are reduced in patient fibroblasts. Top panel, representative immunofluorescence images of primary fibroblasts treated with 10μM <t>MG132</t> for 5 hrs prior to fixation. (Specificity of the anti-PLK4 antibody confirmed by performing RNAi-mediated PLK4 depletion, Fig. S6 ). Scale bar = 10 μm. Bottom panel, quantitation of PLK4 protein levels at the centrosome by immunofluorescence analysis. Exp=3, n=50 cells/exp; error bars, s.e.m of n=150 cells. P value, two-tailed t-test ****p≤0.0001.
    Mg132, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 98/100, based on 842 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Peptide Institute mg132
    The initial endocytosis of mLRP4 is slowed, but not blocked by proteasomal inhibitors. CHO-mLRP4 cells were pretreated with DMSO or DMSO containing <t>MG132</t> (20 μM) for either 30 min (A) or 2 h (B). The initial endocytosis rates of mLRP4 were measured using 125 I-RAP as described in MATERIALS AND METHODS. Data represent triplicate determination with SE values given as error bars.
    Mg132, supplied by Peptide Institute, used in various techniques. Bioz Stars score: 92/100, based on 551 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Enzo Biochem mg132
    Cullin 3 SPOP is the physiological E3 ubiquitin ligase for PD-L1 a–d, IB analysis of WCL derived from C4-2 cells treated with <t>MG132</t> (10 μM) or MLN4924 (1 μM) for 12 hours ( a ), immunoprecipitates (IP) and WCL derived from 293T cells transfected with indicated constructs ( b , d ), or anti-PD-L1 IP and WCL derived from PC3 cells ( e ). Cells were treated with MG132 (10 μM) for 12 hours in b, c . e–g, IB analysis of WCL derived from Spop +/+ versus Spop −/− MEFs ( e ), C4-2 cells depleted SPOP with sgRNAs ( f ), or C4-2 cells stably expressing indicated SPOP WT and mutants ( g ). h, i, IB analysis of IP and WCL derived from 293T cells ( h ), or Ni-NTA pull-down products derived from PC3 cells transfected with indicated constructs and treated with 30 μM MG132 for 6 hours. j–l, FACS analysis for PD-L1 ( j) or CD3 + T-cell population ( l ) of the B16-F10 implanted tumors ectopically expressing SPOP-WT or F102C mutant (n = 6 mice each group). Tumor weight were recorded at the time of sacrifice ( k ) (n = 5 mice each group). m, Representative images of PD-L1 and CD8 immunohistochemistry (IHC) staining in SPOP wild-type or mutant primary human prostate cancer samples. The scale bar: 400 μm or 100 μm. n, o Quantification of IHC analysis for PD-L1 ( n ) and CD8 + T cells ( o ) in SPOP wild-type versus mutant human prostate tumor specimens. (n =15 for SPOP mutant, n = 82 for SPOP WT). Error bars, ± s.d., two-tailed t -test, except ( n ) Mann-Whitney test, * P
    Mg132, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 94/100, based on 986 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck KGaA mg132
    Progerin accumulates in autophagic vacuoles upon MG 132 treatment Immunofluorescence staining of PML (red) and LC3B (green) in HGPS and control fibroblasts both treated with <t>MG132</t> (5 μM) or DMSO (control) for 24 h. Nucleolar and cytoplasmic colocalization of LC3B with PML are shown (arrows). Scale bar, 10 μm. Nucleolar progerin (red) and LC3B (green) accumulation (arrows) in HGPS fibroblasts upon 5 μM MG132 treatment. Scale bar, 5 μm. Progerin (red) accumulated in LC3B (green), p62 (green) and LAMP‐2 (green) positive cytoplasmic vesicles (arrows) after 48 h MG132 (5 μM) exposure of HGPS fibroblasts. Scale bar, 5 μm. The magnifications are shown; scale bar, 200 nm. Data information: Data in (A–C) are representative of five independent experiments. The experiments were performed on fibroblasts of HGPS patients and healthy subjects matched for age and passage.
    Mg132, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 636 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Tocris mg132
    Activation of AMPK enhances the protein levels of both Insig-1 and Insig-2. a – c AMPK agonists increase the protein levels of Insig-1. HEK293 cells were transfected with pcDNA or myc-tagged Insig-1 for 24 h, followed by treatment with 2 mM metformin or 5 μM <t>MG132</t> ( a ), or 1 mM AICAR ( b ) as indicated; and HepG2 cells were transfected with pcDNA or myc-tagged Insig-1 for 24 h, and then infected with adenoviruses encoding constitutive active AMPK (CA-AMPK) or GFP for 48 h ( c ). Immunoblots were performed. d – f AMPK agonists increase the protein levels of Insig-2. HEK293 cells were transfected with pcDNA or FLAG-tagged Insig-2 for 24 h, followed by treatment with 2 mM metformin or 5 μM MG132 ( d ), 1 mM AICAR ( e ); and HepG2 cells were transfected with pcDNA or FLAG-tagged Insig-2 for 24 h, and then infected with adenoviruses encoding CA-AMPK or GFP for 48 h ( f ). Immunoblots were performed
    Mg132, supplied by Tocris, used in various techniques. Bioz Stars score: 99/100, based on 592 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck & Co mg132
    NFκB up-regulates BAG3 and HspB8 expression after various protein aggregation stresses. (A) Control (HeLa) and p65-depleted (p65-KD#2) cell lines were either untreated (NT) or treated with 5 and 15 mM azetidine (Aze) and canavanine (Cana) or 5 μM <t>MG132</t> for 6 h. At 16 h after treatments, total protein extracts were prepared and analyzed by immunoblots probed with antibodies against BAG3, HspB8, and actin (as a loading control). (B) As in A, but with HeLa and p65-KD#2 cell lines transiently transfected with control plasmids (Cont) or plasmids expressing HspB5wt, HspB5R120G, SOD1wt-EGFP, SOD1G93A-EGFP, or SOD1G85R-EGFP. Analysis was performed 35 h posttransfection. Expression of HspB5 and SOD1-EGFP was checked by probing immunoblots with anti-HspB5 and anti-EGFP antibodies. BAG3 and HspB8 levels were quantified; the densitometric measurements were normalized to the corresponding actin bands, and ratios were calculated between HeLa or p65-KD#2-treated cells vs. nontreated counterparts and are reported in the graphs ( n = 3).
    Mg132, supplied by Merck & Co, used in various techniques. Bioz Stars score: 92/100, based on 429 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc mg132
    NFκB up-regulates BAG3 and HspB8 expression after various protein aggregation stresses. (A) Control (HeLa) and p65-depleted (p65-KD#2) cell lines were either untreated (NT) or treated with 5 and 15 mM azetidine (Aze) and canavanine (Cana) or 5 μM <t>MG132</t> for 6 h. At 16 h after treatments, total protein extracts were prepared and analyzed by immunoblots probed with antibodies against BAG3, HspB8, and actin (as a loading control). (B) As in A, but with HeLa and p65-KD#2 cell lines transiently transfected with control plasmids (Cont) or plasmids expressing HspB5wt, HspB5R120G, SOD1wt-EGFP, SOD1G93A-EGFP, or SOD1G85R-EGFP. Analysis was performed 35 h posttransfection. Expression of HspB5 and SOD1-EGFP was checked by probing immunoblots with anti-HspB5 and anti-EGFP antibodies. BAG3 and HspB8 levels were quantified; the densitometric measurements were normalized to the corresponding actin bands, and ratios were calculated between HeLa or p65-KD#2-treated cells vs. nontreated counterparts and are reported in the graphs ( n = 3).
    Mg132, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 276 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    R&D Systems mg132
    STAT3 directly interacts with IKKα in vitro. ( A , C ) IP analysis showing a physical interaction between IKKα and STAT3 in H- Ras MCF-10A ( A ) and HEK293T ( C ) cells. Immunoprecipitation was performed with IKKα (left) or STAT3 (right) antibodies, followed by immunoblotting with antibodies for STAT3 and IKKα, respectively. IgG, a negative control for immunoprecipitation; input, total protein lysate. ( B , D ) Duolink analysis showing the interaction between STAT3 and IKKα in H- Ras MCF-10A ( B ) and MDA-MB-231 ( D ) cells. The representative image was visualized under the fluorescent microscope. ( E , F ) H- Ras MCF-10A ( E ) and HEK293T ( F ) cells were transfected with IKKα wild type (WT) or K44A mutant (K44A) in the presence of STAT3 construct for 48 h, followed by <t>MG132</t> treatment for additional 2 h. Immunoprecipitation analysis showing the interaction between STAT3 and either WT or K44A IKKα. ( G ) Duolink analysis showing the STAT3 binding to IKKα WT or K44A mutant in HEK293T cells.
    Mg132, supplied by R&D Systems, used in various techniques. Bioz Stars score: 92/100, based on 410 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    AG Scientific mg132
    Ribosomal stress leads to RPS2 ubiquitination for MDM2 regulation to induce p53 by dissociation of USP47 and RPS2. ( a ) U2OS cells co-overexpressing either Flag vector or Flag-USP47 were treated with DMSO or 5 nM actinomycin D for 4 h, and immunoprecipitation was performed with anti-Flag agarose. Bound proteins were immunoblotted with the indicated antibodies. α-tubulin was used as a loading control. ( b ) HEK293T cells were co-overexpressed with HA-RPS2, Flag-USP47, and then treated with DMSO or 5 nM actinomycin D for 4 h. Then, cell lysates were immunoprecipitated with anti-Flag M2 agarose and detected with the indicated antibodies. ( c ) U2OS cells were co-overexpressed with Flag-USP47 and HA-RPS2 and then treated with DMSO or 5 nM actinomycin D for 4 h. Cells were immunostained with anti-HA antibody followed by Alexa Fluor 488 anti-mouse IgG and Flag antibodies followed by Alexa Fluor 546 anti-rabbit IgG. Representative immunofluorescence images were captured by confocal microscopy. ( d ) HEK293T cells were overexpressed with HA-RPS2 and His-ubiquitin and then treated with 10 μM <t>MG132</t> for 4 h and 5 nM actinomycin D for indicated times. Ubiquitin conjugates were purified on Ni-NTA-agarose under denaturing conditions, and ubiquitinated RPS2 was detected by anti-HA antibodies. ( e ) U2OS cells were co-transfected with His-ubiquitin, HA-RPS2, and either siControl or siUSP47 and then treated with 10 μM MG132 and either DMSO or 5 nM actinomycin D for 4 h. Ubiquitin conjugates were purified on Ni-NTA agarose under denaturing conditions, and ubiquitinated RPS2 was detected by anti-HA antibodies. ( f ) U2OS cells transfected with siRNA against control or USP47 were treated with either DMSO or 5 nM actinomycin D for 4 h. Cell lysates were immunoblotted with the indicated antibodies. The uncropped blots and molecular weight markers are shown in Figure S7 .
    Mg132, supplied by AG Scientific, used in various techniques. Bioz Stars score: 92/100, based on 194 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam antibody solution
    Ribosomal stress leads to RPS2 ubiquitination for MDM2 regulation to induce p53 by dissociation of USP47 and RPS2. ( a ) U2OS cells co-overexpressing either Flag vector or Flag-USP47 were treated with DMSO or 5 nM actinomycin D for 4 h, and immunoprecipitation was performed with anti-Flag agarose. Bound proteins were immunoblotted with the indicated antibodies. α-tubulin was used as a loading control. ( b ) HEK293T cells were co-overexpressed with HA-RPS2, Flag-USP47, and then treated with DMSO or 5 nM actinomycin D for 4 h. Then, cell lysates were immunoprecipitated with anti-Flag M2 agarose and detected with the indicated antibodies. ( c ) U2OS cells were co-overexpressed with Flag-USP47 and HA-RPS2 and then treated with DMSO or 5 nM actinomycin D for 4 h. Cells were immunostained with anti-HA antibody followed by Alexa Fluor 488 anti-mouse IgG and Flag antibodies followed by Alexa Fluor 546 anti-rabbit IgG. Representative immunofluorescence images were captured by confocal microscopy. ( d ) HEK293T cells were overexpressed with HA-RPS2 and His-ubiquitin and then treated with 10 μM <t>MG132</t> for 4 h and 5 nM actinomycin D for indicated times. Ubiquitin conjugates were purified on Ni-NTA-agarose under denaturing conditions, and ubiquitinated RPS2 was detected by anti-HA antibodies. ( e ) U2OS cells were co-transfected with His-ubiquitin, HA-RPS2, and either siControl or siUSP47 and then treated with 10 μM MG132 and either DMSO or 5 nM actinomycin D for 4 h. Ubiquitin conjugates were purified on Ni-NTA agarose under denaturing conditions, and ubiquitinated RPS2 was detected by anti-HA antibodies. ( f ) U2OS cells transfected with siRNA against control or USP47 were treated with either DMSO or 5 nM actinomycin D for 4 h. Cell lysates were immunoblotted with the indicated antibodies. The uncropped blots and molecular weight markers are shown in Figure S7 .
    Antibody Solution, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 904 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Peptides International mg132
    The degradation of IP3R3 is dependent on FBXL2 localization to cell membranes and on the segregase activity of p97 a , HeLa cells were transfected with GFP-tagged FBXL2 and then treated with either DMSO or GGTI-2418 for 16 h. Live cell imaging was carried out with an LSM510 confocal microscope using a 63× objective. Scale bars, 10 μm. b , NHFs were incubated with GGTi-2418 for 30 h and then with cycloheximide (CHX) and ATP. Cells were subsequently harvested at the indicated times for immunoblotting. The graph shows the quantification of IP3R3 levels from two independent experiments. c , NHFs (passage 3), HeLa and HEK293T cells were incubated with GGTi-2418 for the indicated times. Cells were subsequently harvested for immunoblotting. This experiment was performed once. d , NHFs (passage 3) were serum-starved for 72 h, treated with either DMSO or Eer1, and then re-stimulated with serum (SR) for the indicated times. The graph shows the quantification of IP3R3 levels from two independent experiments. The bracket on the right marks a ladder of bands which, presumably, are ubiquitinated species of IP3R3 that are not degraded when p97 is inhibited. e , During a 72 h serum starvation, NHFs (passage 3 and 4) were transfected with either an siRNA targeting p97 or a non-silencing siRNA (NS). Cells were subsequently stimulated with medium containing serum and harvested at the indicated time points for immunoblotting. The graph shows the quantification of IP3R3 levels from three independent experiments. Error bars indicate s.e.m. These results, together with those shown in d , are in agreement with the findings that IP3Rs are ubiquitinated while they are membrane-associated, and those showing that p97 promotes the degradation of IP3Rs 27 , 28 . Thus, we propose that, after its FBXL2-mediated ubiquitination, IP3R3 is extracted from cell membranes by the segregase activity of p97 to be degraded by the proteasome. f , HEK293T cells were transfected with either an empty vector (EV) or the indicated GFP-tagged and HA-tagged proteins. 24 h post-transfection, cells were treated with <t>MG132</t> for 3 h before harvesting for immunoprecipitation and immunoblotting as indicated. This experiment was performed twice. g, h , HEK293T cells were transfected with Flag-tagged FBXL2 and the indicated versions of tagged IP3R3. After immunopurification with an anti-Flag resin, in vitro ubiquitination of IP3R3 was performed in the presence of UAE1, Ubch3, Ubch5 and ubiquitin (Ub). Where indicated, an excess of methylated ubiquitin (methyl-Ub), which blocks chain extension, was added to the in vitro reactions. The presence of methyl-Ub resulted in the disappearance of the highest molecular weight forms of IP3R3, demonstrating that the high molecular weight forms of IP3R3 are indeed polyubiquitinated species of the protein. Samples were analysed by immunoblotting with the indicated antibodies. The bracket on the right marks a ladder of bands corresponding to ubiquitinated IP3R3. Immunoblots of whole-cell lysates (WCL) are shown at the bottom. Unless otherwise noted, experiments were performed at least three times. For gel source data, see Supplementary Fig. 1 .
    Mg132, supplied by Peptides International, used in various techniques. Bioz Stars score: 92/100, based on 276 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore proteasomal inhibitor mg132
    The degradation of IP3R3 is dependent on FBXL2 localization to cell membranes and on the segregase activity of p97 a , HeLa cells were transfected with GFP-tagged FBXL2 and then treated with either DMSO or GGTI-2418 for 16 h. Live cell imaging was carried out with an LSM510 confocal microscope using a 63× objective. Scale bars, 10 μm. b , NHFs were incubated with GGTi-2418 for 30 h and then with cycloheximide (CHX) and ATP. Cells were subsequently harvested at the indicated times for immunoblotting. The graph shows the quantification of IP3R3 levels from two independent experiments. c , NHFs (passage 3), HeLa and HEK293T cells were incubated with GGTi-2418 for the indicated times. Cells were subsequently harvested for immunoblotting. This experiment was performed once. d , NHFs (passage 3) were serum-starved for 72 h, treated with either DMSO or Eer1, and then re-stimulated with serum (SR) for the indicated times. The graph shows the quantification of IP3R3 levels from two independent experiments. The bracket on the right marks a ladder of bands which, presumably, are ubiquitinated species of IP3R3 that are not degraded when p97 is inhibited. e , During a 72 h serum starvation, NHFs (passage 3 and 4) were transfected with either an siRNA targeting p97 or a non-silencing siRNA (NS). Cells were subsequently stimulated with medium containing serum and harvested at the indicated time points for immunoblotting. The graph shows the quantification of IP3R3 levels from three independent experiments. Error bars indicate s.e.m. These results, together with those shown in d , are in agreement with the findings that IP3Rs are ubiquitinated while they are membrane-associated, and those showing that p97 promotes the degradation of IP3Rs 27 , 28 . Thus, we propose that, after its FBXL2-mediated ubiquitination, IP3R3 is extracted from cell membranes by the segregase activity of p97 to be degraded by the proteasome. f , HEK293T cells were transfected with either an empty vector (EV) or the indicated GFP-tagged and HA-tagged proteins. 24 h post-transfection, cells were treated with <t>MG132</t> for 3 h before harvesting for immunoprecipitation and immunoblotting as indicated. This experiment was performed twice. g, h , HEK293T cells were transfected with Flag-tagged FBXL2 and the indicated versions of tagged IP3R3. After immunopurification with an anti-Flag resin, in vitro ubiquitination of IP3R3 was performed in the presence of UAE1, Ubch3, Ubch5 and ubiquitin (Ub). Where indicated, an excess of methylated ubiquitin (methyl-Ub), which blocks chain extension, was added to the in vitro reactions. The presence of methyl-Ub resulted in the disappearance of the highest molecular weight forms of IP3R3, demonstrating that the high molecular weight forms of IP3R3 are indeed polyubiquitinated species of the protein. Samples were analysed by immunoblotting with the indicated antibodies. The bracket on the right marks a ladder of bands corresponding to ubiquitinated IP3R3. Immunoblots of whole-cell lysates (WCL) are shown at the bottom. Unless otherwise noted, experiments were performed at least three times. For gel source data, see Supplementary Fig. 1 .
    Proteasomal Inhibitor Mg132, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 229 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    beyotime mg132
    Effect of crizotinib treatment on A549 cell metabolism in conjunction with various inhibitors. After pretreatment with <t>MG132,</t> 2-DG and rotenone, the effects of 1 µM crizotinib on (A) cell viability, (B) glucose consumption, (C) lactate content and (D) ATP content were determined in A549 cells. *P
    Mg132, supplied by beyotime, used in various techniques. Bioz Stars score: 92/100, based on 154 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore proteasome inhibitors
    Effect of crizotinib treatment on A549 cell metabolism in conjunction with various inhibitors. After pretreatment with <t>MG132,</t> 2-DG and rotenone, the effects of 1 µM crizotinib on (A) cell viability, (B) glucose consumption, (C) lactate content and (D) ATP content were determined in A549 cells. *P
    Proteasome Inhibitors, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 321 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 321 article reviews
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    92
    ApexBio mg132
    Mutations of residues M260A and L261A partly restrained C protein degradation. a Schematic representation of mutant C proteins, C M260-261 and C M260-263 , in which residues 260-261 and 260-263 in C protein are all mutated to Alanine (A), respectively. Mutant residues are shown above the oblong. b C M260-261 and C M260-263 were expressed in PK-15 cells followed by treatment of <t>MG132</t> or DMSO as described above. Western blot was performed and C M260-261 and C M260-263 were detected with the indicated antibodies. Protein marker is shown on the right
    Mg132, supplied by ApexBio, used in various techniques. Bioz Stars score: 92/100, based on 95 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 95 article reviews
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    Millipore m mg132
    Catalase is neither phosphorylated nor ubiquitinated in breast cancer cells. (a) Catalase protein levels were measured after 5 h incubation with proteasome inhibitor <t>MG132</t> in both MCF-7 and Resox cells. (b) Immunoprecipitation (IP) with anticatalase antibody and immunoblotting (IB) with anti-Flag, antiphosphocatalase, and catalase antibodies. Prior IP, cells were transfected with a plasmid pcDNA3 (1 μ g) coding an ubiquitin-Flag for 72 h.
    M Mg132, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 205 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/m mg132/product/Millipore
    Average 99 stars, based on 205 article reviews
    Price from $9.99 to $1999.99
    m mg132 - by Bioz Stars, 2021-01
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    N/A
    The ubiquitin proteasome pathway plays an integral role in the selective degradation of intracellular proteins While important for clearing damaged or mis folded proteins this proteolytic pathway also regulates the
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    N/A
    The ubiquitin proteasome pathway plays an integral role in the selective degradation of intracellular proteins While important for clearing damaged or misfolded proteins this proteolytic pathway also regulates the availability
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    Image Search Results


    Peli1 binds to and mediates Lys48 ubiquitination of NIK. a Immunoblot analysis of NIK and HSP60 in whole-cell lysates of WT and Peli1 -deficient splenic B cells stimulated with anti-CD40 (αCD40). b Analysis of Lys48 ubiquitination of NIK in WT and KO B cells left unstimulated or stimulated with anti-CD40 (αCD40) for 4 h in the presence of a proteasome inhibitor MG132. IP, immunoprecipitation; IB, immunoblotting. c Ubiquitination of NIK in HEK293 cells transfected with (+) or without (−) indicated expression vectors. d Immunoassays on lysates of WT splenic B cells left unstimulated or stimulated with anti-CD40 (αCD40) for 4 h in the presence of MG132, followed by IP with control IgG or anti-NIK and immunoblot analysis of NIK-associated Peli1. e IP analysis examining the association of NIK with Peli1 in HEK293 cells transfected with (+) or without (−) indicated expression vectors. f Immunoblot analysis of Lys48 ubiquitination of NIK assessed by in vitro ubiquitination assay with a mixture of E1, E2 (UbcH5c), ATP, Flag-NIK, HA-Peli1, or HA-Peli1ΔC after IP with anti-Flag. g Immunoblot analysis of p52, p100, NIK, c-IAP2, Peli1, and HSP60 in total lysis of control and Peli1 -knockdown M12 cells that pretreated with DMSO or smac mimetic BV6 for 4 h, and then stimulated with anti-CD40 (αCD40) (upper panel) or BAFF (lower panel) at the indicated time points. Ctrl, control. h Analysis of Lys48 ubiquitination of NIK in control and Peli1 -knockdown M12 cells that pretreated with DMSO or smac mimetic BV6 for 4 h, then left unstimulated or stimulated with anti-CD40 (αCD40) for 4 h in the presence of MG132. Ctrl, control. Data are shown based on three independent experiments

    Journal: Nature Communications

    Article Title: Peli1 negatively regulates noncanonical NF-κB signaling to restrain systemic lupus erythematosus

    doi: 10.1038/s41467-018-03530-3

    Figure Lengend Snippet: Peli1 binds to and mediates Lys48 ubiquitination of NIK. a Immunoblot analysis of NIK and HSP60 in whole-cell lysates of WT and Peli1 -deficient splenic B cells stimulated with anti-CD40 (αCD40). b Analysis of Lys48 ubiquitination of NIK in WT and KO B cells left unstimulated or stimulated with anti-CD40 (αCD40) for 4 h in the presence of a proteasome inhibitor MG132. IP, immunoprecipitation; IB, immunoblotting. c Ubiquitination of NIK in HEK293 cells transfected with (+) or without (−) indicated expression vectors. d Immunoassays on lysates of WT splenic B cells left unstimulated or stimulated with anti-CD40 (αCD40) for 4 h in the presence of MG132, followed by IP with control IgG or anti-NIK and immunoblot analysis of NIK-associated Peli1. e IP analysis examining the association of NIK with Peli1 in HEK293 cells transfected with (+) or without (−) indicated expression vectors. f Immunoblot analysis of Lys48 ubiquitination of NIK assessed by in vitro ubiquitination assay with a mixture of E1, E2 (UbcH5c), ATP, Flag-NIK, HA-Peli1, or HA-Peli1ΔC after IP with anti-Flag. g Immunoblot analysis of p52, p100, NIK, c-IAP2, Peli1, and HSP60 in total lysis of control and Peli1 -knockdown M12 cells that pretreated with DMSO or smac mimetic BV6 for 4 h, and then stimulated with anti-CD40 (αCD40) (upper panel) or BAFF (lower panel) at the indicated time points. Ctrl, control. h Analysis of Lys48 ubiquitination of NIK in control and Peli1 -knockdown M12 cells that pretreated with DMSO or smac mimetic BV6 for 4 h, then left unstimulated or stimulated with anti-CD40 (αCD40) for 4 h in the presence of MG132. Ctrl, control. Data are shown based on three independent experiments

    Article Snippet: MG132 (C2211) and LPS (L3129) were from Sigma.

    Techniques: Immunoprecipitation, Transfection, Expressing, In Vitro, Ubiquitin Assay, Lysis

    Overexpression of Peli1 prevents lupus-like disease. a Immunoblot analysis of p52, p100, NIK, Peli1, Peli1ΔC and HSP60 in Peli1 -knockdown M12 cells that reconstituted with empty vector (EV), WT full-length Peli1 or Peli1ΔC that stimulated with anti-CD40 (αCD40) at the indicated time points. b Analysis of Lys48 ubiquitination of NIK in Peli1 -knockdown M12 cells reconstituted with EV, WT Peli1, or Peli1ΔC that stimulated with anti-CD40 (αCD40) for 4 h in the presence of MG132. c LISA of NP-specific IgM, IgG2a, IgG2b and IgG3 in the serum of Rag1 −/− mice that transferred with WT T cells plus KO B cells reconstituted with EV, WT Peli1 or Peli1ΔC, and then immunized intraperitoneally with NP-KLH. d – h Peli1 -deficient (KO) mice were immunized with 7.5 million of BM12 CD4 + T cell to induce lupus-like disease, and then injected with pRV-GFP retrovirus encoding Peli1 or Peli1ΔC 12 h post-immunization. The IgG deposits in kidney were examined by staining with Alexa Fluor 488-labeled anti-mouse IgG ( d ). Scale bar, 100 μm. The anti-dsDNA, anti-ssDNA, anti-histone IgG in serum were determined by ELISA ( e ). The percentages of CD19 − CD138 + plasma cells, PD-1 + CXCR5 + Tfh cells ( f , g ) and infected B220 + B cells ( h ) in spleens were assessed by flow cytometry. Data are shown as the mean ± SEM based on three independent experiments. Two-tailed Student’s t- tests were performed. * P

    Journal: Nature Communications

    Article Title: Peli1 negatively regulates noncanonical NF-κB signaling to restrain systemic lupus erythematosus

    doi: 10.1038/s41467-018-03530-3

    Figure Lengend Snippet: Overexpression of Peli1 prevents lupus-like disease. a Immunoblot analysis of p52, p100, NIK, Peli1, Peli1ΔC and HSP60 in Peli1 -knockdown M12 cells that reconstituted with empty vector (EV), WT full-length Peli1 or Peli1ΔC that stimulated with anti-CD40 (αCD40) at the indicated time points. b Analysis of Lys48 ubiquitination of NIK in Peli1 -knockdown M12 cells reconstituted with EV, WT Peli1, or Peli1ΔC that stimulated with anti-CD40 (αCD40) for 4 h in the presence of MG132. c LISA of NP-specific IgM, IgG2a, IgG2b and IgG3 in the serum of Rag1 −/− mice that transferred with WT T cells plus KO B cells reconstituted with EV, WT Peli1 or Peli1ΔC, and then immunized intraperitoneally with NP-KLH. d – h Peli1 -deficient (KO) mice were immunized with 7.5 million of BM12 CD4 + T cell to induce lupus-like disease, and then injected with pRV-GFP retrovirus encoding Peli1 or Peli1ΔC 12 h post-immunization. The IgG deposits in kidney were examined by staining with Alexa Fluor 488-labeled anti-mouse IgG ( d ). Scale bar, 100 μm. The anti-dsDNA, anti-ssDNA, anti-histone IgG in serum were determined by ELISA ( e ). The percentages of CD19 − CD138 + plasma cells, PD-1 + CXCR5 + Tfh cells ( f , g ) and infected B220 + B cells ( h ) in spleens were assessed by flow cytometry. Data are shown as the mean ± SEM based on three independent experiments. Two-tailed Student’s t- tests were performed. * P

    Article Snippet: MG132 (C2211) and LPS (L3129) were from Sigma.

    Techniques: Over Expression, Plasmid Preparation, Mouse Assay, Injection, Staining, Labeling, Enzyme-linked Immunosorbent Assay, Infection, Flow Cytometry, Cytometry, Two Tailed Test

    Analysis of p53 half-life and proteasomal degradation. ( a ) CRFKpCEFL and CRFKE6 were treated with protein synthesis inhibitor cycloheximide (20 µg/mL) and collected at different time points indicated in hours (h). Cells were lysed and analysed by WB for p53, then the blot was stripped and reprobed for β-actin to ensure equal protein loading in each lane and allow normalization. A representative gel demonstrating p53 shorter half-life in presence of FcaPV-2 E6 is shown. ( b ) The graph shows the quantification of p53 bands, normalized to β-actin levels and calibrated against the levels of p53 at time 0 of treatment (set as 100%). Results shown are the means +/− standard deviation (SD) of at least three independent experiments and demonstrate accelerated p53 degradation in CRFKE6 compared to CRFKpCEFL at each time point (Time 1: P = 0.057; Time 2.5: P = 0.0345; Time 5: P = 0,0034). ( c ) CRFKpCEFL and CRFKE6 were treated with the proteasome inhibitor MG132 at 30 µM for 4 h and collected to be analysed by WB for p53. Hela were concomitantly analysed as control. The blot was stripped and reprobed for β-actin to ensure equal protein loading in each lane and allow normalization. ( d ) Accumulation upon MG132 treatment in CRFKpCEFL with respect to untreated cells was calculated by densitometric quantification of p53 bands normalized to β-actin levels and arbitrarily set as 100% (white bar). The same calculation was applied to treated vs untreated CRFKE6 and the difference in the % of accumulation obtained by ratio with the value pointed out in CRFKpCEFL (black bar). Results shown are the means +/− SD of four independent experiments and demonstrate higher % of p53 accumulation in CRFKE6 compared to CRFKpCEFL (P = 0.0152).

    Journal: Scientific Reports

    Article Title: Felis catus papillomavirus type-2 E6 binds to E6AP, promotes E6AP/p53 binding and enhances p53 proteasomal degradation

    doi: 10.1038/s41598-018-35723-7

    Figure Lengend Snippet: Analysis of p53 half-life and proteasomal degradation. ( a ) CRFKpCEFL and CRFKE6 were treated with protein synthesis inhibitor cycloheximide (20 µg/mL) and collected at different time points indicated in hours (h). Cells were lysed and analysed by WB for p53, then the blot was stripped and reprobed for β-actin to ensure equal protein loading in each lane and allow normalization. A representative gel demonstrating p53 shorter half-life in presence of FcaPV-2 E6 is shown. ( b ) The graph shows the quantification of p53 bands, normalized to β-actin levels and calibrated against the levels of p53 at time 0 of treatment (set as 100%). Results shown are the means +/− standard deviation (SD) of at least three independent experiments and demonstrate accelerated p53 degradation in CRFKE6 compared to CRFKpCEFL at each time point (Time 1: P = 0.057; Time 2.5: P = 0.0345; Time 5: P = 0,0034). ( c ) CRFKpCEFL and CRFKE6 were treated with the proteasome inhibitor MG132 at 30 µM for 4 h and collected to be analysed by WB for p53. Hela were concomitantly analysed as control. The blot was stripped and reprobed for β-actin to ensure equal protein loading in each lane and allow normalization. ( d ) Accumulation upon MG132 treatment in CRFKpCEFL with respect to untreated cells was calculated by densitometric quantification of p53 bands normalized to β-actin levels and arbitrarily set as 100% (white bar). The same calculation was applied to treated vs untreated CRFKE6 and the difference in the % of accumulation obtained by ratio with the value pointed out in CRFKpCEFL (black bar). Results shown are the means +/− SD of four independent experiments and demonstrate higher % of p53 accumulation in CRFKE6 compared to CRFKpCEFL (P = 0.0152).

    Article Snippet: For p53 half-life experiments, 2 × 105 cells were seeded in 6-well plates and, after 24 hours (h), treated with the protein synthesis inhibitor cycloheximide (Sigma #C7698-1G) at 20 µg/mL for 0, 0.5, 1, 2.5, 5 h, or with the proteasome inhibitor MG132 (Sigma #C2211-5MG) at 30 µM for 4 h. In control plates, drugs were replaced with sterile water or DMSO.

    Techniques: Western Blot, Standard Deviation

    Photographs of immunoblots of electrophoretically separated proteins from lysates of rabbit skin cells infected with HSV-1(F), HSV-1(vCPc0), or HSV-1(vCPc0)R in the presence or absence of MG132. Replicate cultures in 25-cm 2 flasks of rabbit skin cells were either mock infected or exposed to 5 (A) or 10 to 100 (B) PFU of corresponding virus per cell. At 3 h after infection, one set of cultures was replenished with fresh untreated medium (lanes 1, 3, 5, and 7) whereas a second set was replenished with medium containing 10 μM MG132 (lanes 2, 4, 6, and 8). The cells were harvested at 24 h after infection. Proteins were solubilized in disruption buffer and electrophoretically separated in 11% denaturing polyacrylamide gels, transferred to nitrocellulose sheets, and reacted with monoclonal antibodies to ICP0, ICP4, ICP8, ICP27, αTIF, or U S 11 or polyclonal antibody to ICP22.

    Journal: Journal of Virology

    Article Title: An Early Regulatory Function Required in a Cell Type-Dependent Manner Is Expressed by the Genomic but Not the cDNA Copy of the Herpes Simplex Virus 1 Gene Encoding Infected Cell Protein 0

    doi: 10.1128/JVI.76.19.9744-9755.2002

    Figure Lengend Snippet: Photographs of immunoblots of electrophoretically separated proteins from lysates of rabbit skin cells infected with HSV-1(F), HSV-1(vCPc0), or HSV-1(vCPc0)R in the presence or absence of MG132. Replicate cultures in 25-cm 2 flasks of rabbit skin cells were either mock infected or exposed to 5 (A) or 10 to 100 (B) PFU of corresponding virus per cell. At 3 h after infection, one set of cultures was replenished with fresh untreated medium (lanes 1, 3, 5, and 7) whereas a second set was replenished with medium containing 10 μM MG132 (lanes 2, 4, 6, and 8). The cells were harvested at 24 h after infection. Proteins were solubilized in disruption buffer and electrophoretically separated in 11% denaturing polyacrylamide gels, transferred to nitrocellulose sheets, and reacted with monoclonal antibodies to ICP0, ICP4, ICP8, ICP27, αTIF, or U S 11 or polyclonal antibody to ICP22.

    Article Snippet: The defect in the accumulation of viral proteins in the presence of MG132 maps to the α0 gene inasmuch as the partially rescued virus HSV-1(vCPc0)R cannot be differentiated from the wild-type virus. (ii) The stage of arrest of the HSV-1(vCPc0) mutant in cells exposed to MG132 at 3 h after infection is similar to that of wild-type HSV-1(F) in cells exposed to MG132 before infection. (iii) Even a very high MOI did not overcome the inhibitory effect of MG132 in cells exposed to the drug at 3 h after infection with the HSV-1(vCPc0) mutant.

    Techniques: Western Blot, Infection

    Photographs of immunoblots of electrophoretically separated proteins from Vero, rabbit skin (RSC), 143TK−, and HEp-2 cells infected with HSV-1(F) or HSV-1(vCPc0) in the presence or absence of MG132. Replicate cultures in 25-cm 2 flasks of Vero cells (A), rabbit skin cells (RSC) (B), 143TK− cells (C), or HEp-2 cells (D) were either mock infected or infected with 5 PFU of virus per cell. At 3 h after infection, the medium of one set of cultures was replaced with fresh medium (lanes 1 through 3) whereas a replicate set was replenished with medium supplemented with 10 μM MG132 (lanes 4 through 6). The cells were harvested at 24 h after infection. Proteins were solubilized in disruption buffer and electrophoretically separated in 11% denaturing polyacrylamide gels, transferred to nitrocellulose sheets, and reacted with monoclonal antibodies to ICP0 and U S 11 or polyclonal rabbit antibody to ICP22 as described in Materials and Methods.

    Journal: Journal of Virology

    Article Title: An Early Regulatory Function Required in a Cell Type-Dependent Manner Is Expressed by the Genomic but Not the cDNA Copy of the Herpes Simplex Virus 1 Gene Encoding Infected Cell Protein 0

    doi: 10.1128/JVI.76.19.9744-9755.2002

    Figure Lengend Snippet: Photographs of immunoblots of electrophoretically separated proteins from Vero, rabbit skin (RSC), 143TK−, and HEp-2 cells infected with HSV-1(F) or HSV-1(vCPc0) in the presence or absence of MG132. Replicate cultures in 25-cm 2 flasks of Vero cells (A), rabbit skin cells (RSC) (B), 143TK− cells (C), or HEp-2 cells (D) were either mock infected or infected with 5 PFU of virus per cell. At 3 h after infection, the medium of one set of cultures was replaced with fresh medium (lanes 1 through 3) whereas a replicate set was replenished with medium supplemented with 10 μM MG132 (lanes 4 through 6). The cells were harvested at 24 h after infection. Proteins were solubilized in disruption buffer and electrophoretically separated in 11% denaturing polyacrylamide gels, transferred to nitrocellulose sheets, and reacted with monoclonal antibodies to ICP0 and U S 11 or polyclonal rabbit antibody to ICP22 as described in Materials and Methods.

    Article Snippet: The defect in the accumulation of viral proteins in the presence of MG132 maps to the α0 gene inasmuch as the partially rescued virus HSV-1(vCPc0)R cannot be differentiated from the wild-type virus. (ii) The stage of arrest of the HSV-1(vCPc0) mutant in cells exposed to MG132 at 3 h after infection is similar to that of wild-type HSV-1(F) in cells exposed to MG132 before infection. (iii) Even a very high MOI did not overcome the inhibitory effect of MG132 in cells exposed to the drug at 3 h after infection with the HSV-1(vCPc0) mutant.

    Techniques: Western Blot, Infection

    Effect of expression of α0-intron 1 sequences delivered in trans on the expression of α0 and U S 11 genes in HSV-1(vCPc0) infection in the presence or absence of MG132. (A) Experimental design. Replicate cultures in 25-cm 2 flasks of rabbit skin cells were either mock infected (exposed to insect cell medium [lanes 3 and 10]) or infected with baculoviruses expressing the α0-intron 1 sequence from HSV-1 strains F, KOS, or 17 at MOIs of 40, 25, or 10, respectively (lanes 4 through 6 and 11 through 13) or baculovirus lacking introns at a MOI of 30 (lanes 7 and 14). Cells were maintained at 37°C in Dulbecco's modified Eagle's medium supplemented with 5% newborn calf serum and 7 mM sodium butyrate (Sigma). At 8 h after infection, the cells were infected with 5 PFU of HSV-1(vCPc0) per cell. In addition, replicate cultures not previously exposed to baculoviruses (or insect cell medium) were mock infected (exposed to 199V medium [lanes 1 and 8]) or infected with HSV-1(vCPc0) (lanes 2 and 9) at this time. The cells were maintained in 199V medium supplemented with 7 mM sodium butyrate. Three hours later, the medium of one set of cultures was replaced with fresh medium (C, lanes 8 through 14) whereas a replicate set was replenished with medium supplemented with 10 μM MG132 (B, lanes 1 through 7). The cells were harvested at 24 h after infection with HSV-1(vCPc0). (B and C) Photographs of immunoblots of electrophoretically separated proteins from MG132-treated (B) and untreated (C) cells. Proteins were solubilized in disruption buffer and electrophoretically separated on 11% denaturing polyacrylamide gels, transferred to nitrocellulose sheets, and reacted with monoclonal antibodies to ICP0 and U S 11.

    Journal: Journal of Virology

    Article Title: An Early Regulatory Function Required in a Cell Type-Dependent Manner Is Expressed by the Genomic but Not the cDNA Copy of the Herpes Simplex Virus 1 Gene Encoding Infected Cell Protein 0

    doi: 10.1128/JVI.76.19.9744-9755.2002

    Figure Lengend Snippet: Effect of expression of α0-intron 1 sequences delivered in trans on the expression of α0 and U S 11 genes in HSV-1(vCPc0) infection in the presence or absence of MG132. (A) Experimental design. Replicate cultures in 25-cm 2 flasks of rabbit skin cells were either mock infected (exposed to insect cell medium [lanes 3 and 10]) or infected with baculoviruses expressing the α0-intron 1 sequence from HSV-1 strains F, KOS, or 17 at MOIs of 40, 25, or 10, respectively (lanes 4 through 6 and 11 through 13) or baculovirus lacking introns at a MOI of 30 (lanes 7 and 14). Cells were maintained at 37°C in Dulbecco's modified Eagle's medium supplemented with 5% newborn calf serum and 7 mM sodium butyrate (Sigma). At 8 h after infection, the cells were infected with 5 PFU of HSV-1(vCPc0) per cell. In addition, replicate cultures not previously exposed to baculoviruses (or insect cell medium) were mock infected (exposed to 199V medium [lanes 1 and 8]) or infected with HSV-1(vCPc0) (lanes 2 and 9) at this time. The cells were maintained in 199V medium supplemented with 7 mM sodium butyrate. Three hours later, the medium of one set of cultures was replaced with fresh medium (C, lanes 8 through 14) whereas a replicate set was replenished with medium supplemented with 10 μM MG132 (B, lanes 1 through 7). The cells were harvested at 24 h after infection with HSV-1(vCPc0). (B and C) Photographs of immunoblots of electrophoretically separated proteins from MG132-treated (B) and untreated (C) cells. Proteins were solubilized in disruption buffer and electrophoretically separated on 11% denaturing polyacrylamide gels, transferred to nitrocellulose sheets, and reacted with monoclonal antibodies to ICP0 and U S 11.

    Article Snippet: The defect in the accumulation of viral proteins in the presence of MG132 maps to the α0 gene inasmuch as the partially rescued virus HSV-1(vCPc0)R cannot be differentiated from the wild-type virus. (ii) The stage of arrest of the HSV-1(vCPc0) mutant in cells exposed to MG132 at 3 h after infection is similar to that of wild-type HSV-1(F) in cells exposed to MG132 before infection. (iii) Even a very high MOI did not overcome the inhibitory effect of MG132 in cells exposed to the drug at 3 h after infection with the HSV-1(vCPc0) mutant.

    Techniques: Expressing, Infection, Sequencing, Modification, Western Blot

    HSV-1 blocks apoptosis induced by MG132. (A and B) Wild-type and repaired viruses block apoptosis induced by MG132. (A) Experimental design. Replicate cultures in 25-cm 2 flasks of rabbit skin cells were either mock infected or infected with 10 PFU of HSV-1(F), HSV-1(vCPc0), or HSV-1(vCPc0)R per cell. At 3 h after infection, the medium of one set of cultures was replaced with fresh medium (lanes 1 through 4) whereas that of the second set was replenished with medium supplemented with 10 μM MG132 (lanes 5 through 8). The cells were harvested at 24 h after infection. (B and B′) Photographs of positive and negative images of ethidium bromide-stained low-molecular-weight DNA from mock-infected and HSV-1-infected cells. Cells were harvested and processed as described in Materials and Methods. DNA was separated in 1.5% agarose gels containing 0.5 μg of ethidium bromide/ml. Oligonucleosomal DNA fragments were visualized by UV light transillumination. Photographs were taken with the aid of Eagle Eye II (Stratagene), and both positive and reverse images are shown to help visualize bands of fragmented cellular DNA. (C and D) Minimal interval between time of infection and time of addition of MG132 necessary to block fragmentation of cellular DNA. (C) Experimental design. Replicate cultures of rabbit skin cells grown in 25-cm 2 flasks were exposed to MG132 at a fixed time (zero time). The cultures were infected with 10 PFU per cell of HSV-1(F) either 4, 3, 2, or 1 h before exposure to the drug, simultaneously with the addition of the drug, or at 1 or 2 h after exposure to the drug (D and D′, lower panels). Cells were harvested and processed as described in Materials and Methods. Replicate cultures either mock infected or infected with HSV-1(F) virus and maintained in medium without MG132 (D and D′, top panels, lanes 1 through 6) were processed in a similar manner. (D and D′) Photographs of positive and negative images of ethidium bromide-stained low-molecular-weight DNA from mock-infected and HSV-1(F)-infected cells. Cells were harvested and processed as described in Materials and Methods. DNA was separated and visualized as described above.

    Journal: Journal of Virology

    Article Title: An Early Regulatory Function Required in a Cell Type-Dependent Manner Is Expressed by the Genomic but Not the cDNA Copy of the Herpes Simplex Virus 1 Gene Encoding Infected Cell Protein 0

    doi: 10.1128/JVI.76.19.9744-9755.2002

    Figure Lengend Snippet: HSV-1 blocks apoptosis induced by MG132. (A and B) Wild-type and repaired viruses block apoptosis induced by MG132. (A) Experimental design. Replicate cultures in 25-cm 2 flasks of rabbit skin cells were either mock infected or infected with 10 PFU of HSV-1(F), HSV-1(vCPc0), or HSV-1(vCPc0)R per cell. At 3 h after infection, the medium of one set of cultures was replaced with fresh medium (lanes 1 through 4) whereas that of the second set was replenished with medium supplemented with 10 μM MG132 (lanes 5 through 8). The cells were harvested at 24 h after infection. (B and B′) Photographs of positive and negative images of ethidium bromide-stained low-molecular-weight DNA from mock-infected and HSV-1-infected cells. Cells were harvested and processed as described in Materials and Methods. DNA was separated in 1.5% agarose gels containing 0.5 μg of ethidium bromide/ml. Oligonucleosomal DNA fragments were visualized by UV light transillumination. Photographs were taken with the aid of Eagle Eye II (Stratagene), and both positive and reverse images are shown to help visualize bands of fragmented cellular DNA. (C and D) Minimal interval between time of infection and time of addition of MG132 necessary to block fragmentation of cellular DNA. (C) Experimental design. Replicate cultures of rabbit skin cells grown in 25-cm 2 flasks were exposed to MG132 at a fixed time (zero time). The cultures were infected with 10 PFU per cell of HSV-1(F) either 4, 3, 2, or 1 h before exposure to the drug, simultaneously with the addition of the drug, or at 1 or 2 h after exposure to the drug (D and D′, lower panels). Cells were harvested and processed as described in Materials and Methods. Replicate cultures either mock infected or infected with HSV-1(F) virus and maintained in medium without MG132 (D and D′, top panels, lanes 1 through 6) were processed in a similar manner. (D and D′) Photographs of positive and negative images of ethidium bromide-stained low-molecular-weight DNA from mock-infected and HSV-1(F)-infected cells. Cells were harvested and processed as described in Materials and Methods. DNA was separated and visualized as described above.

    Article Snippet: The defect in the accumulation of viral proteins in the presence of MG132 maps to the α0 gene inasmuch as the partially rescued virus HSV-1(vCPc0)R cannot be differentiated from the wild-type virus. (ii) The stage of arrest of the HSV-1(vCPc0) mutant in cells exposed to MG132 at 3 h after infection is similar to that of wild-type HSV-1(F) in cells exposed to MG132 before infection. (iii) Even a very high MOI did not overcome the inhibitory effect of MG132 in cells exposed to the drug at 3 h after infection with the HSV-1(vCPc0) mutant.

    Techniques: Blocking Assay, Infection, Staining, Molecular Weight

    Effect of the time of MG132 exposure on viral gene expression. Replicate cultures in 25-cm 2 flasks of Vero (A) or rabbit skin (B) cells were either mock infected or exposed to 1, 10, or 100 PFU of HSV-1(F) per cell. Cells were either untreated (lanes 1 through 4) or treated with 10 μM MG132 at 2 h before (lanes 5 through 7) or 3 h after (lanes 8 through 10) virus infection. At 22 h after infection, cells were harvested, solubilized in disruption buffer, electrophoretically separated in 11% denaturing polyacrylamide gels, transferred to nitrocellulose sheets, and reacted with monoclonal antibody to ICP0 or U S 11 or polyclonal antibody to ICP22 as described in Materials and Methods.

    Journal: Journal of Virology

    Article Title: An Early Regulatory Function Required in a Cell Type-Dependent Manner Is Expressed by the Genomic but Not the cDNA Copy of the Herpes Simplex Virus 1 Gene Encoding Infected Cell Protein 0

    doi: 10.1128/JVI.76.19.9744-9755.2002

    Figure Lengend Snippet: Effect of the time of MG132 exposure on viral gene expression. Replicate cultures in 25-cm 2 flasks of Vero (A) or rabbit skin (B) cells were either mock infected or exposed to 1, 10, or 100 PFU of HSV-1(F) per cell. Cells were either untreated (lanes 1 through 4) or treated with 10 μM MG132 at 2 h before (lanes 5 through 7) or 3 h after (lanes 8 through 10) virus infection. At 22 h after infection, cells were harvested, solubilized in disruption buffer, electrophoretically separated in 11% denaturing polyacrylamide gels, transferred to nitrocellulose sheets, and reacted with monoclonal antibody to ICP0 or U S 11 or polyclonal antibody to ICP22 as described in Materials and Methods.

    Article Snippet: The defect in the accumulation of viral proteins in the presence of MG132 maps to the α0 gene inasmuch as the partially rescued virus HSV-1(vCPc0)R cannot be differentiated from the wild-type virus. (ii) The stage of arrest of the HSV-1(vCPc0) mutant in cells exposed to MG132 at 3 h after infection is similar to that of wild-type HSV-1(F) in cells exposed to MG132 before infection. (iii) Even a very high MOI did not overcome the inhibitory effect of MG132 in cells exposed to the drug at 3 h after infection with the HSV-1(vCPc0) mutant.

    Techniques: Expressing, Infection

    Transcriptional and protein consequences of PLK4 mutations a) Schematic of the human PLK4 gene. Coding exons (black), UTRs (white), alternatively spliced region in exon 5 (grey), arrow heads, RT-PCR primer positions. b) PLK4 protein domain structure. Kinase domain (KD)( grey), PEST sequences (1-3) (black), polo-box domains (PBD1-3) (blue/turquoise/green). Middle panel, the c.2811-5G > C mutation creates a new splice acceptor site that leads to retention of 4 bp of intron 15 sequence in the PLK4 mRNA, resulting in premature truncation of the protein at its C-terminus, disrupting the terminal polo-box domain (PB3). Lower panel depicts domain structure for the alternative isoform (ALT) resulting from use of an internal exon 5 splice donor site. c) Sequence electropherograms of the exon 15-16 junction of PLK4 amplified by RT-PCR from patient and control RNA. d) Alternative splicing of an internal exon 5 splice donor site is not detected in other vertebrates by RT-PCR. e) Levels of functional PLK4 are reduced to 25% of normal levels, in patients with the c.1299_1309delTAAAG mutation. Transcript levels plotted from quantitative RT-PCR on RNA extracted from patient primary fibroblast cell lines (n=3 experiments (exp), performed in triplicate; error bars, s.e.m). P value, two-tailed t-test *p≤0.05. Further details and characterization of PLK4 ALT and FL transcript levels, Fig. S5 . f) Immunoblotting demonstrates reduced endogenous PLK4 protein levels in patient fibroblasts. Cell lysates of asynchronous cells. Left two lanes, RNAi of PLK4 in RPE1 cells demonstrating specificity of PLK4 antibody, with two PLK4-specific protein bands visualized. Remaining lanes, patient and control primary fibroblasts, upper panel standard exposure PLK4 immunoblot with middle panel overexposed to demonstrate residual detectable protein in P1 patient. Note also loss of the upper PLK4 band in P6 and P7. Lower panel, loading control, blot probed with anti-Actin antibody. g) PLK4 protein levels at the centrosome are reduced in patient fibroblasts. Top panel, representative immunofluorescence images of primary fibroblasts treated with 10μM MG132 for 5 hrs prior to fixation. (Specificity of the anti-PLK4 antibody confirmed by performing RNAi-mediated PLK4 depletion, Fig. S6 ). Scale bar = 10 μm. Bottom panel, quantitation of PLK4 protein levels at the centrosome by immunofluorescence analysis. Exp=3, n=50 cells/exp; error bars, s.e.m of n=150 cells. P value, two-tailed t-test ****p≤0.0001.

    Journal: Nature genetics

    Article Title: Mutations in PLK4, encoding a master regulator of centriole biogenesis, cause microcephaly, growth failure and retinopathy

    doi: 10.1038/ng.3122

    Figure Lengend Snippet: Transcriptional and protein consequences of PLK4 mutations a) Schematic of the human PLK4 gene. Coding exons (black), UTRs (white), alternatively spliced region in exon 5 (grey), arrow heads, RT-PCR primer positions. b) PLK4 protein domain structure. Kinase domain (KD)( grey), PEST sequences (1-3) (black), polo-box domains (PBD1-3) (blue/turquoise/green). Middle panel, the c.2811-5G > C mutation creates a new splice acceptor site that leads to retention of 4 bp of intron 15 sequence in the PLK4 mRNA, resulting in premature truncation of the protein at its C-terminus, disrupting the terminal polo-box domain (PB3). Lower panel depicts domain structure for the alternative isoform (ALT) resulting from use of an internal exon 5 splice donor site. c) Sequence electropherograms of the exon 15-16 junction of PLK4 amplified by RT-PCR from patient and control RNA. d) Alternative splicing of an internal exon 5 splice donor site is not detected in other vertebrates by RT-PCR. e) Levels of functional PLK4 are reduced to 25% of normal levels, in patients with the c.1299_1309delTAAAG mutation. Transcript levels plotted from quantitative RT-PCR on RNA extracted from patient primary fibroblast cell lines (n=3 experiments (exp), performed in triplicate; error bars, s.e.m). P value, two-tailed t-test *p≤0.05. Further details and characterization of PLK4 ALT and FL transcript levels, Fig. S5 . f) Immunoblotting demonstrates reduced endogenous PLK4 protein levels in patient fibroblasts. Cell lysates of asynchronous cells. Left two lanes, RNAi of PLK4 in RPE1 cells demonstrating specificity of PLK4 antibody, with two PLK4-specific protein bands visualized. Remaining lanes, patient and control primary fibroblasts, upper panel standard exposure PLK4 immunoblot with middle panel overexposed to demonstrate residual detectable protein in P1 patient. Note also loss of the upper PLK4 band in P6 and P7. Lower panel, loading control, blot probed with anti-Actin antibody. g) PLK4 protein levels at the centrosome are reduced in patient fibroblasts. Top panel, representative immunofluorescence images of primary fibroblasts treated with 10μM MG132 for 5 hrs prior to fixation. (Specificity of the anti-PLK4 antibody confirmed by performing RNAi-mediated PLK4 depletion, Fig. S6 ). Scale bar = 10 μm. Bottom panel, quantitation of PLK4 protein levels at the centrosome by immunofluorescence analysis. Exp=3, n=50 cells/exp; error bars, s.e.m of n=150 cells. P value, two-tailed t-test ****p≤0.0001.

    Article Snippet: Where indicated, cells were treated with 10 μM MG132 (Cayman Chemicals) or 1.5 μM staurosporine (Alexis Biochemicals) for 5 hrs.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Sequencing, Amplification, Functional Assay, Quantitative RT-PCR, Two Tailed Test, Immunofluorescence, Quantitation Assay

    The initial endocytosis of mLRP4 is slowed, but not blocked by proteasomal inhibitors. CHO-mLRP4 cells were pretreated with DMSO or DMSO containing MG132 (20 μM) for either 30 min (A) or 2 h (B). The initial endocytosis rates of mLRP4 were measured using 125 I-RAP as described in MATERIALS AND METHODS. Data represent triplicate determination with SE values given as error bars.

    Journal: Molecular Biology of the Cell

    Article Title: Proteasome Regulates the Delivery of LDL Receptor-related Protein into the Degradation Pathway

    doi: 10.1091/mbc.E02-03-0152

    Figure Lengend Snippet: The initial endocytosis of mLRP4 is slowed, but not blocked by proteasomal inhibitors. CHO-mLRP4 cells were pretreated with DMSO or DMSO containing MG132 (20 μM) for either 30 min (A) or 2 h (B). The initial endocytosis rates of mLRP4 were measured using 125 I-RAP as described in MATERIALS AND METHODS. Data represent triplicate determination with SE values given as error bars.

    Article Snippet: MG132 (Z-Leu-leu-leu-H aldehyde) proteasomal inhibitor was from Peptide Institute, Inc (Minosh-shi Osaka, Japan).

    Techniques:

    Proteasome inhibitors increase surface localization of mLRP4 and block the delivery of mLRP4 into MVBs. Cryosections of CHO-mLRP4 cells were immunogold labeled with anti-HA antibodies and 10 nm gold particles. (A, C, and D) control cells; (B and E) MG132-treated cells. In control cells (A) by far the majority of mLRP4 at the plasma membrane is present in clathrin-coated pits, whereas in cells treated with MG132 (B and E) label is also present outside the pits. (C and D) MVBs in control cells with label present at the limiting membrane and associated with the internal vesicles. Note the thick clathrin lattices at the limiting membrane (arrows). (E) Peripheral cytoplasm of a MG132-treated cell with labeled clathrin-coated vesicles and long recycling tubules (arrowheads). All bars represent 200 nm.

    Journal: Molecular Biology of the Cell

    Article Title: Proteasome Regulates the Delivery of LDL Receptor-related Protein into the Degradation Pathway

    doi: 10.1091/mbc.E02-03-0152

    Figure Lengend Snippet: Proteasome inhibitors increase surface localization of mLRP4 and block the delivery of mLRP4 into MVBs. Cryosections of CHO-mLRP4 cells were immunogold labeled with anti-HA antibodies and 10 nm gold particles. (A, C, and D) control cells; (B and E) MG132-treated cells. In control cells (A) by far the majority of mLRP4 at the plasma membrane is present in clathrin-coated pits, whereas in cells treated with MG132 (B and E) label is also present outside the pits. (C and D) MVBs in control cells with label present at the limiting membrane and associated with the internal vesicles. Note the thick clathrin lattices at the limiting membrane (arrows). (E) Peripheral cytoplasm of a MG132-treated cell with labeled clathrin-coated vesicles and long recycling tubules (arrowheads). All bars represent 200 nm.

    Article Snippet: MG132 (Z-Leu-leu-leu-H aldehyde) proteasomal inhibitor was from Peptide Institute, Inc (Minosh-shi Osaka, Japan).

    Techniques: Blocking Assay, Labeling

    The proteasomal inhibitor MG132 increases the half-life of a LRP minireceptor, mLRP4. CHO-mLRP4 cells were metabolically labeled with [ 35 S]cysteine for 30 min and chased for the indicated times, in the absence or presence of MG132 (20 μM). After each chase, cell lysates were immunoprecipitated with anti-HA antibody and analyzed with SDS gels (6%) under reducing conditions. The molecular size markers in this and subsequent figures are given in kilodaltons on the left. The positions of the precursor and the mature forms of mLRP4 are marked. (B) The band intensities in A (includes the precursor and the mature forms) were quantitated and plotted against chase time.

    Journal: Molecular Biology of the Cell

    Article Title: Proteasome Regulates the Delivery of LDL Receptor-related Protein into the Degradation Pathway

    doi: 10.1091/mbc.E02-03-0152

    Figure Lengend Snippet: The proteasomal inhibitor MG132 increases the half-life of a LRP minireceptor, mLRP4. CHO-mLRP4 cells were metabolically labeled with [ 35 S]cysteine for 30 min and chased for the indicated times, in the absence or presence of MG132 (20 μM). After each chase, cell lysates were immunoprecipitated with anti-HA antibody and analyzed with SDS gels (6%) under reducing conditions. The molecular size markers in this and subsequent figures are given in kilodaltons on the left. The positions of the precursor and the mature forms of mLRP4 are marked. (B) The band intensities in A (includes the precursor and the mature forms) were quantitated and plotted against chase time.

    Article Snippet: MG132 (Z-Leu-leu-leu-H aldehyde) proteasomal inhibitor was from Peptide Institute, Inc (Minosh-shi Osaka, Japan).

    Techniques: Metabolic Labelling, Labeling, Immunoprecipitation

    Treatment of CHO-mLRP4 cells with proteasomal inhibitors increases the steady state levels of mLRP4. (A) CHO-mLRP4 cells were treated with either vehicle DMSO or DMSO containing MG132 (20 μM) for various periods of time. Equal quantities of cell lysates were separated via SDS gel (6%) and blotted with anti-HA antibody. Only the precursor form and the ligand-binding subunit of the mature form are detected in the blot because these forms, but not the 85-kDa subunit mature form, contain the HA epitope. Note the increase of the mature form, but not the precursor form of mLRP4 after MG132 treatment. (B) The effects of proteasomal inhibition on steady state levels of mLRP4 were also seen with lactacystin, but not with vehicle DMSO alone.

    Journal: Molecular Biology of the Cell

    Article Title: Proteasome Regulates the Delivery of LDL Receptor-related Protein into the Degradation Pathway

    doi: 10.1091/mbc.E02-03-0152

    Figure Lengend Snippet: Treatment of CHO-mLRP4 cells with proteasomal inhibitors increases the steady state levels of mLRP4. (A) CHO-mLRP4 cells were treated with either vehicle DMSO or DMSO containing MG132 (20 μM) for various periods of time. Equal quantities of cell lysates were separated via SDS gel (6%) and blotted with anti-HA antibody. Only the precursor form and the ligand-binding subunit of the mature form are detected in the blot because these forms, but not the 85-kDa subunit mature form, contain the HA epitope. Note the increase of the mature form, but not the precursor form of mLRP4 after MG132 treatment. (B) The effects of proteasomal inhibition on steady state levels of mLRP4 were also seen with lactacystin, but not with vehicle DMSO alone.

    Article Snippet: MG132 (Z-Leu-leu-leu-H aldehyde) proteasomal inhibitor was from Peptide Institute, Inc (Minosh-shi Osaka, Japan).

    Techniques: SDS-Gel, Ligand Binding Assay, Inhibition

    Proteasomal inhibitors increase cell surface mLRP4. (A) CHO-mLRP4 cells were treated with DMSO or DMSO containing MG132 (20 μM) for 2 h. Cells were then dissociated and immunostained with anti-HA antibody. Cell surface mLRP4 was detected with goat anti-mouse Ig-FITC and FACS analysis. Background fluorescence intensity was assessed in the absence of primary antibody and subtracted from each assay. The mean values from representative triplicate determinations were averaged with the SE value given as error bars. (B) Kinetic analyses of cell surface mLRP4 after treatment with proteasomal inhibitors. CHO-mLRP4 cells were treated with MG132 (20 μM) for various periods of time as indicated. Treatment with the alternative proteasome inhibitor lactacystin (20 μM) or vehicle DMSO alone were also performed at selected time points. Cell surface mLRP4 was determined via FACS analysis as indicated above. Shown in the figure are results of a representative experiment. Note the gradual increase of cell surface mLRP4 upon treatment with proteasomal inhibitors, but not DMSO.

    Journal: Molecular Biology of the Cell

    Article Title: Proteasome Regulates the Delivery of LDL Receptor-related Protein into the Degradation Pathway

    doi: 10.1091/mbc.E02-03-0152

    Figure Lengend Snippet: Proteasomal inhibitors increase cell surface mLRP4. (A) CHO-mLRP4 cells were treated with DMSO or DMSO containing MG132 (20 μM) for 2 h. Cells were then dissociated and immunostained with anti-HA antibody. Cell surface mLRP4 was detected with goat anti-mouse Ig-FITC and FACS analysis. Background fluorescence intensity was assessed in the absence of primary antibody and subtracted from each assay. The mean values from representative triplicate determinations were averaged with the SE value given as error bars. (B) Kinetic analyses of cell surface mLRP4 after treatment with proteasomal inhibitors. CHO-mLRP4 cells were treated with MG132 (20 μM) for various periods of time as indicated. Treatment with the alternative proteasome inhibitor lactacystin (20 μM) or vehicle DMSO alone were also performed at selected time points. Cell surface mLRP4 was determined via FACS analysis as indicated above. Shown in the figure are results of a representative experiment. Note the gradual increase of cell surface mLRP4 upon treatment with proteasomal inhibitors, but not DMSO.

    Article Snippet: MG132 (Z-Leu-leu-leu-H aldehyde) proteasomal inhibitor was from Peptide Institute, Inc (Minosh-shi Osaka, Japan).

    Techniques: FACS, Fluorescence

    A functional ubiquitin conjugation system is not required for mLRP4-mediated endocytosis. CHO-ts20-mLRP4 or CHO-ts20-GHR cells were incubated with Cy3-RAP (20 nM) for 10 min or Cy3-GH for 30 min, respectively, under the conditions indicated. Cells were washed, fixed, and examined with a confocal microscope. At the permissive temperature (30°C), endocytosis of both Cy3-RAP (A) and Cy3-GH (B) is observed by the appearance of punctuate fluorescence representing ligand distribution in endocytic compartments. The endocytosis of Cy3-GH, but not Cy3-RAP, was inhibited at the nonpermissive temperature (41.5°C), and in the presence of the proteasomal inhibitor MG132 (C-F).

    Journal: Molecular Biology of the Cell

    Article Title: Proteasome Regulates the Delivery of LDL Receptor-related Protein into the Degradation Pathway

    doi: 10.1091/mbc.E02-03-0152

    Figure Lengend Snippet: A functional ubiquitin conjugation system is not required for mLRP4-mediated endocytosis. CHO-ts20-mLRP4 or CHO-ts20-GHR cells were incubated with Cy3-RAP (20 nM) for 10 min or Cy3-GH for 30 min, respectively, under the conditions indicated. Cells were washed, fixed, and examined with a confocal microscope. At the permissive temperature (30°C), endocytosis of both Cy3-RAP (A) and Cy3-GH (B) is observed by the appearance of punctuate fluorescence representing ligand distribution in endocytic compartments. The endocytosis of Cy3-GH, but not Cy3-RAP, was inhibited at the nonpermissive temperature (41.5°C), and in the presence of the proteasomal inhibitor MG132 (C-F).

    Article Snippet: MG132 (Z-Leu-leu-leu-H aldehyde) proteasomal inhibitor was from Peptide Institute, Inc (Minosh-shi Osaka, Japan).

    Techniques: Functional Assay, Conjugation Assay, Incubation, Microscopy, Fluorescence

    The proteasomal inhibitor MG132 prolongs the cellular half-life of LRP. (A) HepG2 cells were metabolically labeled with [ 35 S]cysteine for 30 min and chased for the indicated times, in the absence or presence of MG132 (20 μM). After each chase, cell lysates were immunoprecipitated with anti-LRP antibody and analyzed with SDS gels (5%) under reducing conditions. The 200-kDa molecular size marker is indicated. (B) The band intensities in A were quantitated and plotted against chase times.

    Journal: Molecular Biology of the Cell

    Article Title: Proteasome Regulates the Delivery of LDL Receptor-related Protein into the Degradation Pathway

    doi: 10.1091/mbc.E02-03-0152

    Figure Lengend Snippet: The proteasomal inhibitor MG132 prolongs the cellular half-life of LRP. (A) HepG2 cells were metabolically labeled with [ 35 S]cysteine for 30 min and chased for the indicated times, in the absence or presence of MG132 (20 μM). After each chase, cell lysates were immunoprecipitated with anti-LRP antibody and analyzed with SDS gels (5%) under reducing conditions. The 200-kDa molecular size marker is indicated. (B) The band intensities in A were quantitated and plotted against chase times.

    Article Snippet: MG132 (Z-Leu-leu-leu-H aldehyde) proteasomal inhibitor was from Peptide Institute, Inc (Minosh-shi Osaka, Japan).

    Techniques: Metabolic Labelling, Labeling, Immunoprecipitation, Marker

    Cullin 3 SPOP is the physiological E3 ubiquitin ligase for PD-L1 a–d, IB analysis of WCL derived from C4-2 cells treated with MG132 (10 μM) or MLN4924 (1 μM) for 12 hours ( a ), immunoprecipitates (IP) and WCL derived from 293T cells transfected with indicated constructs ( b , d ), or anti-PD-L1 IP and WCL derived from PC3 cells ( e ). Cells were treated with MG132 (10 μM) for 12 hours in b, c . e–g, IB analysis of WCL derived from Spop +/+ versus Spop −/− MEFs ( e ), C4-2 cells depleted SPOP with sgRNAs ( f ), or C4-2 cells stably expressing indicated SPOP WT and mutants ( g ). h, i, IB analysis of IP and WCL derived from 293T cells ( h ), or Ni-NTA pull-down products derived from PC3 cells transfected with indicated constructs and treated with 30 μM MG132 for 6 hours. j–l, FACS analysis for PD-L1 ( j) or CD3 + T-cell population ( l ) of the B16-F10 implanted tumors ectopically expressing SPOP-WT or F102C mutant (n = 6 mice each group). Tumor weight were recorded at the time of sacrifice ( k ) (n = 5 mice each group). m, Representative images of PD-L1 and CD8 immunohistochemistry (IHC) staining in SPOP wild-type or mutant primary human prostate cancer samples. The scale bar: 400 μm or 100 μm. n, o Quantification of IHC analysis for PD-L1 ( n ) and CD8 + T cells ( o ) in SPOP wild-type versus mutant human prostate tumor specimens. (n =15 for SPOP mutant, n = 82 for SPOP WT). Error bars, ± s.d., two-tailed t -test, except ( n ) Mann-Whitney test, * P

    Journal: Nature

    Article Title: Cyclin D-CDK4 kinase destabilizes PD-L1 via Cul3SPOP to control cancer immune surveillance

    doi: 10.1038/nature25015

    Figure Lengend Snippet: Cullin 3 SPOP is the physiological E3 ubiquitin ligase for PD-L1 a–d, IB analysis of WCL derived from C4-2 cells treated with MG132 (10 μM) or MLN4924 (1 μM) for 12 hours ( a ), immunoprecipitates (IP) and WCL derived from 293T cells transfected with indicated constructs ( b , d ), or anti-PD-L1 IP and WCL derived from PC3 cells ( e ). Cells were treated with MG132 (10 μM) for 12 hours in b, c . e–g, IB analysis of WCL derived from Spop +/+ versus Spop −/− MEFs ( e ), C4-2 cells depleted SPOP with sgRNAs ( f ), or C4-2 cells stably expressing indicated SPOP WT and mutants ( g ). h, i, IB analysis of IP and WCL derived from 293T cells ( h ), or Ni-NTA pull-down products derived from PC3 cells transfected with indicated constructs and treated with 30 μM MG132 for 6 hours. j–l, FACS analysis for PD-L1 ( j) or CD3 + T-cell population ( l ) of the B16-F10 implanted tumors ectopically expressing SPOP-WT or F102C mutant (n = 6 mice each group). Tumor weight were recorded at the time of sacrifice ( k ) (n = 5 mice each group). m, Representative images of PD-L1 and CD8 immunohistochemistry (IHC) staining in SPOP wild-type or mutant primary human prostate cancer samples. The scale bar: 400 μm or 100 μm. n, o Quantification of IHC analysis for PD-L1 ( n ) and CD8 + T cells ( o ) in SPOP wild-type versus mutant human prostate tumor specimens. (n =15 for SPOP mutant, n = 82 for SPOP WT). Error bars, ± s.d., two-tailed t -test, except ( n ) Mann-Whitney test, * P

    Article Snippet: MG132 (BML-PI102-0005) was purchased from Enzo life science.

    Techniques: Derivative Assay, Transfection, Construct, Stable Transfection, Expressing, FACS, Mutagenesis, Mouse Assay, Immunohistochemistry, Staining, Two Tailed Test, MANN-WHITNEY

    Depletion of Cdh1 , but not Cdc20 , prolongs SPOP proteins stability, which is simultaneously coupled with a decrease in PD-L1 protein level a–c, Immunoblot (IB) analysis of whole cell lysates (WCL) derived from HeLa depleted SPOP through the CRISPR-Cas9 system (a) or depleted Cdc20 or Cdh1 through multiple independent shRNAs (b, c) . d, IB analysis of WCL and immunoprecipitation (IP) derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 hours before harvesting. e, IB analysis of WCL and IP derived from HeLa cells treated with MG132 (10 μM) for 12 hours before harvesting. f, A sequence comparison of D-box motif (RxxLxxxxN) in SPOP derived from different species. g, IB analysis of WCL derived from HeLa cells transfected with indicated constructs. h, i, IB analysis of WCL derived from 293T cells transfected with indicated constructs. 36 h post transfection, cells were treated with cycloheximide (CHX) as indicated time points before harvesting (h) . The protein abundance of SPOP-WT and deletion of RxxL mutant were quantified by the ImageJ software (i) .

    Journal: Nature

    Article Title: Cyclin D-CDK4 kinase destabilizes PD-L1 via Cul3SPOP to control cancer immune surveillance

    doi: 10.1038/nature25015

    Figure Lengend Snippet: Depletion of Cdh1 , but not Cdc20 , prolongs SPOP proteins stability, which is simultaneously coupled with a decrease in PD-L1 protein level a–c, Immunoblot (IB) analysis of whole cell lysates (WCL) derived from HeLa depleted SPOP through the CRISPR-Cas9 system (a) or depleted Cdc20 or Cdh1 through multiple independent shRNAs (b, c) . d, IB analysis of WCL and immunoprecipitation (IP) derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 hours before harvesting. e, IB analysis of WCL and IP derived from HeLa cells treated with MG132 (10 μM) for 12 hours before harvesting. f, A sequence comparison of D-box motif (RxxLxxxxN) in SPOP derived from different species. g, IB analysis of WCL derived from HeLa cells transfected with indicated constructs. h, i, IB analysis of WCL derived from 293T cells transfected with indicated constructs. 36 h post transfection, cells were treated with cycloheximide (CHX) as indicated time points before harvesting (h) . The protein abundance of SPOP-WT and deletion of RxxL mutant were quantified by the ImageJ software (i) .

    Article Snippet: MG132 (BML-PI102-0005) was purchased from Enzo life science.

    Techniques: Derivative Assay, CRISPR, Immunoprecipitation, Transfection, Construct, Sequencing, Mutagenesis, Software

    Cyclin D/CDK4-mediated phosphorylation of SPOP at the Ser6 residue promotes its binding with 14-3-3γ to reduce its poly-ubiquitination and subsequent degradation by APC/Cdh1 a, A sequence comparison of conserved SP sites and putative 14-3-3γ binding motif in SPOP. b, Immunoblot (IB) analysis of whole cell lysates (WCL) and immunoprecipitation (IP) derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 hours before harvesting. c, d, In vitro kinase assays with recombinant Rb and SPOP as substrates and cyclin D1/CDK4, cyclin D2/CDK4 and cyclin D3/CDK4 as kinase complex were performed. BSA was used as a negative control where indicated. e, IB analysis of WCL and immunoprecipitation (IP) derived from MDA-MB-231 cells transfected with indicated constructs, which were treated with/without palbociclib (1 μM) for 12 hours. f, Streptavidin beads pull-down assay for biotin-labeled SPOP peptide with/without phosphorylation at the Ser6 residue to examine its in vitro association with 14-3-3γ. g, IB analysis of WCL and GST pull-down precipitates derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 hours before harvesting. h, i, IB analysis of WCL and IP derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 hours before harvesting. j, k, IB analysis of WCL derived from 293T cells transfected with indicated constructs. 36 h post transfection, cells were treated with 20 μg/ml cycloheximide (CHX) as indicated time points (j) . The protein abundance of SPOP-WT and S6A mutant were quantified by the ImageJ software and plotted accordingly (k) . l, p, IB of WCL and Ni-NTA pull-down products derived from the lysates of PC3 cells transfected with the indicated constructs. Cells were treated with MG132 (30 μM) for 6 hours before harvesting and lysed in the denaturing buffer for following assay. m–o, IB analysis of WCL and IP derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) and with/without palbociclib (1 μM) for 12 hours before harvesting. q–s, IB of WCLs derived from PC3, BT549 and HeLa cells stably expressing sh14-3-3γ as well as shScr as a negative control. t, IB of WCL derived from HeLa cells stably expressing shScr or sh 14-3-3γ synchronized in M phase by nocodazole treatment prior to releasing back into the cell cycle for the indicated times.

    Journal: Nature

    Article Title: Cyclin D-CDK4 kinase destabilizes PD-L1 via Cul3SPOP to control cancer immune surveillance

    doi: 10.1038/nature25015

    Figure Lengend Snippet: Cyclin D/CDK4-mediated phosphorylation of SPOP at the Ser6 residue promotes its binding with 14-3-3γ to reduce its poly-ubiquitination and subsequent degradation by APC/Cdh1 a, A sequence comparison of conserved SP sites and putative 14-3-3γ binding motif in SPOP. b, Immunoblot (IB) analysis of whole cell lysates (WCL) and immunoprecipitation (IP) derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 hours before harvesting. c, d, In vitro kinase assays with recombinant Rb and SPOP as substrates and cyclin D1/CDK4, cyclin D2/CDK4 and cyclin D3/CDK4 as kinase complex were performed. BSA was used as a negative control where indicated. e, IB analysis of WCL and immunoprecipitation (IP) derived from MDA-MB-231 cells transfected with indicated constructs, which were treated with/without palbociclib (1 μM) for 12 hours. f, Streptavidin beads pull-down assay for biotin-labeled SPOP peptide with/without phosphorylation at the Ser6 residue to examine its in vitro association with 14-3-3γ. g, IB analysis of WCL and GST pull-down precipitates derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 hours before harvesting. h, i, IB analysis of WCL and IP derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 hours before harvesting. j, k, IB analysis of WCL derived from 293T cells transfected with indicated constructs. 36 h post transfection, cells were treated with 20 μg/ml cycloheximide (CHX) as indicated time points (j) . The protein abundance of SPOP-WT and S6A mutant were quantified by the ImageJ software and plotted accordingly (k) . l, p, IB of WCL and Ni-NTA pull-down products derived from the lysates of PC3 cells transfected with the indicated constructs. Cells were treated with MG132 (30 μM) for 6 hours before harvesting and lysed in the denaturing buffer for following assay. m–o, IB analysis of WCL and IP derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) and with/without palbociclib (1 μM) for 12 hours before harvesting. q–s, IB of WCLs derived from PC3, BT549 and HeLa cells stably expressing sh14-3-3γ as well as shScr as a negative control. t, IB of WCL derived from HeLa cells stably expressing shScr or sh 14-3-3γ synchronized in M phase by nocodazole treatment prior to releasing back into the cell cycle for the indicated times.

    Article Snippet: MG132 (BML-PI102-0005) was purchased from Enzo life science.

    Techniques: Binding Assay, Sequencing, Immunoprecipitation, Derivative Assay, Transfection, Construct, In Vitro, Recombinant, Negative Control, Multiple Displacement Amplification, Pull Down Assay, Labeling, Mutagenesis, Software, Stable Transfection, Expressing

    Cyclin D-CDK4-mediated phosphorylation of SPOP stabilizes SPOP largely through recruiting 14-3-3γ to disrupt its binding with Cdh1 a–e, IB of WCL derived from HeLa cells with/without depletion of SPOP ( a ) or Cdh1 ( e ) synchronized in M phase by nocodazole treatment prior to releasing for the indicated times, IP and WCL derived from MDA-MB-231 ( b ) or 293T ( c ) cells, or Ni-NTA pull-down products derived from HeLa cells transfected with the indicated constructs ( d ). Cells were treated with MG132 (30 μM) for 6 hours in b–d . f, In vitro kinase assays showing that cyclin D1/CDK4 phosphorylates recombinant SPOP at Ser6, not Ser222. g–j, IB analysis of IP and WCL derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) or with/without palbociclib (1 μM) for 12 hours ( g–i ), or HeLa cells with/without depletion of SPOP treated with palbociclib (0.5, 1 μM) for 48 hours ( j ). k, CT26 implanted tumor-bearing mice were enrolled in different treatment groups as indicated. Tumor volumes of mice treated with control antibody (n = 13), anti-PD-1 mAb (n = 14), the CDK4/6 inhibitor, palbociclib (n = 12) or combined therapy (n = 12) were measured every three days and plotted individually. We repeated this experiment twice. l, Kaplan-Meier survival curves for each treatment group demonstrate the improved efficacy of combining PD-1 mAb with the CDK4/6 inhibitor, palbociclib. *** P

    Journal: Nature

    Article Title: Cyclin D-CDK4 kinase destabilizes PD-L1 via Cul3SPOP to control cancer immune surveillance

    doi: 10.1038/nature25015

    Figure Lengend Snippet: Cyclin D-CDK4-mediated phosphorylation of SPOP stabilizes SPOP largely through recruiting 14-3-3γ to disrupt its binding with Cdh1 a–e, IB of WCL derived from HeLa cells with/without depletion of SPOP ( a ) or Cdh1 ( e ) synchronized in M phase by nocodazole treatment prior to releasing for the indicated times, IP and WCL derived from MDA-MB-231 ( b ) or 293T ( c ) cells, or Ni-NTA pull-down products derived from HeLa cells transfected with the indicated constructs ( d ). Cells were treated with MG132 (30 μM) for 6 hours in b–d . f, In vitro kinase assays showing that cyclin D1/CDK4 phosphorylates recombinant SPOP at Ser6, not Ser222. g–j, IB analysis of IP and WCL derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) or with/without palbociclib (1 μM) for 12 hours ( g–i ), or HeLa cells with/without depletion of SPOP treated with palbociclib (0.5, 1 μM) for 48 hours ( j ). k, CT26 implanted tumor-bearing mice were enrolled in different treatment groups as indicated. Tumor volumes of mice treated with control antibody (n = 13), anti-PD-1 mAb (n = 14), the CDK4/6 inhibitor, palbociclib (n = 12) or combined therapy (n = 12) were measured every three days and plotted individually. We repeated this experiment twice. l, Kaplan-Meier survival curves for each treatment group demonstrate the improved efficacy of combining PD-1 mAb with the CDK4/6 inhibitor, palbociclib. *** P

    Article Snippet: MG132 (BML-PI102-0005) was purchased from Enzo life science.

    Techniques: Binding Assay, Derivative Assay, Multiple Displacement Amplification, Transfection, Construct, In Vitro, Recombinant, Mouse Assay

    SPOP negatively regulates PD-L1 protein stability in a poly-ubiquitination dependent manner a–c, Immunoblot (IB) analysis of whole cell lysates (WCL) derived from 293T cells transfected with indicated constructs. d, e, IB analysis of WCL derived from 293T cells transfected with indicated constructs. 36 h post transfection, cells were treated with 20 μg/ml cycloheximide (CHX) at indicated time points (d) . The PD-L1 protein abundance were quantified by the ImageJ software and plotted (e) . f, IB of WCL and Ni-NTA pull-down products derived from the lysates of PC3 cells transfected with the indicated constructs. Cells were treated with MG132 (30 μM) for 6 hours before harvesting and lysed in the denaturing buffer. g, A schematic illustration of SPOP with MATH and BTB domain to interact with substrate and Cullin 3, respectively. h, IB analysis of WCL and IP derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 hours before harvesting. i IB analysis of WCL derived from 293T cells transfected with indicated constructs. j, qRT-PCR analysis of relative mRNA levels of PD-L1 from Spop +/+ and Spop −/− MEFs. Data were represented as mean ± s.d, n=5. k, IB analysis of WCL derived from PC3 cells infected with indicated lentiviral shRNAs against SPOP and selected with puromycin (1 μg/ml) for 72 hours before harvesting. l–m, IB analysis of WCL derived from C42 cells with depletion of SPOP using sgRNA and treated with cycloheximide (CHX) for indicated time points before harvesting (l) . The PD-L1 protein abundance were quantified by the ImageJ software and plotted (m) . n, o, IB analysis of WCL derived from LNCaP cells stably expressing shAR or shERG as well as shScr as a negative control. p, q, IB analysis of WCL derived from DU145 cells stably expressing shTrim24 or shDEK as well as shScr as a negative control. r–u, IB analysis of WCL derived from C42 SPOP WT and SPOP −/− cells that stably expressed shAR, shERG, shTrim24, or shDEK as well as shScr, respectively.

    Journal: Nature

    Article Title: Cyclin D-CDK4 kinase destabilizes PD-L1 via Cul3SPOP to control cancer immune surveillance

    doi: 10.1038/nature25015

    Figure Lengend Snippet: SPOP negatively regulates PD-L1 protein stability in a poly-ubiquitination dependent manner a–c, Immunoblot (IB) analysis of whole cell lysates (WCL) derived from 293T cells transfected with indicated constructs. d, e, IB analysis of WCL derived from 293T cells transfected with indicated constructs. 36 h post transfection, cells were treated with 20 μg/ml cycloheximide (CHX) at indicated time points (d) . The PD-L1 protein abundance were quantified by the ImageJ software and plotted (e) . f, IB of WCL and Ni-NTA pull-down products derived from the lysates of PC3 cells transfected with the indicated constructs. Cells were treated with MG132 (30 μM) for 6 hours before harvesting and lysed in the denaturing buffer. g, A schematic illustration of SPOP with MATH and BTB domain to interact with substrate and Cullin 3, respectively. h, IB analysis of WCL and IP derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 hours before harvesting. i IB analysis of WCL derived from 293T cells transfected with indicated constructs. j, qRT-PCR analysis of relative mRNA levels of PD-L1 from Spop +/+ and Spop −/− MEFs. Data were represented as mean ± s.d, n=5. k, IB analysis of WCL derived from PC3 cells infected with indicated lentiviral shRNAs against SPOP and selected with puromycin (1 μg/ml) for 72 hours before harvesting. l–m, IB analysis of WCL derived from C42 cells with depletion of SPOP using sgRNA and treated with cycloheximide (CHX) for indicated time points before harvesting (l) . The PD-L1 protein abundance were quantified by the ImageJ software and plotted (m) . n, o, IB analysis of WCL derived from LNCaP cells stably expressing shAR or shERG as well as shScr as a negative control. p, q, IB analysis of WCL derived from DU145 cells stably expressing shTrim24 or shDEK as well as shScr as a negative control. r–u, IB analysis of WCL derived from C42 SPOP WT and SPOP −/− cells that stably expressed shAR, shERG, shTrim24, or shDEK as well as shScr, respectively.

    Article Snippet: MG132 (BML-PI102-0005) was purchased from Enzo life science.

    Techniques: Derivative Assay, Transfection, Construct, Software, Quantitative RT-PCR, Infection, Stable Transfection, Expressing, Negative Control

    Cullin 3 SPOP promotes PD-L1 ubiquitination and subsequent degradation largely through interaction with the cytoplasmic tail of PD-L1 a, A schematic illustration of PD-L1 with N-terminal signal peptide, extracellular domain, trans-membrane domain, cytoplasmic tail and the potential SPOP-binding motif in PD-L1. b, d, Immunoblot (IB) analysis of whole cell lysates (WCL) and GST pull-down precipitates derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 hours before harvesting. c, IB analysis of WCL derived from PC3 stably expressing shCullin 3. e, g, IB analysis of WCL and immunoprecipitation (IP) derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 hours before harvesting. f, IB of WCL and Ni-NTA pull-down products derived from the lysates of PC3 cells transfected with the indicated constructs. Cells were treated with MG132 (30 μM) for 6 hours before harvesting and lysed in the denature buffer. h, IB analysis of WCL and IP derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 hours before harvesting. i, IB of WCL derived from MDA-MB-231 PD-L1 KO cells stably expressing PD-L1 WT, delta 283-290, T290M as well as EV as a negative control. j, IB analysis of WCL derived from 293T cells transfected with HA-PD-L1 WT and the T290M mutant, which were treated with cycloheximide (CHX) for indicated time points before harvesting. k, IB of WCL and Ni-NTA pull-down products derived from the lysates of PC3 cells transfected with the indicated constructs. Cells were treated with MG132 (30 μM) for 6 hours before harvesting and lysed in the denaturing buffer. l, IB of WCL derived from 293T cells transfected with indicated constructs.

    Journal: Nature

    Article Title: Cyclin D-CDK4 kinase destabilizes PD-L1 via Cul3SPOP to control cancer immune surveillance

    doi: 10.1038/nature25015

    Figure Lengend Snippet: Cullin 3 SPOP promotes PD-L1 ubiquitination and subsequent degradation largely through interaction with the cytoplasmic tail of PD-L1 a, A schematic illustration of PD-L1 with N-terminal signal peptide, extracellular domain, trans-membrane domain, cytoplasmic tail and the potential SPOP-binding motif in PD-L1. b, d, Immunoblot (IB) analysis of whole cell lysates (WCL) and GST pull-down precipitates derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 hours before harvesting. c, IB analysis of WCL derived from PC3 stably expressing shCullin 3. e, g, IB analysis of WCL and immunoprecipitation (IP) derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 hours before harvesting. f, IB of WCL and Ni-NTA pull-down products derived from the lysates of PC3 cells transfected with the indicated constructs. Cells were treated with MG132 (30 μM) for 6 hours before harvesting and lysed in the denature buffer. h, IB analysis of WCL and IP derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 hours before harvesting. i, IB of WCL derived from MDA-MB-231 PD-L1 KO cells stably expressing PD-L1 WT, delta 283-290, T290M as well as EV as a negative control. j, IB analysis of WCL derived from 293T cells transfected with HA-PD-L1 WT and the T290M mutant, which were treated with cycloheximide (CHX) for indicated time points before harvesting. k, IB of WCL and Ni-NTA pull-down products derived from the lysates of PC3 cells transfected with the indicated constructs. Cells were treated with MG132 (30 μM) for 6 hours before harvesting and lysed in the denaturing buffer. l, IB of WCL derived from 293T cells transfected with indicated constructs.

    Article Snippet: MG132 (BML-PI102-0005) was purchased from Enzo life science.

    Techniques: Binding Assay, Derivative Assay, Transfection, Construct, Stable Transfection, Expressing, Immunoprecipitation, Multiple Displacement Amplification, Negative Control, Mutagenesis

    Progerin accumulates in autophagic vacuoles upon MG 132 treatment Immunofluorescence staining of PML (red) and LC3B (green) in HGPS and control fibroblasts both treated with MG132 (5 μM) or DMSO (control) for 24 h. Nucleolar and cytoplasmic colocalization of LC3B with PML are shown (arrows). Scale bar, 10 μm. Nucleolar progerin (red) and LC3B (green) accumulation (arrows) in HGPS fibroblasts upon 5 μM MG132 treatment. Scale bar, 5 μm. Progerin (red) accumulated in LC3B (green), p62 (green) and LAMP‐2 (green) positive cytoplasmic vesicles (arrows) after 48 h MG132 (5 μM) exposure of HGPS fibroblasts. Scale bar, 5 μm. The magnifications are shown; scale bar, 200 nm. Data information: Data in (A–C) are representative of five independent experiments. The experiments were performed on fibroblasts of HGPS patients and healthy subjects matched for age and passage.

    Journal: EMBO Molecular Medicine

    Article Title: MG132‐induced progerin clearance is mediated by autophagy activation and splicing regulation

    doi: 10.15252/emmm.201607315

    Figure Lengend Snippet: Progerin accumulates in autophagic vacuoles upon MG 132 treatment Immunofluorescence staining of PML (red) and LC3B (green) in HGPS and control fibroblasts both treated with MG132 (5 μM) or DMSO (control) for 24 h. Nucleolar and cytoplasmic colocalization of LC3B with PML are shown (arrows). Scale bar, 10 μm. Nucleolar progerin (red) and LC3B (green) accumulation (arrows) in HGPS fibroblasts upon 5 μM MG132 treatment. Scale bar, 5 μm. Progerin (red) accumulated in LC3B (green), p62 (green) and LAMP‐2 (green) positive cytoplasmic vesicles (arrows) after 48 h MG132 (5 μM) exposure of HGPS fibroblasts. Scale bar, 5 μm. The magnifications are shown; scale bar, 200 nm. Data information: Data in (A–C) are representative of five independent experiments. The experiments were performed on fibroblasts of HGPS patients and healthy subjects matched for age and passage.

    Article Snippet: Fibroblasts were cultured in the presence or absence of MG132 (474790, Merck Chemical LTD), MG115 (SCP0005, Sigma), MG262 (I‐120‐200, R & D Systems), bortezomib (S1013, Euromedex), carfilzomib (S2853, Euromedex), chloroquine diphosphate crystalline (C6628, Sigma), bafilomycin A1 (B1793, Sigma), caspase‐6 inhibitor Z‐VEID‐FMK (FMK006, R & D Systems), pan‐caspase inhibitor Z‐VAD‐FMK (FMK001, R & D Systems), leptomycin B (L2913, Sigma), 2‐D08 (SML1052, Sigma), and ginkgolic acid C15:1 (74741, Sigma).

    Techniques: Immunofluorescence, Staining

    MG 132 reduces progerin and SRSF ‐1 levels in patient iPS ‐Derived MSC and VSMC Immunofluorescence images of HGPS‐derived iPS‐VSMC and WT‐derived iPS‐VSMC cultured with DMEM medium containing either indicated MG132 concentrations (0.15, 0.31, 0.62, 1.25, 2.5, 5, and 10 μM) or DMSO (0) for 24 h and stained for progerin (green). Scale bar, 40 μm. ( n = 3). MG132 treatment resulted in a decrease of progerin levels. A representative Western blotting experiment in whole lysates showing progerin, actin, GAPDH, SRSF‐1, LC3B‐I, and LC3B‐II expression in 5 μM MG132‐treated HGPS fibroblasts (+), 2.5 μM MG132‐treated HGPS iPS‐MSC (+), and 1.2 μM MG132‐treated HGPS iPS‐VSMC (+) for 24 h, relative to DMSO‐treated cells (−). ( n = 4). HGPS‐derived iPS‐MSC and iPS‐VSMC viability was measured at 24 h post‐treatment with MG132 at the indicated concentrations using CellTiter‐Glo Luminescent Cell Viability Assay. Results are reported as viability percentage of MG132‐treated cells relative to DMSO‐treated cells. Results are expressed as mean ± SEM ( n = 4). Source data are available online for this figure.

    Journal: EMBO Molecular Medicine

    Article Title: MG132‐induced progerin clearance is mediated by autophagy activation and splicing regulation

    doi: 10.15252/emmm.201607315

    Figure Lengend Snippet: MG 132 reduces progerin and SRSF ‐1 levels in patient iPS ‐Derived MSC and VSMC Immunofluorescence images of HGPS‐derived iPS‐VSMC and WT‐derived iPS‐VSMC cultured with DMEM medium containing either indicated MG132 concentrations (0.15, 0.31, 0.62, 1.25, 2.5, 5, and 10 μM) or DMSO (0) for 24 h and stained for progerin (green). Scale bar, 40 μm. ( n = 3). MG132 treatment resulted in a decrease of progerin levels. A representative Western blotting experiment in whole lysates showing progerin, actin, GAPDH, SRSF‐1, LC3B‐I, and LC3B‐II expression in 5 μM MG132‐treated HGPS fibroblasts (+), 2.5 μM MG132‐treated HGPS iPS‐MSC (+), and 1.2 μM MG132‐treated HGPS iPS‐VSMC (+) for 24 h, relative to DMSO‐treated cells (−). ( n = 4). HGPS‐derived iPS‐MSC and iPS‐VSMC viability was measured at 24 h post‐treatment with MG132 at the indicated concentrations using CellTiter‐Glo Luminescent Cell Viability Assay. Results are reported as viability percentage of MG132‐treated cells relative to DMSO‐treated cells. Results are expressed as mean ± SEM ( n = 4). Source data are available online for this figure.

    Article Snippet: Fibroblasts were cultured in the presence or absence of MG132 (474790, Merck Chemical LTD), MG115 (SCP0005, Sigma), MG262 (I‐120‐200, R & D Systems), bortezomib (S1013, Euromedex), carfilzomib (S2853, Euromedex), chloroquine diphosphate crystalline (C6628, Sigma), bafilomycin A1 (B1793, Sigma), caspase‐6 inhibitor Z‐VEID‐FMK (FMK006, R & D Systems), pan‐caspase inhibitor Z‐VAD‐FMK (FMK001, R & D Systems), leptomycin B (L2913, Sigma), 2‐D08 (SML1052, Sigma), and ginkgolic acid C15:1 (74741, Sigma).

    Techniques: Derivative Assay, Immunofluorescence, Cell Culture, Staining, Western Blot, Expressing, Cell Viability Assay

    MG 132 improves cellular HGPS phenotypes Immunofluorescence images of HGPS fibroblasts cultured with DMEM medium containing 5 μM MG132, or the same volume of vehicle (DMSO, 0.025% v/v) for 48 h and stained for lamin A/C (red) and DAPI (blue). Scale bar, 5 μm. ( n = 3). The percentage of normal nuclei (nuclei with a smooth oval shape) and abnormal nuclei (nuclei with blebs, irregular shape, or multiple folds) was calculated using the Nuclear Irregularity Index (NII) plugin of the ImageJ software (version 1.6.0, NIH, USA). At least 200 fibroblast nuclei were randomly selected for each cell line. A representative image and the mean values of three different experiments are shown. Senescence rate in 2 HGPS fibroblasts and two control fibroblasts treated with 5 μM MG132 for 48 h relative to DMSO‐treated cells. Each experiment was performed on cells at the same passage level. Senescence is determined as relative light units (RLU). ( n = 4). HGPS fibroblasts were untreated, vehicle control (DMSO) or 5 μM MG132 treated up to 10 days. The medium was replaced with new drug every 48 h. ( n = 3). Senescence rate in HGPS fibroblasts (untreated or DMSO‐treated cells at each time point vs. Day 0. MG132‐treated cells vs. DMSO‐treated cells at each time point) (D). Cell proliferation rate based on the incorporation of bromodeoxyuridine (BrdU) into the DNA was expressed as absorbance OD450‐550. (MG132‐treated cells vs. DMSO‐treated cells at each time point). Proliferation rates in normal fibroblasts (WT fibroblasts) are presented (E). Viability (MG132‐treated cells vs. DMSO‐treated cells at each time point) (F) and cytotoxicity (MG132‐treated cells vs. DMSO‐treated cells at each time point) (G). Immunofluorescence microscopy on primary dermal fibroblasts from a healthy individual (control) and an individual with HGPS treated with DMSO (MG132 vehicle control), 5 μM MG132, 20 μM control scrambled morpholino antisense oligonucleotides (Scr‐AON), or 20 μM specific AON for 10 days. The medium was replaced with new drug every 48 h. Cells were stained with DAPI (blue) and antibodies to the indicated proteins. The percentage of positive staining is indicated, at least 200 fibroblast nuclei were randomly selected for each cell line ( n = 3) and examined using ImageJ software. Scale bar, 40 μm. Heatmap of RNAseq data (ArrayExpress accession number: E‐MTAB‐5807) from HGPS fibroblasts treated with DMSO (vehicle control) or with 5 μM MG132 for 6 h. This analysis represents the fold change, in MG132‐treated relative to DMSO‐treated HGPS fibroblasts, of the most characteristic transcripts of the cellular HGPS phenotype. ( n = 2). Data information: Results are expressed as mean ± SEM, Student's t ‐test, * P

    Journal: EMBO Molecular Medicine

    Article Title: MG132‐induced progerin clearance is mediated by autophagy activation and splicing regulation

    doi: 10.15252/emmm.201607315

    Figure Lengend Snippet: MG 132 improves cellular HGPS phenotypes Immunofluorescence images of HGPS fibroblasts cultured with DMEM medium containing 5 μM MG132, or the same volume of vehicle (DMSO, 0.025% v/v) for 48 h and stained for lamin A/C (red) and DAPI (blue). Scale bar, 5 μm. ( n = 3). The percentage of normal nuclei (nuclei with a smooth oval shape) and abnormal nuclei (nuclei with blebs, irregular shape, or multiple folds) was calculated using the Nuclear Irregularity Index (NII) plugin of the ImageJ software (version 1.6.0, NIH, USA). At least 200 fibroblast nuclei were randomly selected for each cell line. A representative image and the mean values of three different experiments are shown. Senescence rate in 2 HGPS fibroblasts and two control fibroblasts treated with 5 μM MG132 for 48 h relative to DMSO‐treated cells. Each experiment was performed on cells at the same passage level. Senescence is determined as relative light units (RLU). ( n = 4). HGPS fibroblasts were untreated, vehicle control (DMSO) or 5 μM MG132 treated up to 10 days. The medium was replaced with new drug every 48 h. ( n = 3). Senescence rate in HGPS fibroblasts (untreated or DMSO‐treated cells at each time point vs. Day 0. MG132‐treated cells vs. DMSO‐treated cells at each time point) (D). Cell proliferation rate based on the incorporation of bromodeoxyuridine (BrdU) into the DNA was expressed as absorbance OD450‐550. (MG132‐treated cells vs. DMSO‐treated cells at each time point). Proliferation rates in normal fibroblasts (WT fibroblasts) are presented (E). Viability (MG132‐treated cells vs. DMSO‐treated cells at each time point) (F) and cytotoxicity (MG132‐treated cells vs. DMSO‐treated cells at each time point) (G). Immunofluorescence microscopy on primary dermal fibroblasts from a healthy individual (control) and an individual with HGPS treated with DMSO (MG132 vehicle control), 5 μM MG132, 20 μM control scrambled morpholino antisense oligonucleotides (Scr‐AON), or 20 μM specific AON for 10 days. The medium was replaced with new drug every 48 h. Cells were stained with DAPI (blue) and antibodies to the indicated proteins. The percentage of positive staining is indicated, at least 200 fibroblast nuclei were randomly selected for each cell line ( n = 3) and examined using ImageJ software. Scale bar, 40 μm. Heatmap of RNAseq data (ArrayExpress accession number: E‐MTAB‐5807) from HGPS fibroblasts treated with DMSO (vehicle control) or with 5 μM MG132 for 6 h. This analysis represents the fold change, in MG132‐treated relative to DMSO‐treated HGPS fibroblasts, of the most characteristic transcripts of the cellular HGPS phenotype. ( n = 2). Data information: Results are expressed as mean ± SEM, Student's t ‐test, * P

    Article Snippet: Fibroblasts were cultured in the presence or absence of MG132 (474790, Merck Chemical LTD), MG115 (SCP0005, Sigma), MG262 (I‐120‐200, R & D Systems), bortezomib (S1013, Euromedex), carfilzomib (S2853, Euromedex), chloroquine diphosphate crystalline (C6628, Sigma), bafilomycin A1 (B1793, Sigma), caspase‐6 inhibitor Z‐VEID‐FMK (FMK006, R & D Systems), pan‐caspase inhibitor Z‐VAD‐FMK (FMK001, R & D Systems), leptomycin B (L2913, Sigma), 2‐D08 (SML1052, Sigma), and ginkgolic acid C15:1 (74741, Sigma).

    Techniques: Immunofluorescence, Cell Culture, Staining, Software, Microscopy

    MG 132 reduces progerin levels in HGPS fibroblasts Immunofluorescence images of HGPS fibroblasts treated with MG132 for 24 h and stained for progerin (green). Scale bar, 40 μm. ( n = 4). HGPS fibroblasts viability was measured at 24 h post‐treatment with MG132 at the indicated concentrations using CellTiter‐Glo Luminescent Cell Viability Assay. Results are reported as viability percentage of MG132‐treated cells relative to DMSO‐treated cells. ( n = 4). MG132 treatment results in a decrease of progerin levels, (Ctrl: untreated HGPS cells). Lower panel, progerin expression levels in MG132‐treated cells were normalized to those treated with DMSO and to tubulin values (loading control). Fibroblasts isolated from biopsies of healthy subjects (Control) were used as negative controls to validate the specificity of anti‐progerin antibody. ( n = 5). Upper panels, lamin A/C, progerin, GAPDH, LC3B‐I and LC3B‐II expression, in whole lysates from HGPS fibroblasts untreated (Ctrl) or treated with DMSO (48 h), 5 μM MG132 (36 and 48 h), or both 5 μM MG132 and chloroquine added simultaneously (48 h). The images were cropped from the same Western blot experiment. Lower panels, progerin expression levels (relative to DMSO‐treated cells: “*”, or relative to MG132‐treated cells for 48H: “§”, and LC3BII/LC3BI ratios relative to DMSO‐treated cells: “*”) were normalized to GAPDH values. ( n = 5). Data information: Results are expressed as mean ± SEM, Student's t ‐test, * P

    Journal: EMBO Molecular Medicine

    Article Title: MG132‐induced progerin clearance is mediated by autophagy activation and splicing regulation

    doi: 10.15252/emmm.201607315

    Figure Lengend Snippet: MG 132 reduces progerin levels in HGPS fibroblasts Immunofluorescence images of HGPS fibroblasts treated with MG132 for 24 h and stained for progerin (green). Scale bar, 40 μm. ( n = 4). HGPS fibroblasts viability was measured at 24 h post‐treatment with MG132 at the indicated concentrations using CellTiter‐Glo Luminescent Cell Viability Assay. Results are reported as viability percentage of MG132‐treated cells relative to DMSO‐treated cells. ( n = 4). MG132 treatment results in a decrease of progerin levels, (Ctrl: untreated HGPS cells). Lower panel, progerin expression levels in MG132‐treated cells were normalized to those treated with DMSO and to tubulin values (loading control). Fibroblasts isolated from biopsies of healthy subjects (Control) were used as negative controls to validate the specificity of anti‐progerin antibody. ( n = 5). Upper panels, lamin A/C, progerin, GAPDH, LC3B‐I and LC3B‐II expression, in whole lysates from HGPS fibroblasts untreated (Ctrl) or treated with DMSO (48 h), 5 μM MG132 (36 and 48 h), or both 5 μM MG132 and chloroquine added simultaneously (48 h). The images were cropped from the same Western blot experiment. Lower panels, progerin expression levels (relative to DMSO‐treated cells: “*”, or relative to MG132‐treated cells for 48H: “§”, and LC3BII/LC3BI ratios relative to DMSO‐treated cells: “*”) were normalized to GAPDH values. ( n = 5). Data information: Results are expressed as mean ± SEM, Student's t ‐test, * P

    Article Snippet: Fibroblasts were cultured in the presence or absence of MG132 (474790, Merck Chemical LTD), MG115 (SCP0005, Sigma), MG262 (I‐120‐200, R & D Systems), bortezomib (S1013, Euromedex), carfilzomib (S2853, Euromedex), chloroquine diphosphate crystalline (C6628, Sigma), bafilomycin A1 (B1793, Sigma), caspase‐6 inhibitor Z‐VEID‐FMK (FMK006, R & D Systems), pan‐caspase inhibitor Z‐VAD‐FMK (FMK001, R & D Systems), leptomycin B (L2913, Sigma), 2‐D08 (SML1052, Sigma), and ginkgolic acid C15:1 (74741, Sigma).

    Techniques: Immunofluorescence, Staining, Cell Viability Assay, Expressing, Isolation, Western Blot

    MG 132 reduces progerin transcripts and splicing factor SRSF ‐1 protein expression, while SRSF ‐5 protein levels are increased Upper panel, lamin A/C, progerin, and GAPDH expressions in whole lysates from HGPS fibroblasts treated for 48 h either with DMSO control (−) or with (+) the indicated drug (s) (MG132 (5 μM), chloroquine (50 μM), bafilomycin A1 (Baf. A1) (100 nM), caspase‐6 inhibitor (Casp‐6 inh.) (50 μM) or leptomycin B (Lept. B) (20 ng/ml). Lower panels, progerin expression levels in drug (s)‐treated cells relative to DMSO‐treated cells (well no. 1) were normalized to GAPDH values. ( n = 5). Downregulation of progerin transcripts in response to MG132. Quantitative real‐time PCR analyses of progerin mRNA levels in HGPS fibroblasts treated with 5 μM MG132 relative to DMSO‐treated cells (Control: “C”). ( n = 3). A representative Western blotting experiment in whole lysates of HGPS fibroblasts showing progerin, lamin A, lamin C, actin, GAPDH and SRSF‐1 expression in MG132‐treated HGPS cells up to 96 h and relative to DMSO‐treated cells “C”. Urea was used to lyse cells. ( n = 4). Proteasome activities in HGPS fibroblasts treated with 5 μM MG132 relative to DMSO‐treated cells, (Control: untreated cells). ( n = 3). MG132‐mediated SRSF‐5 accumulation and SRSF‐1 downregulation correlate with progerin clearance. A representative Western blotting experiment in whole lysates of HGPS fibroblasts showing progerin, SRSF‐5, GAPDH, SRSF‐1, LC3BI, and LC3BII expression in MG132‐treated HGPS cells up to 96 h and relative to DMSO‐treated cells (Control: “C”). The medium was replaced with new drug every 48 h. Urea was used to lyse cells. ( n = 4). siRNA inactivation of SRSF‐1 reduces progerin levels in HGPS fibroblasts. HGPS fibroblasts were transfected with control siRNA or with siRNA specific for SRSF‐1 and 48 h later cells were treated for 24 h with DMSO control (−) or with (+) MG132 (5 μM). ( n = 3). Left panels, caspase‐mediated downregulation of SRSF‐1, in addition to autophagy, contribute to progerin clearance. Western blotting evaluation of lamin A/C, progerin, actin and SRSF‐1 in whole lysates from HGPS fibroblasts treated for 48 h either with DMSO control (−) or with (+) the indicated drug (s) [MG132 (5 μM), chloroquine (50 μM), bafilomycin A1 (100 nM) or pan‐caspase inhibitor (50 μM)]. Rihgt panels, progerin and SRSF‐1 expression levels relative to DMSO‐treated cells (well no. 1) were normalized to actin values using ImageJ software. ( n = 6). Data information: Results are expressed as mean ± SEM, Student's t ‐test, * P

    Journal: EMBO Molecular Medicine

    Article Title: MG132‐induced progerin clearance is mediated by autophagy activation and splicing regulation

    doi: 10.15252/emmm.201607315

    Figure Lengend Snippet: MG 132 reduces progerin transcripts and splicing factor SRSF ‐1 protein expression, while SRSF ‐5 protein levels are increased Upper panel, lamin A/C, progerin, and GAPDH expressions in whole lysates from HGPS fibroblasts treated for 48 h either with DMSO control (−) or with (+) the indicated drug (s) (MG132 (5 μM), chloroquine (50 μM), bafilomycin A1 (Baf. A1) (100 nM), caspase‐6 inhibitor (Casp‐6 inh.) (50 μM) or leptomycin B (Lept. B) (20 ng/ml). Lower panels, progerin expression levels in drug (s)‐treated cells relative to DMSO‐treated cells (well no. 1) were normalized to GAPDH values. ( n = 5). Downregulation of progerin transcripts in response to MG132. Quantitative real‐time PCR analyses of progerin mRNA levels in HGPS fibroblasts treated with 5 μM MG132 relative to DMSO‐treated cells (Control: “C”). ( n = 3). A representative Western blotting experiment in whole lysates of HGPS fibroblasts showing progerin, lamin A, lamin C, actin, GAPDH and SRSF‐1 expression in MG132‐treated HGPS cells up to 96 h and relative to DMSO‐treated cells “C”. Urea was used to lyse cells. ( n = 4). Proteasome activities in HGPS fibroblasts treated with 5 μM MG132 relative to DMSO‐treated cells, (Control: untreated cells). ( n = 3). MG132‐mediated SRSF‐5 accumulation and SRSF‐1 downregulation correlate with progerin clearance. A representative Western blotting experiment in whole lysates of HGPS fibroblasts showing progerin, SRSF‐5, GAPDH, SRSF‐1, LC3BI, and LC3BII expression in MG132‐treated HGPS cells up to 96 h and relative to DMSO‐treated cells (Control: “C”). The medium was replaced with new drug every 48 h. Urea was used to lyse cells. ( n = 4). siRNA inactivation of SRSF‐1 reduces progerin levels in HGPS fibroblasts. HGPS fibroblasts were transfected with control siRNA or with siRNA specific for SRSF‐1 and 48 h later cells were treated for 24 h with DMSO control (−) or with (+) MG132 (5 μM). ( n = 3). Left panels, caspase‐mediated downregulation of SRSF‐1, in addition to autophagy, contribute to progerin clearance. Western blotting evaluation of lamin A/C, progerin, actin and SRSF‐1 in whole lysates from HGPS fibroblasts treated for 48 h either with DMSO control (−) or with (+) the indicated drug (s) [MG132 (5 μM), chloroquine (50 μM), bafilomycin A1 (100 nM) or pan‐caspase inhibitor (50 μM)]. Rihgt panels, progerin and SRSF‐1 expression levels relative to DMSO‐treated cells (well no. 1) were normalized to actin values using ImageJ software. ( n = 6). Data information: Results are expressed as mean ± SEM, Student's t ‐test, * P

    Article Snippet: Fibroblasts were cultured in the presence or absence of MG132 (474790, Merck Chemical LTD), MG115 (SCP0005, Sigma), MG262 (I‐120‐200, R & D Systems), bortezomib (S1013, Euromedex), carfilzomib (S2853, Euromedex), chloroquine diphosphate crystalline (C6628, Sigma), bafilomycin A1 (B1793, Sigma), caspase‐6 inhibitor Z‐VEID‐FMK (FMK006, R & D Systems), pan‐caspase inhibitor Z‐VAD‐FMK (FMK001, R & D Systems), leptomycin B (L2913, Sigma), 2‐D08 (SML1052, Sigma), and ginkgolic acid C15:1 (74741, Sigma).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Software

    Summary diagram showing MG 132 effects on progerin localizations and putative involvement of caspases, splicing, and autophagy systems in progerin clearance In fibroblasts from HGPS patients, progerin accumulates in thread‐like PML‐NBs. MG132 treatment resulted in a caspase‐mediated reduction of SRSF‐1 levels as well as SRSF‐5 accumulation, leading to a decrease in progerin transcript levels. In parallel, MG132 at first induces nucleolar translocation of progerin and then its accumulation and degradation in autophagic vacuoles.

    Journal: EMBO Molecular Medicine

    Article Title: MG132‐induced progerin clearance is mediated by autophagy activation and splicing regulation

    doi: 10.15252/emmm.201607315

    Figure Lengend Snippet: Summary diagram showing MG 132 effects on progerin localizations and putative involvement of caspases, splicing, and autophagy systems in progerin clearance In fibroblasts from HGPS patients, progerin accumulates in thread‐like PML‐NBs. MG132 treatment resulted in a caspase‐mediated reduction of SRSF‐1 levels as well as SRSF‐5 accumulation, leading to a decrease in progerin transcript levels. In parallel, MG132 at first induces nucleolar translocation of progerin and then its accumulation and degradation in autophagic vacuoles.

    Article Snippet: Fibroblasts were cultured in the presence or absence of MG132 (474790, Merck Chemical LTD), MG115 (SCP0005, Sigma), MG262 (I‐120‐200, R & D Systems), bortezomib (S1013, Euromedex), carfilzomib (S2853, Euromedex), chloroquine diphosphate crystalline (C6628, Sigma), bafilomycin A1 (B1793, Sigma), caspase‐6 inhibitor Z‐VEID‐FMK (FMK006, R & D Systems), pan‐caspase inhibitor Z‐VAD‐FMK (FMK001, R & D Systems), leptomycin B (L2913, Sigma), 2‐D08 (SML1052, Sigma), and ginkgolic acid C15:1 (74741, Sigma).

    Techniques: Translocation Assay

    MG 132 reduces progerin and SRSF ‐1 levels in Lmna G609G /G609G mice muscle Western blotting evaluation of lamin A/C, progerin, actin, GAPDH, and SRSF‐1 in gastrocnemius muscle lysate of Lmna G609G/G609G mice treated with 1 μg/kg (mice 1 and 2) or 10 μg/kg (mice 3 and 4) MG132. The results are shown as four successive pairs (left: DMSO‐treated (0 μg/kg) and right: MG132‐treated (1 or 10 μg/kg) muscles, each corresponding to a single mouse). Lamin A‐, progerin‐, lamin C‐, and SRSF‐1‐specific bands, corresponding to DMSO‐treated left muscle (−) and 1 μg/kg MG132‐treated right muscle (+), were quantified by ImageJ software and their expression levels were normalized to GAPDH values. Lamin A‐, progerin‐, lamin C‐, and SRSF‐1‐specific bands, corresponding to DMSO‐treated left muscle (−) and 10 μg/kg MG132‐treated right muscle (+), were quantified by ImageJ software, and their expression levels were normalized to GAPDH values. Data information: Results are expressed as mean ± SEM, n = 5, Student's t ‐test, * P

    Journal: EMBO Molecular Medicine

    Article Title: MG132‐induced progerin clearance is mediated by autophagy activation and splicing regulation

    doi: 10.15252/emmm.201607315

    Figure Lengend Snippet: MG 132 reduces progerin and SRSF ‐1 levels in Lmna G609G /G609G mice muscle Western blotting evaluation of lamin A/C, progerin, actin, GAPDH, and SRSF‐1 in gastrocnemius muscle lysate of Lmna G609G/G609G mice treated with 1 μg/kg (mice 1 and 2) or 10 μg/kg (mice 3 and 4) MG132. The results are shown as four successive pairs (left: DMSO‐treated (0 μg/kg) and right: MG132‐treated (1 or 10 μg/kg) muscles, each corresponding to a single mouse). Lamin A‐, progerin‐, lamin C‐, and SRSF‐1‐specific bands, corresponding to DMSO‐treated left muscle (−) and 1 μg/kg MG132‐treated right muscle (+), were quantified by ImageJ software and their expression levels were normalized to GAPDH values. Lamin A‐, progerin‐, lamin C‐, and SRSF‐1‐specific bands, corresponding to DMSO‐treated left muscle (−) and 10 μg/kg MG132‐treated right muscle (+), were quantified by ImageJ software, and their expression levels were normalized to GAPDH values. Data information: Results are expressed as mean ± SEM, n = 5, Student's t ‐test, * P

    Article Snippet: Fibroblasts were cultured in the presence or absence of MG132 (474790, Merck Chemical LTD), MG115 (SCP0005, Sigma), MG262 (I‐120‐200, R & D Systems), bortezomib (S1013, Euromedex), carfilzomib (S2853, Euromedex), chloroquine diphosphate crystalline (C6628, Sigma), bafilomycin A1 (B1793, Sigma), caspase‐6 inhibitor Z‐VEID‐FMK (FMK006, R & D Systems), pan‐caspase inhibitor Z‐VAD‐FMK (FMK001, R & D Systems), leptomycin B (L2913, Sigma), 2‐D08 (SML1052, Sigma), and ginkgolic acid C15:1 (74741, Sigma).

    Techniques: Mouse Assay, Western Blot, Software, Expressing

    MG 132 treatment resulted in a decrease in the mRNA levels but not the protein levels of lamin A and C MG132 reduces lamin A and lamin C transcript levels. Quantitative real‐time PCR analyses of lamin A and lamin C mRNA levels in HGPS fibroblasts treated with 5 μM MG132. Both lamin A and lamin C protein levels are increased upon HGPS fibroblasts treatment with 5 μM MG132. Data information: All experiments were performed in triplicate. Results are expressed as mean ± SEM. * P

    Journal: EMBO Molecular Medicine

    Article Title: MG132‐induced progerin clearance is mediated by autophagy activation and splicing regulation

    doi: 10.15252/emmm.201607315

    Figure Lengend Snippet: MG 132 treatment resulted in a decrease in the mRNA levels but not the protein levels of lamin A and C MG132 reduces lamin A and lamin C transcript levels. Quantitative real‐time PCR analyses of lamin A and lamin C mRNA levels in HGPS fibroblasts treated with 5 μM MG132. Both lamin A and lamin C protein levels are increased upon HGPS fibroblasts treatment with 5 μM MG132. Data information: All experiments were performed in triplicate. Results are expressed as mean ± SEM. * P

    Article Snippet: Fibroblasts were cultured in the presence or absence of MG132 (474790, Merck Chemical LTD), MG115 (SCP0005, Sigma), MG262 (I‐120‐200, R & D Systems), bortezomib (S1013, Euromedex), carfilzomib (S2853, Euromedex), chloroquine diphosphate crystalline (C6628, Sigma), bafilomycin A1 (B1793, Sigma), caspase‐6 inhibitor Z‐VEID‐FMK (FMK006, R & D Systems), pan‐caspase inhibitor Z‐VAD‐FMK (FMK001, R & D Systems), leptomycin B (L2913, Sigma), 2‐D08 (SML1052, Sigma), and ginkgolic acid C15:1 (74741, Sigma).

    Techniques: Real-time Polymerase Chain Reaction

    Nucleolar translocation of progerin upon MG 132 treatment Subnuclear distribution of progerin (red) and PML (green) after MG132 treatment of HGPS fibroblasts. Cells cultured with 5 μM MG132 for 24 h show staining of progerin and PML in intranuclear foci. MG132 induces the translocation of PML (green), emerin (green), p53 (red), 26S proteasome subunit (red), and ubiquitin (red) into nucleoli of HGPS and control fibroblasts cultured in the presence of 5 μM MG132 for 24 h (relative to DMSO‐treated cells: control). Nucleoli were labeled with fibrillarin antibodies and nuclei with DAPI (blue). The merged images are shown. Data information: Data in (A,B) are representative of six independent experiments. The experiments were performed on fibroblasts of HGPS patients and healthy subjects matched for age and passage. Scale bars, 5 μm.

    Journal: EMBO Molecular Medicine

    Article Title: MG132‐induced progerin clearance is mediated by autophagy activation and splicing regulation

    doi: 10.15252/emmm.201607315

    Figure Lengend Snippet: Nucleolar translocation of progerin upon MG 132 treatment Subnuclear distribution of progerin (red) and PML (green) after MG132 treatment of HGPS fibroblasts. Cells cultured with 5 μM MG132 for 24 h show staining of progerin and PML in intranuclear foci. MG132 induces the translocation of PML (green), emerin (green), p53 (red), 26S proteasome subunit (red), and ubiquitin (red) into nucleoli of HGPS and control fibroblasts cultured in the presence of 5 μM MG132 for 24 h (relative to DMSO‐treated cells: control). Nucleoli were labeled with fibrillarin antibodies and nuclei with DAPI (blue). The merged images are shown. Data information: Data in (A,B) are representative of six independent experiments. The experiments were performed on fibroblasts of HGPS patients and healthy subjects matched for age and passage. Scale bars, 5 μm.

    Article Snippet: Fibroblasts were cultured in the presence or absence of MG132 (474790, Merck Chemical LTD), MG115 (SCP0005, Sigma), MG262 (I‐120‐200, R & D Systems), bortezomib (S1013, Euromedex), carfilzomib (S2853, Euromedex), chloroquine diphosphate crystalline (C6628, Sigma), bafilomycin A1 (B1793, Sigma), caspase‐6 inhibitor Z‐VEID‐FMK (FMK006, R & D Systems), pan‐caspase inhibitor Z‐VAD‐FMK (FMK001, R & D Systems), leptomycin B (L2913, Sigma), 2‐D08 (SML1052, Sigma), and ginkgolic acid C15:1 (74741, Sigma).

    Techniques: Translocation Assay, Cell Culture, Staining, Labeling

    Peptide aldehyde proteasome inhibitor ( MG 132, MG 115), and peptide boronate ( MG 262) but not other analogs (bortezomib and carfilzomib) elicited efficient progerin clearance MG132 and MG262 induced clearance of progerin. A representative Western blotting experiment in whole lysates of HGPS fibroblasts showing progerin and GAPDH expression in MG132‐ and MG262‐treated HGPS cells at the indicated concentrations relative to DMSO‐treated (−) cells for 24 h (Control). ( n = 5). Western blotting evaluation of lamin A/C, progerin and GAPDH in whole lysates from HGPS fibroblasts untreated (−) or treated with bortezomib or carfilzomib for 24 h at indicated concentrations. ( n = 4). Left panels: progerin, actin, and GAPDH expressions in whole lysates from HGPS fibroblasts treated for 48 h with DMSO (Ctrl) or 5 μM MG132, MG115, MG262, bortezomib (BTZ), or carfilzomib (CFZ). Right panels: Progerin expression levels relative to DMSO‐treated cells were normalized to actin values using ImageJ software. ( n = 6). Proteasome activities (trypsin: Z‐LRR aminoluciferin, chymotrypsin: Suc‐LLVY aminoluciferin and caspase‐like: Z‐nLPnLD‐aminoluciferin) in HGPS fibroblasts upon 24 h MG132, bortezomib, or carfilzomib treatment, used at 5 μM. ( n = 4). Data information: Results in (C, D) are expressed as mean ± SEM, Student's t ‐test, ** P

    Journal: EMBO Molecular Medicine

    Article Title: MG132‐induced progerin clearance is mediated by autophagy activation and splicing regulation

    doi: 10.15252/emmm.201607315

    Figure Lengend Snippet: Peptide aldehyde proteasome inhibitor ( MG 132, MG 115), and peptide boronate ( MG 262) but not other analogs (bortezomib and carfilzomib) elicited efficient progerin clearance MG132 and MG262 induced clearance of progerin. A representative Western blotting experiment in whole lysates of HGPS fibroblasts showing progerin and GAPDH expression in MG132‐ and MG262‐treated HGPS cells at the indicated concentrations relative to DMSO‐treated (−) cells for 24 h (Control). ( n = 5). Western blotting evaluation of lamin A/C, progerin and GAPDH in whole lysates from HGPS fibroblasts untreated (−) or treated with bortezomib or carfilzomib for 24 h at indicated concentrations. ( n = 4). Left panels: progerin, actin, and GAPDH expressions in whole lysates from HGPS fibroblasts treated for 48 h with DMSO (Ctrl) or 5 μM MG132, MG115, MG262, bortezomib (BTZ), or carfilzomib (CFZ). Right panels: Progerin expression levels relative to DMSO‐treated cells were normalized to actin values using ImageJ software. ( n = 6). Proteasome activities (trypsin: Z‐LRR aminoluciferin, chymotrypsin: Suc‐LLVY aminoluciferin and caspase‐like: Z‐nLPnLD‐aminoluciferin) in HGPS fibroblasts upon 24 h MG132, bortezomib, or carfilzomib treatment, used at 5 μM. ( n = 4). Data information: Results in (C, D) are expressed as mean ± SEM, Student's t ‐test, ** P

    Article Snippet: Fibroblasts were cultured in the presence or absence of MG132 (474790, Merck Chemical LTD), MG115 (SCP0005, Sigma), MG262 (I‐120‐200, R & D Systems), bortezomib (S1013, Euromedex), carfilzomib (S2853, Euromedex), chloroquine diphosphate crystalline (C6628, Sigma), bafilomycin A1 (B1793, Sigma), caspase‐6 inhibitor Z‐VEID‐FMK (FMK006, R & D Systems), pan‐caspase inhibitor Z‐VAD‐FMK (FMK001, R & D Systems), leptomycin B (L2913, Sigma), 2‐D08 (SML1052, Sigma), and ginkgolic acid C15:1 (74741, Sigma).

    Techniques: Western Blot, Expressing, Software

    Activation of AMPK enhances the protein levels of both Insig-1 and Insig-2. a – c AMPK agonists increase the protein levels of Insig-1. HEK293 cells were transfected with pcDNA or myc-tagged Insig-1 for 24 h, followed by treatment with 2 mM metformin or 5 μM MG132 ( a ), or 1 mM AICAR ( b ) as indicated; and HepG2 cells were transfected with pcDNA or myc-tagged Insig-1 for 24 h, and then infected with adenoviruses encoding constitutive active AMPK (CA-AMPK) or GFP for 48 h ( c ). Immunoblots were performed. d – f AMPK agonists increase the protein levels of Insig-2. HEK293 cells were transfected with pcDNA or FLAG-tagged Insig-2 for 24 h, followed by treatment with 2 mM metformin or 5 μM MG132 ( d ), 1 mM AICAR ( e ); and HepG2 cells were transfected with pcDNA or FLAG-tagged Insig-2 for 24 h, and then infected with adenoviruses encoding CA-AMPK or GFP for 48 h ( f ). Immunoblots were performed

    Journal: Nature Communications

    Article Title: Post-translational regulation of lipogenesis via AMPK-dependent phosphorylation of insulin-induced gene

    doi: 10.1038/s41467-019-08585-4

    Figure Lengend Snippet: Activation of AMPK enhances the protein levels of both Insig-1 and Insig-2. a – c AMPK agonists increase the protein levels of Insig-1. HEK293 cells were transfected with pcDNA or myc-tagged Insig-1 for 24 h, followed by treatment with 2 mM metformin or 5 μM MG132 ( a ), or 1 mM AICAR ( b ) as indicated; and HepG2 cells were transfected with pcDNA or myc-tagged Insig-1 for 24 h, and then infected with adenoviruses encoding constitutive active AMPK (CA-AMPK) or GFP for 48 h ( c ). Immunoblots were performed. d – f AMPK agonists increase the protein levels of Insig-2. HEK293 cells were transfected with pcDNA or FLAG-tagged Insig-2 for 24 h, followed by treatment with 2 mM metformin or 5 μM MG132 ( d ), 1 mM AICAR ( e ); and HepG2 cells were transfected with pcDNA or FLAG-tagged Insig-2 for 24 h, and then infected with adenoviruses encoding CA-AMPK or GFP for 48 h ( f ). Immunoblots were performed

    Article Snippet: MG132 (cat. 1748) was from TOCRIS Bioscience (Bristol, UK).

    Techniques: Activation Assay, Transfection, Infection, Western Blot

    NFκB up-regulates BAG3 and HspB8 expression after various protein aggregation stresses. (A) Control (HeLa) and p65-depleted (p65-KD#2) cell lines were either untreated (NT) or treated with 5 and 15 mM azetidine (Aze) and canavanine (Cana) or 5 μM MG132 for 6 h. At 16 h after treatments, total protein extracts were prepared and analyzed by immunoblots probed with antibodies against BAG3, HspB8, and actin (as a loading control). (B) As in A, but with HeLa and p65-KD#2 cell lines transiently transfected with control plasmids (Cont) or plasmids expressing HspB5wt, HspB5R120G, SOD1wt-EGFP, SOD1G93A-EGFP, or SOD1G85R-EGFP. Analysis was performed 35 h posttransfection. Expression of HspB5 and SOD1-EGFP was checked by probing immunoblots with anti-HspB5 and anti-EGFP antibodies. BAG3 and HspB8 levels were quantified; the densitometric measurements were normalized to the corresponding actin bands, and ratios were calculated between HeLa or p65-KD#2-treated cells vs. nontreated counterparts and are reported in the graphs ( n = 3).

    Journal: Molecular Biology of the Cell

    Article Title: NFκB is a central regulator of protein quality control in response to protein aggregation stresses via autophagy modulation

    doi: 10.1091/mbc.E15-12-0835

    Figure Lengend Snippet: NFκB up-regulates BAG3 and HspB8 expression after various protein aggregation stresses. (A) Control (HeLa) and p65-depleted (p65-KD#2) cell lines were either untreated (NT) or treated with 5 and 15 mM azetidine (Aze) and canavanine (Cana) or 5 μM MG132 for 6 h. At 16 h after treatments, total protein extracts were prepared and analyzed by immunoblots probed with antibodies against BAG3, HspB8, and actin (as a loading control). (B) As in A, but with HeLa and p65-KD#2 cell lines transiently transfected with control plasmids (Cont) or plasmids expressing HspB5wt, HspB5R120G, SOD1wt-EGFP, SOD1G93A-EGFP, or SOD1G85R-EGFP. Analysis was performed 35 h posttransfection. Expression of HspB5 and SOD1-EGFP was checked by probing immunoblots with anti-HspB5 and anti-EGFP antibodies. BAG3 and HspB8 levels were quantified; the densitometric measurements were normalized to the corresponding actin bands, and ratios were calculated between HeLa or p65-KD#2-treated cells vs. nontreated counterparts and are reported in the graphs ( n = 3).

    Article Snippet: MG132 was from Merck (Fontenay sous Bois, France).

    Techniques: Expressing, Western Blot, Transfection

    p65 depletion alters autophagy induction by protein aggregation stresses. Control (HeLa) and p65-depleted cell lines (p65-KD#2) were left untreated or subjected to amino acid analogue (azetidine and canavanine; A) or MG132 treatment (B) as described in Figure 3 . (C, D) Cell lines were untreated (NT) or transiently transfected with control plasmids (Cont) or plasmid expressing wild-type or mutated forms of HspB5 (C) and SOD1-EGFP (D) as described in Figure 3 . Total protein extracts were analyzed in immunoblots probed with LC3, HSPB5, EGFP, and actin antibodies. The histograms show LC3-II/Actin ratios calculated as described in Figure 3 . Results are representative of four independent experiments.

    Journal: Molecular Biology of the Cell

    Article Title: NFκB is a central regulator of protein quality control in response to protein aggregation stresses via autophagy modulation

    doi: 10.1091/mbc.E15-12-0835

    Figure Lengend Snippet: p65 depletion alters autophagy induction by protein aggregation stresses. Control (HeLa) and p65-depleted cell lines (p65-KD#2) were left untreated or subjected to amino acid analogue (azetidine and canavanine; A) or MG132 treatment (B) as described in Figure 3 . (C, D) Cell lines were untreated (NT) or transiently transfected with control plasmids (Cont) or plasmid expressing wild-type or mutated forms of HspB5 (C) and SOD1-EGFP (D) as described in Figure 3 . Total protein extracts were analyzed in immunoblots probed with LC3, HSPB5, EGFP, and actin antibodies. The histograms show LC3-II/Actin ratios calculated as described in Figure 3 . Results are representative of four independent experiments.

    Article Snippet: MG132 was from Merck (Fontenay sous Bois, France).

    Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot

    Amino acid analogue or MG132 treatment and overexpression of mutated forms of HspB5 and SOD1 induce the formation of protein aggregates. HeLa cells were untreated (NT) or subjected to 5 and 15 mM azetidine (A) or canavanine (B) treatment or 2.5 and 5 μM MG132 treatment (C) for 6 h and allowed to recover in fresh culture medium for 16 h. HeLa cells were transiently transfected with control (Cont) plasmids (see Materials and Methods ) or plasmids expressing wild-type HspB5 or its mutated form, HspB5R120G (D), or wild-type SOD1-EGFP or its mutated forms, SOD1G93A-EGFP and SOD1G85R-EGFP (E), and were analyzed 48 h after transfection. Cells were fixed, permeabilized, and stained with antibodies against multiubiquitin (red staining) and pericentrin (indicator of MTOC localization; green staining; A–C) or HspB5 (red staining; D) and pericentrin (green staining; D). Nuclei were stained with Hoechst (blue), and cells were analyzed with a fluorescence microscope. The percentage of cells containing aggregates larger or smaller than 3 μm is reported in the histograms. n = 4.

    Journal: Molecular Biology of the Cell

    Article Title: NFκB is a central regulator of protein quality control in response to protein aggregation stresses via autophagy modulation

    doi: 10.1091/mbc.E15-12-0835

    Figure Lengend Snippet: Amino acid analogue or MG132 treatment and overexpression of mutated forms of HspB5 and SOD1 induce the formation of protein aggregates. HeLa cells were untreated (NT) or subjected to 5 and 15 mM azetidine (A) or canavanine (B) treatment or 2.5 and 5 μM MG132 treatment (C) for 6 h and allowed to recover in fresh culture medium for 16 h. HeLa cells were transiently transfected with control (Cont) plasmids (see Materials and Methods ) or plasmids expressing wild-type HspB5 or its mutated form, HspB5R120G (D), or wild-type SOD1-EGFP or its mutated forms, SOD1G93A-EGFP and SOD1G85R-EGFP (E), and were analyzed 48 h after transfection. Cells were fixed, permeabilized, and stained with antibodies against multiubiquitin (red staining) and pericentrin (indicator of MTOC localization; green staining; A–C) or HspB5 (red staining; D) and pericentrin (green staining; D). Nuclei were stained with Hoechst (blue), and cells were analyzed with a fluorescence microscope. The percentage of cells containing aggregates larger or smaller than 3 μm is reported in the histograms. n = 4.

    Article Snippet: MG132 was from Merck (Fontenay sous Bois, France).

    Techniques: Over Expression, Transfection, Expressing, Staining, Fluorescence, Microscopy

    Amino acid analogue or MG132 treatment and overexpression of mutated forms of HspB5 and SOD1 increase autophagic flux. HeLa cells were either nontreated (NT) or subjected to azetidine or canavanine (5 or 15 mM) treatment (A) or MG132 (0.5–5 μM) treatment (B) for 6 h. (C, D) Cells were left untreated (NT) or transiently transfected with control plasmids (Cont) or plasmids expressing wild-type HspB5 and its mutated form, HspB5R120G (C), or wild-type SOD1-EGFP and its mutated forms, SOD1G93A-EGFP and SOD1G85R-EGFP (D). (E–G) HeLa cells were subjected to the same treatments as described in the presence of 5 μM cathepsin inhibitors pepstatin A (PepA) and E64d. The inhibitors were added concomitantly to the treatment or just after transfection and were not removed until protein extraction. Total protein extracts were prepared 16 h after treatment and 24 h after transfection and subjected to SDS–PAGE. Immunoblots were probed with antibodies against LC3-I and -II, HspB5, EGFP, and actin (as a loading control). The histograms show LC3-II/actin ratios, which were calculated from quantifications of LC3-II and actin bands of Western blots by ImageJ ( n = 3; ratio was set at 1.0 for NT or Cont-transfected cells). Results are representative of three independent experiments.

    Journal: Molecular Biology of the Cell

    Article Title: NFκB is a central regulator of protein quality control in response to protein aggregation stresses via autophagy modulation

    doi: 10.1091/mbc.E15-12-0835

    Figure Lengend Snippet: Amino acid analogue or MG132 treatment and overexpression of mutated forms of HspB5 and SOD1 increase autophagic flux. HeLa cells were either nontreated (NT) or subjected to azetidine or canavanine (5 or 15 mM) treatment (A) or MG132 (0.5–5 μM) treatment (B) for 6 h. (C, D) Cells were left untreated (NT) or transiently transfected with control plasmids (Cont) or plasmids expressing wild-type HspB5 and its mutated form, HspB5R120G (C), or wild-type SOD1-EGFP and its mutated forms, SOD1G93A-EGFP and SOD1G85R-EGFP (D). (E–G) HeLa cells were subjected to the same treatments as described in the presence of 5 μM cathepsin inhibitors pepstatin A (PepA) and E64d. The inhibitors were added concomitantly to the treatment or just after transfection and were not removed until protein extraction. Total protein extracts were prepared 16 h after treatment and 24 h after transfection and subjected to SDS–PAGE. Immunoblots were probed with antibodies against LC3-I and -II, HspB5, EGFP, and actin (as a loading control). The histograms show LC3-II/actin ratios, which were calculated from quantifications of LC3-II and actin bands of Western blots by ImageJ ( n = 3; ratio was set at 1.0 for NT or Cont-transfected cells). Results are representative of three independent experiments.

    Article Snippet: MG132 was from Merck (Fontenay sous Bois, France).

    Techniques: Over Expression, Transfection, Expressing, Protein Extraction, SDS Page, Western Blot

    p65-deficient cells show increased protein aggregation in response to various protein aggregation stresses. Control (HeLa) and p65-depleted cell lines (p65-KD#2) were either untreated (NT) or treated for 6 h with 15 mM azetidine (Aze) or canavanine (Cana; A) or with 2.5 and 5 μM MG132 (B). (C, D) Cell lines were untreated or transiently transfected with plasmids expressing HspB5wt and HspB5R120G (C) or SOD1wt-EGFP, SOD1G93A-EGFP, and SOD1G85R-EGFP (D). Immunofluorescence analyses were performed 16 h after treatment or 48 h after transfection. Cells were fixed, permeabilized, and stained with multiubiquitin (red staining) and pericentrin (green staining) antibodies (A, B) or with HspB5 antibody (red staining; C). Fluorescent antibodies, SOD1-EGFP proteins, and nuclei stained with Hoechst (blue staining) were visualized under a fluorescence microscope. Percentage of cells containing aggregates larger or smaller than 3 μm is shown in the histograms (mean ± SD); n = 3.

    Journal: Molecular Biology of the Cell

    Article Title: NFκB is a central regulator of protein quality control in response to protein aggregation stresses via autophagy modulation

    doi: 10.1091/mbc.E15-12-0835

    Figure Lengend Snippet: p65-deficient cells show increased protein aggregation in response to various protein aggregation stresses. Control (HeLa) and p65-depleted cell lines (p65-KD#2) were either untreated (NT) or treated for 6 h with 15 mM azetidine (Aze) or canavanine (Cana; A) or with 2.5 and 5 μM MG132 (B). (C, D) Cell lines were untreated or transiently transfected with plasmids expressing HspB5wt and HspB5R120G (C) or SOD1wt-EGFP, SOD1G93A-EGFP, and SOD1G85R-EGFP (D). Immunofluorescence analyses were performed 16 h after treatment or 48 h after transfection. Cells were fixed, permeabilized, and stained with multiubiquitin (red staining) and pericentrin (green staining) antibodies (A, B) or with HspB5 antibody (red staining; C). Fluorescent antibodies, SOD1-EGFP proteins, and nuclei stained with Hoechst (blue staining) were visualized under a fluorescence microscope. Percentage of cells containing aggregates larger or smaller than 3 μm is shown in the histograms (mean ± SD); n = 3.

    Article Snippet: MG132 was from Merck (Fontenay sous Bois, France).

    Techniques: Transfection, Expressing, Immunofluorescence, Staining, Fluorescence, Microscopy

    Protein aggregation stresses stimulate NFκB activity. pNFκBLuc stable HeLa transformants were untreated (NT) or treated with 5 or 15 mM azetidine (Aze) or canavanine (Cana; A) or with increasing concentration (0.5–10 μM) of MG132 (B) for 6 h. As a positive control for NFκB induction, cells were also subjected to 90-min heat shock treatment (HS) at 43°C (A). (C, D) HeLa cells were transiently transfected or not (NT) with control plasmids (Cont) or plasmids expressing wild-type HspB5 or its mutated form, HspB5R120G (C), or wild- type SOD1-EGFP or its mutated forms, SOD1G93A-EGFP and SOD1G85R-EGFP (D). (E) pNFκBLuc HeLa cells were either transfected with an empty vector or with expression vectors for dominant-negative mutants of IκBα (pLXSN-IκBα) or of IKKβ (pRK5-IKKβ(K44A)). The next day, cells were treated with 2000 U/ml TNFα for 4 h, followed by 16 h of recovery or subjected to the same treatments as described (15 mM Aze or Cana, 5 μM MG132, or expression of wild-type and mutated forms of HspB5 and SOD1). Insets, immunoblot analysis of HspB5 and SOD1-EGFP levels in transfected HeLa cells 48 h after transfection. Luciferase activity was measured 16 h after treatment or 48 h after transfection. Results are presented as fold stimulation (ratio between luciferase activity after treatment vs. luciferase activity in NT counterpart). Results are presented as mean ± SD. n ≥ 3.

    Journal: Molecular Biology of the Cell

    Article Title: NFκB is a central regulator of protein quality control in response to protein aggregation stresses via autophagy modulation

    doi: 10.1091/mbc.E15-12-0835

    Figure Lengend Snippet: Protein aggregation stresses stimulate NFκB activity. pNFκBLuc stable HeLa transformants were untreated (NT) or treated with 5 or 15 mM azetidine (Aze) or canavanine (Cana; A) or with increasing concentration (0.5–10 μM) of MG132 (B) for 6 h. As a positive control for NFκB induction, cells were also subjected to 90-min heat shock treatment (HS) at 43°C (A). (C, D) HeLa cells were transiently transfected or not (NT) with control plasmids (Cont) or plasmids expressing wild-type HspB5 or its mutated form, HspB5R120G (C), or wild- type SOD1-EGFP or its mutated forms, SOD1G93A-EGFP and SOD1G85R-EGFP (D). (E) pNFκBLuc HeLa cells were either transfected with an empty vector or with expression vectors for dominant-negative mutants of IκBα (pLXSN-IκBα) or of IKKβ (pRK5-IKKβ(K44A)). The next day, cells were treated with 2000 U/ml TNFα for 4 h, followed by 16 h of recovery or subjected to the same treatments as described (15 mM Aze or Cana, 5 μM MG132, or expression of wild-type and mutated forms of HspB5 and SOD1). Insets, immunoblot analysis of HspB5 and SOD1-EGFP levels in transfected HeLa cells 48 h after transfection. Luciferase activity was measured 16 h after treatment or 48 h after transfection. Results are presented as fold stimulation (ratio between luciferase activity after treatment vs. luciferase activity in NT counterpart). Results are presented as mean ± SD. n ≥ 3.

    Article Snippet: MG132 was from Merck (Fontenay sous Bois, France).

    Techniques: Activity Assay, Concentration Assay, Positive Control, Transfection, Expressing, Plasmid Preparation, Dominant Negative Mutation, Luciferase

    BAG3 colocalizes with protein aggregates induced by various aggregation stresses. (A–D) HeLa cells were either nontreated (A) or treated with 15 mM azetidine (B) and canavanine (C) or with 5 μM MG132 (D) for 6 h. At 16 h after treatment, cells were fixed and analyzed by confocal microscopy for BAG3 and multiubiquitin localization. (E–I) Cells were transiently transfected with plasmids expressing HspB5wt (E) or HspB5R120G (F) or SOD1 wt-EGFP (G), SOD1G85R-EGFP (H), and SOD1G93A-EGFP (I). At 48 h after transfection, cells were fixed and analyzed by confocal microscopy for BAG3, HspB5, and SOD1-EGFP localization. Single channels and channel overlays (merge) are shown. The intensity of red and green fluorescence measured along portions (white lines in inset pictures) that cross over aggregates is plotted for each condition. Each table indicates the r 2 and the percentage of aggregates colocalizing with BAG3, where r 2 is the mean of the squares of Pearson’s correlation coefficient r (mean ± SD) in aggregate-containing and random aggregate-devoid portions of the cells; a value close to 0 indicates a lack of colocalization, and a value close to 1 indicates complete colocalization, because all values are positive. The r 2 values of the aggregate-containing vs. aggregate-devoid portions are statistically different with p

    Journal: Molecular Biology of the Cell

    Article Title: NFκB is a central regulator of protein quality control in response to protein aggregation stresses via autophagy modulation

    doi: 10.1091/mbc.E15-12-0835

    Figure Lengend Snippet: BAG3 colocalizes with protein aggregates induced by various aggregation stresses. (A–D) HeLa cells were either nontreated (A) or treated with 15 mM azetidine (B) and canavanine (C) or with 5 μM MG132 (D) for 6 h. At 16 h after treatment, cells were fixed and analyzed by confocal microscopy for BAG3 and multiubiquitin localization. (E–I) Cells were transiently transfected with plasmids expressing HspB5wt (E) or HspB5R120G (F) or SOD1 wt-EGFP (G), SOD1G85R-EGFP (H), and SOD1G93A-EGFP (I). At 48 h after transfection, cells were fixed and analyzed by confocal microscopy for BAG3, HspB5, and SOD1-EGFP localization. Single channels and channel overlays (merge) are shown. The intensity of red and green fluorescence measured along portions (white lines in inset pictures) that cross over aggregates is plotted for each condition. Each table indicates the r 2 and the percentage of aggregates colocalizing with BAG3, where r 2 is the mean of the squares of Pearson’s correlation coefficient r (mean ± SD) in aggregate-containing and random aggregate-devoid portions of the cells; a value close to 0 indicates a lack of colocalization, and a value close to 1 indicates complete colocalization, because all values are positive. The r 2 values of the aggregate-containing vs. aggregate-devoid portions are statistically different with p

    Article Snippet: MG132 was from Merck (Fontenay sous Bois, France).

    Techniques: Confocal Microscopy, Transfection, Expressing, Fluorescence

    STAT3 directly interacts with IKKα in vitro. ( A , C ) IP analysis showing a physical interaction between IKKα and STAT3 in H- Ras MCF-10A ( A ) and HEK293T ( C ) cells. Immunoprecipitation was performed with IKKα (left) or STAT3 (right) antibodies, followed by immunoblotting with antibodies for STAT3 and IKKα, respectively. IgG, a negative control for immunoprecipitation; input, total protein lysate. ( B , D ) Duolink analysis showing the interaction between STAT3 and IKKα in H- Ras MCF-10A ( B ) and MDA-MB-231 ( D ) cells. The representative image was visualized under the fluorescent microscope. ( E , F ) H- Ras MCF-10A ( E ) and HEK293T ( F ) cells were transfected with IKKα wild type (WT) or K44A mutant (K44A) in the presence of STAT3 construct for 48 h, followed by MG132 treatment for additional 2 h. Immunoprecipitation analysis showing the interaction between STAT3 and either WT or K44A IKKα. ( G ) Duolink analysis showing the STAT3 binding to IKKα WT or K44A mutant in HEK293T cells.

    Journal: Cancers

    Article Title: STAT3 Stabilizes IKKα Protein through Direct Interaction in Transformed and Cancerous Human Breast Epithelial Cells

    doi: 10.3390/cancers13010082

    Figure Lengend Snippet: STAT3 directly interacts with IKKα in vitro. ( A , C ) IP analysis showing a physical interaction between IKKα and STAT3 in H- Ras MCF-10A ( A ) and HEK293T ( C ) cells. Immunoprecipitation was performed with IKKα (left) or STAT3 (right) antibodies, followed by immunoblotting with antibodies for STAT3 and IKKα, respectively. IgG, a negative control for immunoprecipitation; input, total protein lysate. ( B , D ) Duolink analysis showing the interaction between STAT3 and IKKα in H- Ras MCF-10A ( B ) and MDA-MB-231 ( D ) cells. The representative image was visualized under the fluorescent microscope. ( E , F ) H- Ras MCF-10A ( E ) and HEK293T ( F ) cells were transfected with IKKα wild type (WT) or K44A mutant (K44A) in the presence of STAT3 construct for 48 h, followed by MG132 treatment for additional 2 h. Immunoprecipitation analysis showing the interaction between STAT3 and either WT or K44A IKKα. ( G ) Duolink analysis showing the STAT3 binding to IKKα WT or K44A mutant in HEK293T cells.

    Article Snippet: Reagents and Antibodies Recombinant human IL-6 and MG132 were purchased from R & D Systems, Inc. (Minneapolis, MN, USA).

    Techniques: In Vitro, Immunoprecipitation, Negative Control, Multiple Displacement Amplification, Microscopy, Transfection, Mutagenesis, Construct, Binding Assay

    Ribosomal stress leads to RPS2 ubiquitination for MDM2 regulation to induce p53 by dissociation of USP47 and RPS2. ( a ) U2OS cells co-overexpressing either Flag vector or Flag-USP47 were treated with DMSO or 5 nM actinomycin D for 4 h, and immunoprecipitation was performed with anti-Flag agarose. Bound proteins were immunoblotted with the indicated antibodies. α-tubulin was used as a loading control. ( b ) HEK293T cells were co-overexpressed with HA-RPS2, Flag-USP47, and then treated with DMSO or 5 nM actinomycin D for 4 h. Then, cell lysates were immunoprecipitated with anti-Flag M2 agarose and detected with the indicated antibodies. ( c ) U2OS cells were co-overexpressed with Flag-USP47 and HA-RPS2 and then treated with DMSO or 5 nM actinomycin D for 4 h. Cells were immunostained with anti-HA antibody followed by Alexa Fluor 488 anti-mouse IgG and Flag antibodies followed by Alexa Fluor 546 anti-rabbit IgG. Representative immunofluorescence images were captured by confocal microscopy. ( d ) HEK293T cells were overexpressed with HA-RPS2 and His-ubiquitin and then treated with 10 μM MG132 for 4 h and 5 nM actinomycin D for indicated times. Ubiquitin conjugates were purified on Ni-NTA-agarose under denaturing conditions, and ubiquitinated RPS2 was detected by anti-HA antibodies. ( e ) U2OS cells were co-transfected with His-ubiquitin, HA-RPS2, and either siControl or siUSP47 and then treated with 10 μM MG132 and either DMSO or 5 nM actinomycin D for 4 h. Ubiquitin conjugates were purified on Ni-NTA agarose under denaturing conditions, and ubiquitinated RPS2 was detected by anti-HA antibodies. ( f ) U2OS cells transfected with siRNA against control or USP47 were treated with either DMSO or 5 nM actinomycin D for 4 h. Cell lysates were immunoblotted with the indicated antibodies. The uncropped blots and molecular weight markers are shown in Figure S7 .

    Journal: Cancers

    Article Title: USP47 Promotes Tumorigenesis by Negative Regulation of p53 through Deubiquitinating Ribosomal Protein S2

    doi: 10.3390/cancers12051137

    Figure Lengend Snippet: Ribosomal stress leads to RPS2 ubiquitination for MDM2 regulation to induce p53 by dissociation of USP47 and RPS2. ( a ) U2OS cells co-overexpressing either Flag vector or Flag-USP47 were treated with DMSO or 5 nM actinomycin D for 4 h, and immunoprecipitation was performed with anti-Flag agarose. Bound proteins were immunoblotted with the indicated antibodies. α-tubulin was used as a loading control. ( b ) HEK293T cells were co-overexpressed with HA-RPS2, Flag-USP47, and then treated with DMSO or 5 nM actinomycin D for 4 h. Then, cell lysates were immunoprecipitated with anti-Flag M2 agarose and detected with the indicated antibodies. ( c ) U2OS cells were co-overexpressed with Flag-USP47 and HA-RPS2 and then treated with DMSO or 5 nM actinomycin D for 4 h. Cells were immunostained with anti-HA antibody followed by Alexa Fluor 488 anti-mouse IgG and Flag antibodies followed by Alexa Fluor 546 anti-rabbit IgG. Representative immunofluorescence images were captured by confocal microscopy. ( d ) HEK293T cells were overexpressed with HA-RPS2 and His-ubiquitin and then treated with 10 μM MG132 for 4 h and 5 nM actinomycin D for indicated times. Ubiquitin conjugates were purified on Ni-NTA-agarose under denaturing conditions, and ubiquitinated RPS2 was detected by anti-HA antibodies. ( e ) U2OS cells were co-transfected with His-ubiquitin, HA-RPS2, and either siControl or siUSP47 and then treated with 10 μM MG132 and either DMSO or 5 nM actinomycin D for 4 h. Ubiquitin conjugates were purified on Ni-NTA agarose under denaturing conditions, and ubiquitinated RPS2 was detected by anti-HA antibodies. ( f ) U2OS cells transfected with siRNA against control or USP47 were treated with either DMSO or 5 nM actinomycin D for 4 h. Cell lysates were immunoblotted with the indicated antibodies. The uncropped blots and molecular weight markers are shown in Figure S7 .

    Article Snippet: MG132 (A.G scientific, San Diego, CA, USA) was treated at a 10 μM concentration to inhibit proteasomal degradation.

    Techniques: Plasmid Preparation, Immunoprecipitation, Immunofluorescence, Confocal Microscopy, Purification, Transfection, Molecular Weight

    USP47 regulates the ubiquitination of p53 and MDM2. ( a ) HEK293T cells were co-overexpressed with His-ubiquitin, p53, MDM2, HA-RPS2, myc-USP47, or myc-USP47 C109S for 24 h and then treated with MG132 (10 μM) for 4 h. Ubiquitin conjugates were purified on NiNTA-agarose under denaturing conditions, and ubiquitinated p53 was detected with anti-p53 antibody. ( b ) U2OS cells co-overexpressing His-ubiquitin, HA-RPS2, MDM2, Flag-USP47, or Flag-USP47 C109S as indicated were treated with MG132 (10 μM) for 4 h. Ubiquitin conjugates were purified on Ni-NTA agarose under denaturing conditions, and ubiquitinated MDM2 was detected with the anti-MDM2 antibody. The non-specific band is denoted by an asterisk. ( c ) H1299 cells were co-overexpressed with MDM2, His-ubiquitin, HA-RPS2, Flag-USP47, or Flag-USP47 C109S and then treated with 10 μM MG132 for 4 h. Ubiquitin conjugates were purified on Ni-NTA agarose under denaturing conditions, and ubiquitinated MDM2 was detected by anti-MDM2 antibodies. ( d ) HEK293T cells were overexpressed with MDM2, His-ubiquitin, and Flag-USP7 or Flag-USP47 and then treated with 10 μM MG132 for 4 h. Ubiquitin conjugates were purified on Ni-NTA agarose under denaturing conditions, and ubiquitinated MDM2 was detected by anti-MDM2 antibodies. The uncropped blots and molecular weight markers are shown in Figure S7 .

    Journal: Cancers

    Article Title: USP47 Promotes Tumorigenesis by Negative Regulation of p53 through Deubiquitinating Ribosomal Protein S2

    doi: 10.3390/cancers12051137

    Figure Lengend Snippet: USP47 regulates the ubiquitination of p53 and MDM2. ( a ) HEK293T cells were co-overexpressed with His-ubiquitin, p53, MDM2, HA-RPS2, myc-USP47, or myc-USP47 C109S for 24 h and then treated with MG132 (10 μM) for 4 h. Ubiquitin conjugates were purified on NiNTA-agarose under denaturing conditions, and ubiquitinated p53 was detected with anti-p53 antibody. ( b ) U2OS cells co-overexpressing His-ubiquitin, HA-RPS2, MDM2, Flag-USP47, or Flag-USP47 C109S as indicated were treated with MG132 (10 μM) for 4 h. Ubiquitin conjugates were purified on Ni-NTA agarose under denaturing conditions, and ubiquitinated MDM2 was detected with the anti-MDM2 antibody. The non-specific band is denoted by an asterisk. ( c ) H1299 cells were co-overexpressed with MDM2, His-ubiquitin, HA-RPS2, Flag-USP47, or Flag-USP47 C109S and then treated with 10 μM MG132 for 4 h. Ubiquitin conjugates were purified on Ni-NTA agarose under denaturing conditions, and ubiquitinated MDM2 was detected by anti-MDM2 antibodies. ( d ) HEK293T cells were overexpressed with MDM2, His-ubiquitin, and Flag-USP7 or Flag-USP47 and then treated with 10 μM MG132 for 4 h. Ubiquitin conjugates were purified on Ni-NTA agarose under denaturing conditions, and ubiquitinated MDM2 was detected by anti-MDM2 antibodies. The uncropped blots and molecular weight markers are shown in Figure S7 .

    Article Snippet: MG132 (A.G scientific, San Diego, CA, USA) was treated at a 10 μM concentration to inhibit proteasomal degradation.

    Techniques: Purification, Molecular Weight

    USP47 deubiquitinates RPS2 and regulates the interaction between RPS2 and MDM2. ( a ) HeLa cells were transfected with ubiquitin, HA-RPS2, Myc-MDM2, Flag-USP47, or Flag-USP47 C109S and then treated with MG132 (10 μM) for 4 h. Cells were harvested and immunoblotted with the indicated antibodies. The non-specific band is denoted by an asterisk. ( b ) HEK293T cells were co-overexpressed with His-ubiquitin, HA-RPS2, MDM2, Flag-USP47, or Flag-USP47 C109S and then treated with MG132 (10 μM) for 4 h. Ubiquitin conjugates were purified on Ni-NTA agarose under denaturing conditions, and ubiquitinated RPS2 was detected with the anti-HA antibody. ( c ) HEK293T cells were overexpressed with MDM2, HA-RPS2, and Flag-USP47, and then immunoprecipitation was performed with anti-HA agarose. Bound proteins were immunoblotted with the indicated antibodies. The non-specific band is denoted by an asterisk. ( d ) HEK293T cells were overexpressed with Myc-USP47, HA-RPS2, and MDM2, and then immunoprecipitation was performed with anti-HA agarose. Bound proteins were immunoblotted with the indicated antibodies. The uncropped blots and molecular weight markers are shown in Figure S7 .

    Journal: Cancers

    Article Title: USP47 Promotes Tumorigenesis by Negative Regulation of p53 through Deubiquitinating Ribosomal Protein S2

    doi: 10.3390/cancers12051137

    Figure Lengend Snippet: USP47 deubiquitinates RPS2 and regulates the interaction between RPS2 and MDM2. ( a ) HeLa cells were transfected with ubiquitin, HA-RPS2, Myc-MDM2, Flag-USP47, or Flag-USP47 C109S and then treated with MG132 (10 μM) for 4 h. Cells were harvested and immunoblotted with the indicated antibodies. The non-specific band is denoted by an asterisk. ( b ) HEK293T cells were co-overexpressed with His-ubiquitin, HA-RPS2, MDM2, Flag-USP47, or Flag-USP47 C109S and then treated with MG132 (10 μM) for 4 h. Ubiquitin conjugates were purified on Ni-NTA agarose under denaturing conditions, and ubiquitinated RPS2 was detected with the anti-HA antibody. ( c ) HEK293T cells were overexpressed with MDM2, HA-RPS2, and Flag-USP47, and then immunoprecipitation was performed with anti-HA agarose. Bound proteins were immunoblotted with the indicated antibodies. The non-specific band is denoted by an asterisk. ( d ) HEK293T cells were overexpressed with Myc-USP47, HA-RPS2, and MDM2, and then immunoprecipitation was performed with anti-HA agarose. Bound proteins were immunoblotted with the indicated antibodies. The uncropped blots and molecular weight markers are shown in Figure S7 .

    Article Snippet: MG132 (A.G scientific, San Diego, CA, USA) was treated at a 10 μM concentration to inhibit proteasomal degradation.

    Techniques: Transfection, Purification, Immunoprecipitation, Molecular Weight

    USP47 regulates p53 in an MDM2-dependent manner. ( a ) HeLa cells transfected with siRNA against control or USP47 were treated with either DMSO (control) or MG132 (10 μM) for 4 h. Cell lysates were immunoblotted with the indicated antibodies. β-actin was used as a loading control. The band intensity was measured by Image J and then normalized by β-actin. ( b ) U2OS cells overexpressed with Flag vector or Flag-USP47 were treated with either DMSO (control) or MG132 (10 μM) for 4 h. Cell lysates were immunoblotted with the indicated antibodies. ( c ) U2OS cells were overexpressed with increasing amounts of Myc-USP47 (0, 1, and 2 μg). After 24 h, cell lysates were immunoblotted with the indicated antibodies. ( d ) U2OS cells transfected with siRNA against control or USP47 were treated with 100 μg/mL cycloheximide and harvested at the indicated times. Cell lysates were immunoblotted with the indicated antibodies. HSP90 was used as a loading control. The band intensity was normalized by HSP90. ( e ) MDM2 +/+ p53 −/− and MDM2 −/− p53 −/− Mouse Embryonic Fibroblast (MEF) cell lines were co-overexpressed with p53, Myc-USP47, and then cell lysates were immunoblotted with the indicated antibodies. The non-specific band is denoted by an asterisk. ( f ) MDM2 +/+ p53 −/− and MDM2 −/− p53 −/− Mouse Embryonic Fibroblast (MEF) cell lines were overexpressed with p53 for 24 h and then transfected with siRNA against control or USP47 for 48 h. Cell lysates were immunoblotted with the indicated antibodies. The non-specific band is denoted by an asterisk. ( g ) The real-time PCR analysis of p53 downstream genes in A549 cells transfected with siRNA against control or USP47 for 48 h. Relative expression values are normalized against β-actin RNA levels, and graphs are shown as fold induction over control siRNA transfected cells. Data from three independent experiments represented by mean ± SD (* p

    Journal: Cancers

    Article Title: USP47 Promotes Tumorigenesis by Negative Regulation of p53 through Deubiquitinating Ribosomal Protein S2

    doi: 10.3390/cancers12051137

    Figure Lengend Snippet: USP47 regulates p53 in an MDM2-dependent manner. ( a ) HeLa cells transfected with siRNA against control or USP47 were treated with either DMSO (control) or MG132 (10 μM) for 4 h. Cell lysates were immunoblotted with the indicated antibodies. β-actin was used as a loading control. The band intensity was measured by Image J and then normalized by β-actin. ( b ) U2OS cells overexpressed with Flag vector or Flag-USP47 were treated with either DMSO (control) or MG132 (10 μM) for 4 h. Cell lysates were immunoblotted with the indicated antibodies. ( c ) U2OS cells were overexpressed with increasing amounts of Myc-USP47 (0, 1, and 2 μg). After 24 h, cell lysates were immunoblotted with the indicated antibodies. ( d ) U2OS cells transfected with siRNA against control or USP47 were treated with 100 μg/mL cycloheximide and harvested at the indicated times. Cell lysates were immunoblotted with the indicated antibodies. HSP90 was used as a loading control. The band intensity was normalized by HSP90. ( e ) MDM2 +/+ p53 −/− and MDM2 −/− p53 −/− Mouse Embryonic Fibroblast (MEF) cell lines were co-overexpressed with p53, Myc-USP47, and then cell lysates were immunoblotted with the indicated antibodies. The non-specific band is denoted by an asterisk. ( f ) MDM2 +/+ p53 −/− and MDM2 −/− p53 −/− Mouse Embryonic Fibroblast (MEF) cell lines were overexpressed with p53 for 24 h and then transfected with siRNA against control or USP47 for 48 h. Cell lysates were immunoblotted with the indicated antibodies. The non-specific band is denoted by an asterisk. ( g ) The real-time PCR analysis of p53 downstream genes in A549 cells transfected with siRNA against control or USP47 for 48 h. Relative expression values are normalized against β-actin RNA levels, and graphs are shown as fold induction over control siRNA transfected cells. Data from three independent experiments represented by mean ± SD (* p

    Article Snippet: MG132 (A.G scientific, San Diego, CA, USA) was treated at a 10 μM concentration to inhibit proteasomal degradation.

    Techniques: Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Expressing

    The degradation of IP3R3 is dependent on FBXL2 localization to cell membranes and on the segregase activity of p97 a , HeLa cells were transfected with GFP-tagged FBXL2 and then treated with either DMSO or GGTI-2418 for 16 h. Live cell imaging was carried out with an LSM510 confocal microscope using a 63× objective. Scale bars, 10 μm. b , NHFs were incubated with GGTi-2418 for 30 h and then with cycloheximide (CHX) and ATP. Cells were subsequently harvested at the indicated times for immunoblotting. The graph shows the quantification of IP3R3 levels from two independent experiments. c , NHFs (passage 3), HeLa and HEK293T cells were incubated with GGTi-2418 for the indicated times. Cells were subsequently harvested for immunoblotting. This experiment was performed once. d , NHFs (passage 3) were serum-starved for 72 h, treated with either DMSO or Eer1, and then re-stimulated with serum (SR) for the indicated times. The graph shows the quantification of IP3R3 levels from two independent experiments. The bracket on the right marks a ladder of bands which, presumably, are ubiquitinated species of IP3R3 that are not degraded when p97 is inhibited. e , During a 72 h serum starvation, NHFs (passage 3 and 4) were transfected with either an siRNA targeting p97 or a non-silencing siRNA (NS). Cells were subsequently stimulated with medium containing serum and harvested at the indicated time points for immunoblotting. The graph shows the quantification of IP3R3 levels from three independent experiments. Error bars indicate s.e.m. These results, together with those shown in d , are in agreement with the findings that IP3Rs are ubiquitinated while they are membrane-associated, and those showing that p97 promotes the degradation of IP3Rs 27 , 28 . Thus, we propose that, after its FBXL2-mediated ubiquitination, IP3R3 is extracted from cell membranes by the segregase activity of p97 to be degraded by the proteasome. f , HEK293T cells were transfected with either an empty vector (EV) or the indicated GFP-tagged and HA-tagged proteins. 24 h post-transfection, cells were treated with MG132 for 3 h before harvesting for immunoprecipitation and immunoblotting as indicated. This experiment was performed twice. g, h , HEK293T cells were transfected with Flag-tagged FBXL2 and the indicated versions of tagged IP3R3. After immunopurification with an anti-Flag resin, in vitro ubiquitination of IP3R3 was performed in the presence of UAE1, Ubch3, Ubch5 and ubiquitin (Ub). Where indicated, an excess of methylated ubiquitin (methyl-Ub), which blocks chain extension, was added to the in vitro reactions. The presence of methyl-Ub resulted in the disappearance of the highest molecular weight forms of IP3R3, demonstrating that the high molecular weight forms of IP3R3 are indeed polyubiquitinated species of the protein. Samples were analysed by immunoblotting with the indicated antibodies. The bracket on the right marks a ladder of bands corresponding to ubiquitinated IP3R3. Immunoblots of whole-cell lysates (WCL) are shown at the bottom. Unless otherwise noted, experiments were performed at least three times. For gel source data, see Supplementary Fig. 1 .

    Journal: Nature

    Article Title: PTEN counteracts FBXL2 to promote IP3R3– and Ca2+–mediated apoptosis limiting tumour growth

    doi: 10.1038/nature22965

    Figure Lengend Snippet: The degradation of IP3R3 is dependent on FBXL2 localization to cell membranes and on the segregase activity of p97 a , HeLa cells were transfected with GFP-tagged FBXL2 and then treated with either DMSO or GGTI-2418 for 16 h. Live cell imaging was carried out with an LSM510 confocal microscope using a 63× objective. Scale bars, 10 μm. b , NHFs were incubated with GGTi-2418 for 30 h and then with cycloheximide (CHX) and ATP. Cells were subsequently harvested at the indicated times for immunoblotting. The graph shows the quantification of IP3R3 levels from two independent experiments. c , NHFs (passage 3), HeLa and HEK293T cells were incubated with GGTi-2418 for the indicated times. Cells were subsequently harvested for immunoblotting. This experiment was performed once. d , NHFs (passage 3) were serum-starved for 72 h, treated with either DMSO or Eer1, and then re-stimulated with serum (SR) for the indicated times. The graph shows the quantification of IP3R3 levels from two independent experiments. The bracket on the right marks a ladder of bands which, presumably, are ubiquitinated species of IP3R3 that are not degraded when p97 is inhibited. e , During a 72 h serum starvation, NHFs (passage 3 and 4) were transfected with either an siRNA targeting p97 or a non-silencing siRNA (NS). Cells were subsequently stimulated with medium containing serum and harvested at the indicated time points for immunoblotting. The graph shows the quantification of IP3R3 levels from three independent experiments. Error bars indicate s.e.m. These results, together with those shown in d , are in agreement with the findings that IP3Rs are ubiquitinated while they are membrane-associated, and those showing that p97 promotes the degradation of IP3Rs 27 , 28 . Thus, we propose that, after its FBXL2-mediated ubiquitination, IP3R3 is extracted from cell membranes by the segregase activity of p97 to be degraded by the proteasome. f , HEK293T cells were transfected with either an empty vector (EV) or the indicated GFP-tagged and HA-tagged proteins. 24 h post-transfection, cells were treated with MG132 for 3 h before harvesting for immunoprecipitation and immunoblotting as indicated. This experiment was performed twice. g, h , HEK293T cells were transfected with Flag-tagged FBXL2 and the indicated versions of tagged IP3R3. After immunopurification with an anti-Flag resin, in vitro ubiquitination of IP3R3 was performed in the presence of UAE1, Ubch3, Ubch5 and ubiquitin (Ub). Where indicated, an excess of methylated ubiquitin (methyl-Ub), which blocks chain extension, was added to the in vitro reactions. The presence of methyl-Ub resulted in the disappearance of the highest molecular weight forms of IP3R3, demonstrating that the high molecular weight forms of IP3R3 are indeed polyubiquitinated species of the protein. Samples were analysed by immunoblotting with the indicated antibodies. The bracket on the right marks a ladder of bands corresponding to ubiquitinated IP3R3. Immunoblots of whole-cell lysates (WCL) are shown at the bottom. Unless otherwise noted, experiments were performed at least three times. For gel source data, see Supplementary Fig. 1 .

    Article Snippet: Where indicated, cells were treated with 10 μM MG132 (Peptides International), 100 μM cycloheximide (Sigma), 10-15 μM GGTi-2418 (Moffit Cancer Center), 10 μM Eeyarestatin 1 (Eer1, TOCRIS bioscience), 100 μM histamine (Sigma), 10 μM MRS2578 (Sigma), 100 μM ATP (Sigma), and 1 μM cyclosporin-A (Sigma).

    Techniques: Activity Assay, Transfection, Live Cell Imaging, Microscopy, Incubation, Plasmid Preparation, Immunoprecipitation, Immu-Puri, In Vitro, Methylation, Molecular Weight, Western Blot

    FBXL2-mediated degradation of IP3R3 controls Ca 2+ flux and sensitivity to apoptosis a , Concentrations of cytosolic Ca 2+ ([Ca 2+ ] c ) were measured with aequorin in response to agonist stimulation (ATP) in NHFs (passage 2 and 3) exponentially growing (Exp), serum-starved (SS), or re-stimulated with serum (SR), which were transfected with an siRNA targeting FBXL2 or a non-silencing (NS) siRNA. Left, two representative traces. Right, quantification of three independent experiments. P values were calculated by one-way ANOVA and multiple-comparisons test. Error bars indicate s.e.m. b-d , Apoptosis was evaluated after treatment with H 2 O 2 using automated nuclei count analysis of twenty randomly chosen fields following a 16 h treatment ( b ), immunoblot detection of cleaved PARP and cleaved caspase-3 following a 3 h treatment ( c ), and automated analysis of cells with released cytochrome c (Cyt c ) from 80 randomly chosen fields following a 3 h treatment ( d ). NHFs were transfected with the indicated siRNAs. Where indicated, cells were pre-treated for 30 min with cyclosporin A (CsA). P values were calculated by one-way ANOVA and multiple-comparisons test. Error bars indicate s.e.m. e , COS-7 cells were transfected with either GFP-tagged IP3R3 or GFP-tagged IP3R3(Q-FR/A-AA). 16 h post-transfection, cells were serum-starved for 48 h, and then re-stimulated with serum for the indicated times. Cells were harvested, and whole-cell lysates (WCLs) were immunoblotted as indicated. The graph shows the quantification of IP3R3 levels from two independent experiments. f , COS-7 cells transfected with the indicated constructs were serum-starved for 20 h and then re-stimulated with serum for 4 h. WCLs were immunoblotted as indicated. g , COS-7 cells transfected with the indicated constructs were serum-starved for 20 h, re-stimulated for 4 h with or without MG132, and treated with ATP. Left, representative traces show concentrations of cytosolic Ca 2+ measured with aequorin. Right, quantification of three independent experiments. P values were calculated by one-way ANOVA and multiple-comparisons test. Error bars indicate s.e.m. h , i , COS-7 cells transfected with the indicated constructs were serum-starved for 20 h, re-stimulated with serum for 4 h, and then treated with H 2 O 2 . Apoptosis shown in h was evaluated as in b , except that H 2 O 2 treatment was for 5 h. Analysis of cytochrome c release shown in i was evaluated as in d . P values were calculated by unpaired t- test. Error bars indicate s.e.m. Unless otherwise noted, experiments were performed at least three times. For gel source data, see Supplementary Fig. 1 .

    Journal: Nature

    Article Title: PTEN counteracts FBXL2 to promote IP3R3– and Ca2+–mediated apoptosis limiting tumour growth

    doi: 10.1038/nature22965

    Figure Lengend Snippet: FBXL2-mediated degradation of IP3R3 controls Ca 2+ flux and sensitivity to apoptosis a , Concentrations of cytosolic Ca 2+ ([Ca 2+ ] c ) were measured with aequorin in response to agonist stimulation (ATP) in NHFs (passage 2 and 3) exponentially growing (Exp), serum-starved (SS), or re-stimulated with serum (SR), which were transfected with an siRNA targeting FBXL2 or a non-silencing (NS) siRNA. Left, two representative traces. Right, quantification of three independent experiments. P values were calculated by one-way ANOVA and multiple-comparisons test. Error bars indicate s.e.m. b-d , Apoptosis was evaluated after treatment with H 2 O 2 using automated nuclei count analysis of twenty randomly chosen fields following a 16 h treatment ( b ), immunoblot detection of cleaved PARP and cleaved caspase-3 following a 3 h treatment ( c ), and automated analysis of cells with released cytochrome c (Cyt c ) from 80 randomly chosen fields following a 3 h treatment ( d ). NHFs were transfected with the indicated siRNAs. Where indicated, cells were pre-treated for 30 min with cyclosporin A (CsA). P values were calculated by one-way ANOVA and multiple-comparisons test. Error bars indicate s.e.m. e , COS-7 cells were transfected with either GFP-tagged IP3R3 or GFP-tagged IP3R3(Q-FR/A-AA). 16 h post-transfection, cells were serum-starved for 48 h, and then re-stimulated with serum for the indicated times. Cells were harvested, and whole-cell lysates (WCLs) were immunoblotted as indicated. The graph shows the quantification of IP3R3 levels from two independent experiments. f , COS-7 cells transfected with the indicated constructs were serum-starved for 20 h and then re-stimulated with serum for 4 h. WCLs were immunoblotted as indicated. g , COS-7 cells transfected with the indicated constructs were serum-starved for 20 h, re-stimulated for 4 h with or without MG132, and treated with ATP. Left, representative traces show concentrations of cytosolic Ca 2+ measured with aequorin. Right, quantification of three independent experiments. P values were calculated by one-way ANOVA and multiple-comparisons test. Error bars indicate s.e.m. h , i , COS-7 cells transfected with the indicated constructs were serum-starved for 20 h, re-stimulated with serum for 4 h, and then treated with H 2 O 2 . Apoptosis shown in h was evaluated as in b , except that H 2 O 2 treatment was for 5 h. Analysis of cytochrome c release shown in i was evaluated as in d . P values were calculated by unpaired t- test. Error bars indicate s.e.m. Unless otherwise noted, experiments were performed at least three times. For gel source data, see Supplementary Fig. 1 .

    Article Snippet: Where indicated, cells were treated with 10 μM MG132 (Peptides International), 100 μM cycloheximide (Sigma), 10-15 μM GGTi-2418 (Moffit Cancer Center), 10 μM Eeyarestatin 1 (Eer1, TOCRIS bioscience), 100 μM histamine (Sigma), 10 μM MRS2578 (Sigma), 100 μM ATP (Sigma), and 1 μM cyclosporin-A (Sigma).

    Techniques: Transfection, Construct

    FBXL2 controls Ca 2+ mobilization and Ca 2+ -mediated apoptosis a , Concentrations of mitochondrial Ca 2+ were measured with mitochondria-targeted aequorin in response to agonist stimulation (ATP, a purinergic GPCR (G-protein-coupled receptor) agonist) in exponentially growing (EXP), serum-starved (SS), and serum re-stimulated (SR) NHFs (passage 4 or 5). Quantification of three independent experiments is shown and represented as percentage change compared to EXP cells, which were set as 100%. P values were calculated by one-way ANOVA and multiple-comparisons test. Error bars indicate s.e.m. b , Concentrations of mitochondrial Ca 2+ were measured as in a in exponentially growing (EXP), serum-starved (SS), and serum re-stimulated for one hour (SR) NHFs (passage 5) transfected with either an siRNA targeting FBXL2 (#1) or a non-silencing siRNA (NS). Quantifications and P value analyses was performed as in a . c , Concentrations of cytosolic Ca 2+ were measured with aequorin in response to agonist stimulation (ATP) in NHFs (passage 2) re-stimulated with serum for one hour (SR) in the absence or presence of MG132 or GGTi2418. On the left, representative traces. On the right, quantification of three independent experiments. P values were calculated by a one-way ANOVA and multiple-comparisons test. Error bars indicate s.e.m. d, e , HeLa cells were transfected with either an empty vector (EV), FBXL2 or FBXL2(CaaX/SaaX). Concentrations of cytosolic ( d ) and mitochondrial ( e ) Ca 2+ were measured with the appropriate aequorin in response to agonist stimulation (ATP). On the left, representative traces. On the right, quantifications of three independent experiments. P values were calculated by one-way ANOVA and multiple-comparisons test. Error bars indicate s.e.m. f , Concentrations of mitochondrial Ca 2+ were measured with mitochondrial targeted aequorin upon treatment with H 2 O 2 in NHFs (passage 2) transfected with either an siRNA targeting FBXL2 (#1) or a non-silencing siRNA (NS). On the left, representative traces. On the right, quantifications of areas under the curve (AUC) are represented as percentage increase compared to NS-transfected cells, which were set as 100%. P values were calculated by unpaired t -test. Error bars indicate s.e.m. At the bottom, immunoblots of cell lysates of a representative experiment upon treatment with H 2 O 2 for 3 h. g , HeLa cells transfected with either an empty vector (EV), FBXL2 or FBXL2(CaaX/SaaX) were treated with either H 2 O 2 (for 5 h) or etoposide (for 5 h). Induction of cell death was evaluated using automated nuclei count analysis of twenty randomly chosen fields. P values were calculated by one-way ANOVA and multiple-comparisons test. Error bars indicate s.e.m. h , COS-7 cells expressing either IP3R3 or IP3R3(Q-FR/A-AA) in combination with Flag-tagged FBXL2 were serum-starved for 20 h, re-stimulated with serum (SR) for 4 h with or without MG132, as indicated, and treated with ATP. Left, representative traces showing concentrations of mitochondrial Ca 2+ measured with mitochondrial targeted aequorin. Right, quantification of three independent experiments. P values were calculated by one-way ANOVA and multiple-comparisons test. Error bars indicate s.e.m. i , Concentrations of mitochondrial Ca 2+ were measured with mitochondria-targeted aequorin upon treatment with H 2 O 2 in COS-7 cells expressing either IP3R3 or IP3R3(Q-FR/A-AA) in combination with Flag-tagged FBXL2. Left, representative traces. Middle, quantifications of areas under the curve represented as percentage increase compared to NS-transfected cells, which were set as 100%. P values were calculated using an unpaired t -test. Error bars indicate s.e.m. Right, immunoblots of cell lysates of a representative experiment upon treatment with H 2 O 2 for 3 h. Unless otherwise noted, experiments were performed at least three times. For gel source data, see Supplementary Fig. 1 .

    Journal: Nature

    Article Title: PTEN counteracts FBXL2 to promote IP3R3– and Ca2+–mediated apoptosis limiting tumour growth

    doi: 10.1038/nature22965

    Figure Lengend Snippet: FBXL2 controls Ca 2+ mobilization and Ca 2+ -mediated apoptosis a , Concentrations of mitochondrial Ca 2+ were measured with mitochondria-targeted aequorin in response to agonist stimulation (ATP, a purinergic GPCR (G-protein-coupled receptor) agonist) in exponentially growing (EXP), serum-starved (SS), and serum re-stimulated (SR) NHFs (passage 4 or 5). Quantification of three independent experiments is shown and represented as percentage change compared to EXP cells, which were set as 100%. P values were calculated by one-way ANOVA and multiple-comparisons test. Error bars indicate s.e.m. b , Concentrations of mitochondrial Ca 2+ were measured as in a in exponentially growing (EXP), serum-starved (SS), and serum re-stimulated for one hour (SR) NHFs (passage 5) transfected with either an siRNA targeting FBXL2 (#1) or a non-silencing siRNA (NS). Quantifications and P value analyses was performed as in a . c , Concentrations of cytosolic Ca 2+ were measured with aequorin in response to agonist stimulation (ATP) in NHFs (passage 2) re-stimulated with serum for one hour (SR) in the absence or presence of MG132 or GGTi2418. On the left, representative traces. On the right, quantification of three independent experiments. P values were calculated by a one-way ANOVA and multiple-comparisons test. Error bars indicate s.e.m. d, e , HeLa cells were transfected with either an empty vector (EV), FBXL2 or FBXL2(CaaX/SaaX). Concentrations of cytosolic ( d ) and mitochondrial ( e ) Ca 2+ were measured with the appropriate aequorin in response to agonist stimulation (ATP). On the left, representative traces. On the right, quantifications of three independent experiments. P values were calculated by one-way ANOVA and multiple-comparisons test. Error bars indicate s.e.m. f , Concentrations of mitochondrial Ca 2+ were measured with mitochondrial targeted aequorin upon treatment with H 2 O 2 in NHFs (passage 2) transfected with either an siRNA targeting FBXL2 (#1) or a non-silencing siRNA (NS). On the left, representative traces. On the right, quantifications of areas under the curve (AUC) are represented as percentage increase compared to NS-transfected cells, which were set as 100%. P values were calculated by unpaired t -test. Error bars indicate s.e.m. At the bottom, immunoblots of cell lysates of a representative experiment upon treatment with H 2 O 2 for 3 h. g , HeLa cells transfected with either an empty vector (EV), FBXL2 or FBXL2(CaaX/SaaX) were treated with either H 2 O 2 (for 5 h) or etoposide (for 5 h). Induction of cell death was evaluated using automated nuclei count analysis of twenty randomly chosen fields. P values were calculated by one-way ANOVA and multiple-comparisons test. Error bars indicate s.e.m. h , COS-7 cells expressing either IP3R3 or IP3R3(Q-FR/A-AA) in combination with Flag-tagged FBXL2 were serum-starved for 20 h, re-stimulated with serum (SR) for 4 h with or without MG132, as indicated, and treated with ATP. Left, representative traces showing concentrations of mitochondrial Ca 2+ measured with mitochondrial targeted aequorin. Right, quantification of three independent experiments. P values were calculated by one-way ANOVA and multiple-comparisons test. Error bars indicate s.e.m. i , Concentrations of mitochondrial Ca 2+ were measured with mitochondria-targeted aequorin upon treatment with H 2 O 2 in COS-7 cells expressing either IP3R3 or IP3R3(Q-FR/A-AA) in combination with Flag-tagged FBXL2. Left, representative traces. Middle, quantifications of areas under the curve represented as percentage increase compared to NS-transfected cells, which were set as 100%. P values were calculated using an unpaired t -test. Error bars indicate s.e.m. Right, immunoblots of cell lysates of a representative experiment upon treatment with H 2 O 2 for 3 h. Unless otherwise noted, experiments were performed at least three times. For gel source data, see Supplementary Fig. 1 .

    Article Snippet: Where indicated, cells were treated with 10 μM MG132 (Peptides International), 100 μM cycloheximide (Sigma), 10-15 μM GGTi-2418 (Moffit Cancer Center), 10 μM Eeyarestatin 1 (Eer1, TOCRIS bioscience), 100 μM histamine (Sigma), 10 μM MRS2578 (Sigma), 100 μM ATP (Sigma), and 1 μM cyclosporin-A (Sigma).

    Techniques: Transfection, Plasmid Preparation, Western Blot, Expressing

    Mapping of the FBXL2-binding domain in IP3R3, and evidence that the N-terminal suppressor domain inhibits the IP3R3-FBXL2 interaction a , Schematic representation of IP3R3 mutants. Binding of IP3R3 to FBXL2 is indicated with the symbol (+). b , HEK293T cells were transfected with GFP-tagged FBXL2 and the indicated Flag-tagged IP3R3 truncated mutants. Whole-cell lysates (WCL) were immunoprecipitated (IP) with anti-Flag resin and proteins were immunoblotted as indicated. This experiment was performed twice. c , HEK293T cells were transfected with GFP-tagged FBXL2 and HA-tagged In IP3R3(1-602) constructs. Sixteen hours after transfection, cells were incubated with MG132 for 3 h before stimulation with ATP for 30 min. Whole-cell lysates were immunoprecipitated (IP) with an anti-HA resin and proteins were immunoblotted as indicated. This experiment was performed twice. d , HeLa cells stably transfected with Flag-tagged FBXL2 under the control of a doxycycline-inducible promoter were treated with doxycycline (0.4 μ g ml −1 ) for 16 h and incubated with or without MG132 (during the last 3 h), in the presence or absence of MRS 2578, an antagonist of P2Y6 receptor (during the last 90 min), as indicated. Cells were subsequently treated with or without ATP for 30 min. Whole-cell lysates were immunoprecipitated (IP) with an anti-Flag resin and proteins were immunoblotted as indicated. Right panel shows quantifications of IP3R3 levels compared to untreated cells (UT), which were set as 100%. P values were calculated by one-way ANOVA and multiple-comparisons test. Error bars indicate s.e.m. The fact (shown in b ) that fragments encoding IP3R3(436–587) and IP3R3(227–602) interact with FBXL2 better than the IP3R3(1–602) fragment suggests that the N terminus of IP3R3 inhibits the interaction between FBXL2 and IP3R3. It has been shown that removal of the N-terminal suppressor domain (amino acids 1–226) increases the binding of IP3 to the IP3 binding core domain (amino acids 227–579), and IP3 binding evokes conformational changes that open the suppressor domain 29 – 32 . These conf ormational changes may allow FBXL2 to access the degron of IP3R3 (that is, the amino acid region required for binding to FBXL2). Thus, in agreement with previous data indicating that IP3 causes the ubiquitination and downregulation of IP3Rs ( ref. 27 ), our results suggest that IP3 promotes the binding of FBXL2 to IP3R3 and that FBXL2 preferentially binds IP3R3 in its open conformation (upon IP3 binding). Accordingly, treatment of cells with ATP, which induces IP3 production and repositioning of the N-terminal suppressor domain, increased the binding between FBXL2 and IP3R3, particularly if proteasomal degradation was inhibited by MG132. This suggests that once IP3 (produced after ATP stimulation) unmasks the IP3R3 degron, FBXL2 binds IP3R3 and this interaction is preserved when FBXL2-mediated degradation of IP3R3 is inhibited. So, ATP and MG132 appear to synergize with each other in promoting and preserving the FBXL2–IP3R3 interaction, respectively. e , Serum-starved (SS) and exponentially (EXP) growing NHFs (passage 2 and 3) were treated with or without ATP as indicated. Cells were subsequently harvested at the indicated time points for immunoblotting. The graph shows the quantification of IP3R3 levels. Error bars indicate s.e.m. f , HEK293T cells were transfected with GFP-tagged FBXL2 and the indicated Flag-tagged IP3R3 deletion mutants (in the context of the 436–587 domain of IP3R3). Whole-cell lysates (WCL) were immunoprecipitated (IP) with anti-Flag resin, and proteins were immunoblotted as indicated. This experiment was performed twice. g , The experiment was performed twice as in f , except that different IP3R3 mutants were used. h , Alignment of the amino acid regions containing the FBXL2-binding motif in IP3R3 orthologues. i , Alignment of the amino acid regions containing the FBXL2-binding motif in human IP3R3 with the corresponding region in IP3R1 and IP3R2. j , HEK293T cells were transfected with the indicated constructs. Whole-cell lysates (WCL) were immunoprecipitated (IP) with anti-Flag resin and immunoblotted as indicated. The bracket on the right marks a ladder of bands corresponding to polyubiquitinated IP3R3. This experiment was performed twice. Unless otherwise noted, experiments were performed at least three times. For gel source data, see Supplementary Fig. 1 .

    Journal: Nature

    Article Title: PTEN counteracts FBXL2 to promote IP3R3– and Ca2+–mediated apoptosis limiting tumour growth

    doi: 10.1038/nature22965

    Figure Lengend Snippet: Mapping of the FBXL2-binding domain in IP3R3, and evidence that the N-terminal suppressor domain inhibits the IP3R3-FBXL2 interaction a , Schematic representation of IP3R3 mutants. Binding of IP3R3 to FBXL2 is indicated with the symbol (+). b , HEK293T cells were transfected with GFP-tagged FBXL2 and the indicated Flag-tagged IP3R3 truncated mutants. Whole-cell lysates (WCL) were immunoprecipitated (IP) with anti-Flag resin and proteins were immunoblotted as indicated. This experiment was performed twice. c , HEK293T cells were transfected with GFP-tagged FBXL2 and HA-tagged In IP3R3(1-602) constructs. Sixteen hours after transfection, cells were incubated with MG132 for 3 h before stimulation with ATP for 30 min. Whole-cell lysates were immunoprecipitated (IP) with an anti-HA resin and proteins were immunoblotted as indicated. This experiment was performed twice. d , HeLa cells stably transfected with Flag-tagged FBXL2 under the control of a doxycycline-inducible promoter were treated with doxycycline (0.4 μ g ml −1 ) for 16 h and incubated with or without MG132 (during the last 3 h), in the presence or absence of MRS 2578, an antagonist of P2Y6 receptor (during the last 90 min), as indicated. Cells were subsequently treated with or without ATP for 30 min. Whole-cell lysates were immunoprecipitated (IP) with an anti-Flag resin and proteins were immunoblotted as indicated. Right panel shows quantifications of IP3R3 levels compared to untreated cells (UT), which were set as 100%. P values were calculated by one-way ANOVA and multiple-comparisons test. Error bars indicate s.e.m. The fact (shown in b ) that fragments encoding IP3R3(436–587) and IP3R3(227–602) interact with FBXL2 better than the IP3R3(1–602) fragment suggests that the N terminus of IP3R3 inhibits the interaction between FBXL2 and IP3R3. It has been shown that removal of the N-terminal suppressor domain (amino acids 1–226) increases the binding of IP3 to the IP3 binding core domain (amino acids 227–579), and IP3 binding evokes conformational changes that open the suppressor domain 29 – 32 . These conf ormational changes may allow FBXL2 to access the degron of IP3R3 (that is, the amino acid region required for binding to FBXL2). Thus, in agreement with previous data indicating that IP3 causes the ubiquitination and downregulation of IP3Rs ( ref. 27 ), our results suggest that IP3 promotes the binding of FBXL2 to IP3R3 and that FBXL2 preferentially binds IP3R3 in its open conformation (upon IP3 binding). Accordingly, treatment of cells with ATP, which induces IP3 production and repositioning of the N-terminal suppressor domain, increased the binding between FBXL2 and IP3R3, particularly if proteasomal degradation was inhibited by MG132. This suggests that once IP3 (produced after ATP stimulation) unmasks the IP3R3 degron, FBXL2 binds IP3R3 and this interaction is preserved when FBXL2-mediated degradation of IP3R3 is inhibited. So, ATP and MG132 appear to synergize with each other in promoting and preserving the FBXL2–IP3R3 interaction, respectively. e , Serum-starved (SS) and exponentially (EXP) growing NHFs (passage 2 and 3) were treated with or without ATP as indicated. Cells were subsequently harvested at the indicated time points for immunoblotting. The graph shows the quantification of IP3R3 levels. Error bars indicate s.e.m. f , HEK293T cells were transfected with GFP-tagged FBXL2 and the indicated Flag-tagged IP3R3 deletion mutants (in the context of the 436–587 domain of IP3R3). Whole-cell lysates (WCL) were immunoprecipitated (IP) with anti-Flag resin, and proteins were immunoblotted as indicated. This experiment was performed twice. g , The experiment was performed twice as in f , except that different IP3R3 mutants were used. h , Alignment of the amino acid regions containing the FBXL2-binding motif in IP3R3 orthologues. i , Alignment of the amino acid regions containing the FBXL2-binding motif in human IP3R3 with the corresponding region in IP3R1 and IP3R2. j , HEK293T cells were transfected with the indicated constructs. Whole-cell lysates (WCL) were immunoprecipitated (IP) with anti-Flag resin and immunoblotted as indicated. The bracket on the right marks a ladder of bands corresponding to polyubiquitinated IP3R3. This experiment was performed twice. Unless otherwise noted, experiments were performed at least three times. For gel source data, see Supplementary Fig. 1 .

    Article Snippet: Where indicated, cells were treated with 10 μM MG132 (Peptides International), 100 μM cycloheximide (Sigma), 10-15 μM GGTi-2418 (Moffit Cancer Center), 10 μM Eeyarestatin 1 (Eer1, TOCRIS bioscience), 100 μM histamine (Sigma), 10 μM MRS2578 (Sigma), 100 μM ATP (Sigma), and 1 μM cyclosporin-A (Sigma).

    Techniques: Binding Assay, Transfection, Immunoprecipitation, Construct, Incubation, Stable Transfection, Produced, Preserving

    IP3R3 is targeted for proteasomal degradation by FBXL2 a , HEK293T cells were transfected with the indicated Flag-tagged F-box proteins (FBPs). 24 h post-transfection, cells were treated with MG132 for 3 h before harvesting for immunoprecipitation and immunoblotting as indicated. WCL, whole-cell lysate from untransfected cells. b , Flag-tagged FBXL2 was stably expressed in HeLa cells. After harvesting, cells were lysed, and cell membranes (CM) were isolated and immunoprecipitated with either a normal mouse IgG or an anti-Flag antibody. Subsequently, immunoprecipitates were analysed by immunoblotting as indicated. The results of these experiments ( n = 2) indicate that FBXL2 is incorporated into a complete SCF ligase that binds its substrate(s) at cellular membranes. c , Flag-tagged FBXL2 or Flag-tagged FBXL2(CaaX/SaaX) were transiently expressed in HeLa cells. After harvesting, cells were lysed and cytoplasmic (CYTO) and cell membrane (CM) fractions were isolated and analysed by immunoblotting as indicated. Actin and calnexin are used as markers for cytoplasmic and cell membrane fractions, respectively. This experiment was performed twice. d , HEK293T cells were transfected with either an empty vector (EV) or the indicated Flag-tagged proteins. 24 h post-transfection, where indicated, cells were treated with MG132 for 3 h before harvesting for immunoprecipitation and immunoblotting. Ned. CUL1, neddylated CUL1. This experiment shows that FBXL2, but not FBXL2(CaaX/SaaX), interacts with endogenous, neddylated CUL1. Since the covalent linkage of NEDD8 to CUL1 stimulates the ubiquitin ligase activity of SCFs and is promoted by the binding of the substrate to the F-box protein subunit, this result suggests that FBXL2 localization to cell membranes is required for substrate binding, which in turn stimulates CUL1 neddylation and SCF activation. Moreover, FBXL2(ΔF-box) bound more IP3R3 than wild-type FBXL2, and this difference could be abolished by treatment with MG132, supporting the hypothesis that FBXL2(ΔF-box) cannot mediate the degradation of IP3R3 since it does not form an active SCF complex. e , HEK293T cells were transfected with either an empty vector or the indicated Flag-tagged proteins. The experiment was performed in the presence or absence of MG132 as indicated. Whole-cell lysates were immunoblotted as indicated. The asterisk indicates a non-specific band. This experiment was performed twice. The fact that proteasome inhibitors prevented the decrease of IP3R3 levels upon re-addition of serum suggests that IP3R3 is degraded by the proteasome in response to mitogens, in agreement with previous studies reporting IP3Rs as substrates of the proteasome 26 , 27 . f , Normal, non-transformed, non-immortalized, human diploid fibroblasts (NHFs) (passage 2) were serum-starved for 72 h and then re-stimulated with serum (SR) for 30 min in the absence or presence of either MG132 (a proteasome inhibitor) or lactacystin (another proteasome inhibitor) as indicated. The graph on the right shows the quantification of IP3R3 levels from three independent experiments. P values were calculated by one-way ANOVA. Error bars indicate s.e.m. g , NHFs (passage 2) were transfected with either three different siRNAs targeting FBXL2 (each independently) or a non-silencing siRNA (NS). The graph shows FBXL2 mRNA levels analysed using realtime PCR in triplicate measurements. Error bars indicate s.e.m. The values represent the ratios between FBXL2 and GAPDH mRNAs. h , During a 72 h serum starvation, NHFs (passage 2 or 3) were transfected with either an siRNA targeting FBXL2 (#1) or a non-silencing siRNA (NS). Cells were subsequently stimulated with medium containing serum and harvested at the indicated times for immunoblotting. The graph shows the quantification of IP3R3 levels from three independent experiments. Error bars indicate s.e.m. i , During a 72 h serum starvation, NHFs (passage 3 or 4) were transfected with either siRNAs targeting FBXL2 (oligo #2 or #3) or a non-silencing siRNA (NS). Cells were subsequently stimulated with medium containing serum and harvested at the indicated time points for immunoblotting. The graph shows the quantification of IP3R3 levels from three independent experiments. Error bars indicate s.e.m. j , Schematic representation of the FBXL2 genomic locus and gRNAs target location. Exon 2 and exon 3 refer to the human FBXL2 gene in NC_000003.12 (GRCh38.p7 (Gene Bank ID: 3129728)). k , Schematic representation of FBXL2 CRISPR–Cas9 mutagenesis outcomes. A first round of CRISPR– Cas9 gene editing yielded no homozygous FBXL2 -knockout clones. A secondary round of CRISPR–Cas9 gene editing was carried out in three FBXL2 +/− hTERT RPE-1 clones, which resulted in cell death, suggesting that FBXL2 is required for cell fitness and it is not possible to generate FBXL2 -knockout cells. Similar results were obtained in A549 cells (data not shown). l , FBXL2 +/+ and FBXL2 +/− RPE-1-hTERT cells (clones 2 and 3) were serum-starved for 72 h and subsequently stimulated with medium containing serum for 90 min, after which cell extracts were immunoblotted for the indicated proteins. The graph shows the quantification of IP3R3 levels from three independent experiments. Unless otherwise noted, experiments were performed at least three times. For gel source data, see Supplementary Fig. 1 .

    Journal: Nature

    Article Title: PTEN counteracts FBXL2 to promote IP3R3– and Ca2+–mediated apoptosis limiting tumour growth

    doi: 10.1038/nature22965

    Figure Lengend Snippet: IP3R3 is targeted for proteasomal degradation by FBXL2 a , HEK293T cells were transfected with the indicated Flag-tagged F-box proteins (FBPs). 24 h post-transfection, cells were treated with MG132 for 3 h before harvesting for immunoprecipitation and immunoblotting as indicated. WCL, whole-cell lysate from untransfected cells. b , Flag-tagged FBXL2 was stably expressed in HeLa cells. After harvesting, cells were lysed, and cell membranes (CM) were isolated and immunoprecipitated with either a normal mouse IgG or an anti-Flag antibody. Subsequently, immunoprecipitates were analysed by immunoblotting as indicated. The results of these experiments ( n = 2) indicate that FBXL2 is incorporated into a complete SCF ligase that binds its substrate(s) at cellular membranes. c , Flag-tagged FBXL2 or Flag-tagged FBXL2(CaaX/SaaX) were transiently expressed in HeLa cells. After harvesting, cells were lysed and cytoplasmic (CYTO) and cell membrane (CM) fractions were isolated and analysed by immunoblotting as indicated. Actin and calnexin are used as markers for cytoplasmic and cell membrane fractions, respectively. This experiment was performed twice. d , HEK293T cells were transfected with either an empty vector (EV) or the indicated Flag-tagged proteins. 24 h post-transfection, where indicated, cells were treated with MG132 for 3 h before harvesting for immunoprecipitation and immunoblotting. Ned. CUL1, neddylated CUL1. This experiment shows that FBXL2, but not FBXL2(CaaX/SaaX), interacts with endogenous, neddylated CUL1. Since the covalent linkage of NEDD8 to CUL1 stimulates the ubiquitin ligase activity of SCFs and is promoted by the binding of the substrate to the F-box protein subunit, this result suggests that FBXL2 localization to cell membranes is required for substrate binding, which in turn stimulates CUL1 neddylation and SCF activation. Moreover, FBXL2(ΔF-box) bound more IP3R3 than wild-type FBXL2, and this difference could be abolished by treatment with MG132, supporting the hypothesis that FBXL2(ΔF-box) cannot mediate the degradation of IP3R3 since it does not form an active SCF complex. e , HEK293T cells were transfected with either an empty vector or the indicated Flag-tagged proteins. The experiment was performed in the presence or absence of MG132 as indicated. Whole-cell lysates were immunoblotted as indicated. The asterisk indicates a non-specific band. This experiment was performed twice. The fact that proteasome inhibitors prevented the decrease of IP3R3 levels upon re-addition of serum suggests that IP3R3 is degraded by the proteasome in response to mitogens, in agreement with previous studies reporting IP3Rs as substrates of the proteasome 26 , 27 . f , Normal, non-transformed, non-immortalized, human diploid fibroblasts (NHFs) (passage 2) were serum-starved for 72 h and then re-stimulated with serum (SR) for 30 min in the absence or presence of either MG132 (a proteasome inhibitor) or lactacystin (another proteasome inhibitor) as indicated. The graph on the right shows the quantification of IP3R3 levels from three independent experiments. P values were calculated by one-way ANOVA. Error bars indicate s.e.m. g , NHFs (passage 2) were transfected with either three different siRNAs targeting FBXL2 (each independently) or a non-silencing siRNA (NS). The graph shows FBXL2 mRNA levels analysed using realtime PCR in triplicate measurements. Error bars indicate s.e.m. The values represent the ratios between FBXL2 and GAPDH mRNAs. h , During a 72 h serum starvation, NHFs (passage 2 or 3) were transfected with either an siRNA targeting FBXL2 (#1) or a non-silencing siRNA (NS). Cells were subsequently stimulated with medium containing serum and harvested at the indicated times for immunoblotting. The graph shows the quantification of IP3R3 levels from three independent experiments. Error bars indicate s.e.m. i , During a 72 h serum starvation, NHFs (passage 3 or 4) were transfected with either siRNAs targeting FBXL2 (oligo #2 or #3) or a non-silencing siRNA (NS). Cells were subsequently stimulated with medium containing serum and harvested at the indicated time points for immunoblotting. The graph shows the quantification of IP3R3 levels from three independent experiments. Error bars indicate s.e.m. j , Schematic representation of the FBXL2 genomic locus and gRNAs target location. Exon 2 and exon 3 refer to the human FBXL2 gene in NC_000003.12 (GRCh38.p7 (Gene Bank ID: 3129728)). k , Schematic representation of FBXL2 CRISPR–Cas9 mutagenesis outcomes. A first round of CRISPR– Cas9 gene editing yielded no homozygous FBXL2 -knockout clones. A secondary round of CRISPR–Cas9 gene editing was carried out in three FBXL2 +/− hTERT RPE-1 clones, which resulted in cell death, suggesting that FBXL2 is required for cell fitness and it is not possible to generate FBXL2 -knockout cells. Similar results were obtained in A549 cells (data not shown). l , FBXL2 +/+ and FBXL2 +/− RPE-1-hTERT cells (clones 2 and 3) were serum-starved for 72 h and subsequently stimulated with medium containing serum for 90 min, after which cell extracts were immunoblotted for the indicated proteins. The graph shows the quantification of IP3R3 levels from three independent experiments. Unless otherwise noted, experiments were performed at least three times. For gel source data, see Supplementary Fig. 1 .

    Article Snippet: Where indicated, cells were treated with 10 μM MG132 (Peptides International), 100 μM cycloheximide (Sigma), 10-15 μM GGTi-2418 (Moffit Cancer Center), 10 μM Eeyarestatin 1 (Eer1, TOCRIS bioscience), 100 μM histamine (Sigma), 10 μM MRS2578 (Sigma), 100 μM ATP (Sigma), and 1 μM cyclosporin-A (Sigma).

    Techniques: Transfection, Immunoprecipitation, Stable Transfection, Isolation, Plasmid Preparation, Activity Assay, Binding Assay, Activation Assay, Transformation Assay, Polymerase Chain Reaction, CRISPR, Mutagenesis, Knock-Out, Clone Assay

    Effect of crizotinib treatment on A549 cell metabolism in conjunction with various inhibitors. After pretreatment with MG132, 2-DG and rotenone, the effects of 1 µM crizotinib on (A) cell viability, (B) glucose consumption, (C) lactate content and (D) ATP content were determined in A549 cells. *P

    Journal: Oncology Letters

    Article Title: Crizotinib changes the metabolic pattern and inhibits ATP production in A549 non-small cell lung cancer cells

    doi: 10.3892/ol.2020.12323

    Figure Lengend Snippet: Effect of crizotinib treatment on A549 cell metabolism in conjunction with various inhibitors. After pretreatment with MG132, 2-DG and rotenone, the effects of 1 µM crizotinib on (A) cell viability, (B) glucose consumption, (C) lactate content and (D) ATP content were determined in A549 cells. *P

    Article Snippet: Cell viabilityAfter cells were treated with 0–100 µM crizotinib or other inhibitors, including 10 nM rotenone, 10 mM 2-DG or 10 nM MG132 for 24 h, 10 µl MTS solution (Promega Corporation) was added to the supernatant.

    Techniques:

    Mutations of residues M260A and L261A partly restrained C protein degradation. a Schematic representation of mutant C proteins, C M260-261 and C M260-263 , in which residues 260-261 and 260-263 in C protein are all mutated to Alanine (A), respectively. Mutant residues are shown above the oblong. b C M260-261 and C M260-263 were expressed in PK-15 cells followed by treatment of MG132 or DMSO as described above. Western blot was performed and C M260-261 and C M260-263 were detected with the indicated antibodies. Protein marker is shown on the right

    Journal: Virology Journal

    Article Title: Important roles of C-terminal residues in degradation of capsid protein of classical swine fever virus

    doi: 10.1186/s12985-019-1238-1

    Figure Lengend Snippet: Mutations of residues M260A and L261A partly restrained C protein degradation. a Schematic representation of mutant C proteins, C M260-261 and C M260-263 , in which residues 260-261 and 260-263 in C protein are all mutated to Alanine (A), respectively. Mutant residues are shown above the oblong. b C M260-261 and C M260-263 were expressed in PK-15 cells followed by treatment of MG132 or DMSO as described above. Western blot was performed and C M260-261 and C M260-263 were detected with the indicated antibodies. Protein marker is shown on the right

    Article Snippet: Biochemical intervention PK-15 or 3D4/2 cells are treated with 10 μM MG132 (A2585, Apexbio) diluted in dimethyl sulfoxide (DMSO) or 5 mM 3-methyladenine (3-MA) (A8353, Apexbio) diluted in sterile ddH2 O for 16 h. The same amount of DMSO or ddH2 O was added to the inoculum as controls.

    Techniques: Mutagenesis, Western Blot, Marker

    C protein is not ubiquitinated. Plasmids encoding proteins C Δ2 , C M260-263 and EGFP were transiently transfected in PK-15 cells followed by treatment of MG132 (10 μM) for 16 h. Cells were lysed and immunoprecipitation was performed. Proteins in cell lysis and proteins pulled-down are detected by Western blot using anti-EGFP and anti-Ub antibodies

    Journal: Virology Journal

    Article Title: Important roles of C-terminal residues in degradation of capsid protein of classical swine fever virus

    doi: 10.1186/s12985-019-1238-1

    Figure Lengend Snippet: C protein is not ubiquitinated. Plasmids encoding proteins C Δ2 , C M260-263 and EGFP were transiently transfected in PK-15 cells followed by treatment of MG132 (10 μM) for 16 h. Cells were lysed and immunoprecipitation was performed. Proteins in cell lysis and proteins pulled-down are detected by Western blot using anti-EGFP and anti-Ub antibodies

    Article Snippet: Biochemical intervention PK-15 or 3D4/2 cells are treated with 10 μM MG132 (A2585, Apexbio) diluted in dimethyl sulfoxide (DMSO) or 5 mM 3-methyladenine (3-MA) (A8353, Apexbio) diluted in sterile ddH2 O for 16 h. The same amount of DMSO or ddH2 O was added to the inoculum as controls.

    Techniques: Transfection, Immunoprecipitation, Lysis, Western Blot

    MG132 upregulated the level of EGFP-tagged C protein. a PK-15 cells were transfected with plasmid pEGFP-N1-C followed by treatment of 10 μM MG132 or 5 mM 3-MA or both of them for 16 h. DMSO or ddH 2 O were used as controls. At 20 hpt, cells were lysed and subjected to Western blot analysis with the indicated antibodies. Tubulin was used as a control. b Schematic representation of EGFP-C protein and its cleaved form. Black frame represents C protein and green frame represents EGFP protein. The arrow indicates the cleavage site of C protein by SPP. PK-15 cells were transfected with plasmid pEGFP-C1-C and were treated with or without MG132 (10 μM). Empty vector pEGFP-C1 was used as a control. Cells were lysed at 20 hpt and Western blot was performed with the indicated antibodies. c PK-15 cells were transfected with plasmid pEGFP-N1-C and treated with or without MG132 as described above. Empty vector pEGFP-N1 was used as a control. Western blot was performed as described above. Relative mRNA levels of EGFP ( d ) and C ( e ) in cells were detected by qRT-PCR. Data was analyzed by Student’s t-test. f 3D4/2 cells were transfected with plasmid pEGFP-N1-C or pEGFP-N1 followed by treatment of 10 μM MG132 or same volume of DMSO for 16 h. The fluorescence in cells was observed at 20 hpt. Scale bar, 100 μm. g Cells in ( f ) were then lysed and analysed by Western blot with the indicated antibodies. Protein marker is shown on the right. Data in all bar plots are shown as mean ± SD and representative of 3 biological replicates. ***, P

    Journal: Virology Journal

    Article Title: Important roles of C-terminal residues in degradation of capsid protein of classical swine fever virus

    doi: 10.1186/s12985-019-1238-1

    Figure Lengend Snippet: MG132 upregulated the level of EGFP-tagged C protein. a PK-15 cells were transfected with plasmid pEGFP-N1-C followed by treatment of 10 μM MG132 or 5 mM 3-MA or both of them for 16 h. DMSO or ddH 2 O were used as controls. At 20 hpt, cells were lysed and subjected to Western blot analysis with the indicated antibodies. Tubulin was used as a control. b Schematic representation of EGFP-C protein and its cleaved form. Black frame represents C protein and green frame represents EGFP protein. The arrow indicates the cleavage site of C protein by SPP. PK-15 cells were transfected with plasmid pEGFP-C1-C and were treated with or without MG132 (10 μM). Empty vector pEGFP-C1 was used as a control. Cells were lysed at 20 hpt and Western blot was performed with the indicated antibodies. c PK-15 cells were transfected with plasmid pEGFP-N1-C and treated with or without MG132 as described above. Empty vector pEGFP-N1 was used as a control. Western blot was performed as described above. Relative mRNA levels of EGFP ( d ) and C ( e ) in cells were detected by qRT-PCR. Data was analyzed by Student’s t-test. f 3D4/2 cells were transfected with plasmid pEGFP-N1-C or pEGFP-N1 followed by treatment of 10 μM MG132 or same volume of DMSO for 16 h. The fluorescence in cells was observed at 20 hpt. Scale bar, 100 μm. g Cells in ( f ) were then lysed and analysed by Western blot with the indicated antibodies. Protein marker is shown on the right. Data in all bar plots are shown as mean ± SD and representative of 3 biological replicates. ***, P

    Article Snippet: Biochemical intervention PK-15 or 3D4/2 cells are treated with 10 μM MG132 (A2585, Apexbio) diluted in dimethyl sulfoxide (DMSO) or 5 mM 3-methyladenine (3-MA) (A8353, Apexbio) diluted in sterile ddH2 O for 16 h. The same amount of DMSO or ddH2 O was added to the inoculum as controls.

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

    C-terminal residues 260 to 267, especially 260-262, are responsible for the degradation of C protein. a Schematic representation of a series of truncated fusion proteins with various residues deletions at C-terminal of C proteins. The numbers in dark oblong showed the remaining residues of C protein. b PK-15 cells were transfected with plasmids encoding C Δ8 , C Δ7 , C Δ5 and C 2 proteins, respectively. Cells were treated with MG132 or DMSO for 16 h and observed under a fluorescence microscope . Scale bar, 100 μm. c Cells were then lysed and analysed by Western blot with the indicated antibodies. Protein marker is shown on the right

    Journal: Virology Journal

    Article Title: Important roles of C-terminal residues in degradation of capsid protein of classical swine fever virus

    doi: 10.1186/s12985-019-1238-1

    Figure Lengend Snippet: C-terminal residues 260 to 267, especially 260-262, are responsible for the degradation of C protein. a Schematic representation of a series of truncated fusion proteins with various residues deletions at C-terminal of C proteins. The numbers in dark oblong showed the remaining residues of C protein. b PK-15 cells were transfected with plasmids encoding C Δ8 , C Δ7 , C Δ5 and C 2 proteins, respectively. Cells were treated with MG132 or DMSO for 16 h and observed under a fluorescence microscope . Scale bar, 100 μm. c Cells were then lysed and analysed by Western blot with the indicated antibodies. Protein marker is shown on the right

    Article Snippet: Biochemical intervention PK-15 or 3D4/2 cells are treated with 10 μM MG132 (A2585, Apexbio) diluted in dimethyl sulfoxide (DMSO) or 5 mM 3-methyladenine (3-MA) (A8353, Apexbio) diluted in sterile ddH2 O for 16 h. The same amount of DMSO or ddH2 O was added to the inoculum as controls.

    Techniques: Transfection, Fluorescence, Microscopy, Western Blot, Marker

    C-terminal residues are more responsible for the degradation of C protein. a Schematic representation of C-EGFP, C ΔC and C ΔN proteins. The numbers in dark oblong showed the remaining residues of C protein. C ΔC is constructed by fusing the first 35 amino acids at N-termianl of C protein to EGFP protein. C ΔN is constructed by fusing the last 64 amino acids at C-termianl of C protein to EGFP protein. b PK-15 cells were transfected with plasmids encoding C ΔC and C ΔN proteins followed by treatment of MG132 or DMSO as described above. Cells were lysed and analysed by Western blot with the indicated antibodies. Protein marker is shown on the right

    Journal: Virology Journal

    Article Title: Important roles of C-terminal residues in degradation of capsid protein of classical swine fever virus

    doi: 10.1186/s12985-019-1238-1

    Figure Lengend Snippet: C-terminal residues are more responsible for the degradation of C protein. a Schematic representation of C-EGFP, C ΔC and C ΔN proteins. The numbers in dark oblong showed the remaining residues of C protein. C ΔC is constructed by fusing the first 35 amino acids at N-termianl of C protein to EGFP protein. C ΔN is constructed by fusing the last 64 amino acids at C-termianl of C protein to EGFP protein. b PK-15 cells were transfected with plasmids encoding C ΔC and C ΔN proteins followed by treatment of MG132 or DMSO as described above. Cells were lysed and analysed by Western blot with the indicated antibodies. Protein marker is shown on the right

    Article Snippet: Biochemical intervention PK-15 or 3D4/2 cells are treated with 10 μM MG132 (A2585, Apexbio) diluted in dimethyl sulfoxide (DMSO) or 5 mM 3-methyladenine (3-MA) (A8353, Apexbio) diluted in sterile ddH2 O for 16 h. The same amount of DMSO or ddH2 O was added to the inoculum as controls.

    Techniques: Construct, Transfection, Western Blot, Marker

    Catalase is neither phosphorylated nor ubiquitinated in breast cancer cells. (a) Catalase protein levels were measured after 5 h incubation with proteasome inhibitor MG132 in both MCF-7 and Resox cells. (b) Immunoprecipitation (IP) with anticatalase antibody and immunoblotting (IB) with anti-Flag, antiphosphocatalase, and catalase antibodies. Prior IP, cells were transfected with a plasmid pcDNA3 (1 μ g) coding an ubiquitin-Flag for 72 h.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Evaluation of Potential Mechanisms Controlling the Catalase Expression in Breast Cancer Cells

    doi: 10.1155/2018/5351967

    Figure Lengend Snippet: Catalase is neither phosphorylated nor ubiquitinated in breast cancer cells. (a) Catalase protein levels were measured after 5 h incubation with proteasome inhibitor MG132 in both MCF-7 and Resox cells. (b) Immunoprecipitation (IP) with anticatalase antibody and immunoblotting (IB) with anti-Flag, antiphosphocatalase, and catalase antibodies. Prior IP, cells were transfected with a plasmid pcDNA3 (1 μ g) coding an ubiquitin-Flag for 72 h.

    Article Snippet: Twenty-four hours posttransfection, cells were treated with 25 μ M MG132 for 5 h. Cells were washed twice with ice-cold PBS and then resuspended in the lysis buffer in the presence of proteases (Protease Inhibitor Cocktail, Sigma, St. Louis, MO, USA) and phosphatase inhibitors (Phosphatase Inhibitor Cocktail, Calbiochem, Merck KGaA, Darmstadt, Germany).

    Techniques: Incubation, Immunoprecipitation, Transfection, Plasmid Preparation