e0554  (New England Biolabs)


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    New England Biolabs e0554
    E0554, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs pcmv flag abce1
    Mechanism of C-I30-u Formation and Aggregation in Human Cells (A) Immunoblots showing CCCP-induced CTE forms (red *) of C-I30 and ATP5a in HeLa cells but not HeLa/GFP-Parkin cells. Blue * indicate preprotein of SDHA that accumulates in both HeLa and HeLa/GFP-Parkin cells upon CCCP treatment. (B) Immunostaining of HeLa cells transfected with C-I30-Flag and treated with CCCP or CCCP+anisomycin. Arrowheads: aggregates outside (white) or inside (yellow) mitochondria. (C) Immunoblots of showing effect of anisomycin treatment on the CTE forms of endogenous ATP5a and C-I30 or C-I30-Flag in HeLa cell. (D) Immunoblots showing effects of the various genetic manipulations or VCP inhibitor treatment on C-I30-Flag-u formation. (E) Immunostaining of HeLa cells co-transfected with C-I30-FLAG and various modifiers, showing effects on CCCP-induced FLAG+ aggregates. Arrowheads: aggregates. (F) Immunostaining of HeLa cells showing effects of ANKZF1 RNAi or OE on C-I30-Flag aggregation. (G) Immunoblots showing effect of PINK1 on ANKZF1 and NEMF interaction with <t>ABCE1</t> in co-IP assays. Scale bar, 3µm in B, E, F. See also Figure S7.
    Pcmv Flag Abce1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Mechanism of C-I30-u Formation and Aggregation in Human Cells (A) Immunoblots showing CCCP-induced CTE forms (red *) of C-I30 and ATP5a in HeLa cells but not HeLa/GFP-Parkin cells. Blue * indicate preprotein of SDHA that accumulates in both HeLa and HeLa/GFP-Parkin cells upon CCCP treatment. (B) Immunostaining of HeLa cells transfected with C-I30-Flag and treated with CCCP or CCCP+anisomycin. Arrowheads: aggregates outside (white) or inside (yellow) mitochondria. (C) Immunoblots of showing effect of anisomycin treatment on the CTE forms of endogenous ATP5a and C-I30 or C-I30-Flag in HeLa cell. (D) Immunoblots showing effects of the various genetic manipulations or VCP inhibitor treatment on C-I30-Flag-u formation. (E) Immunostaining of HeLa cells co-transfected with C-I30-FLAG and various modifiers, showing effects on CCCP-induced FLAG+ aggregates. Arrowheads: aggregates. (F) Immunostaining of HeLa cells showing effects of ANKZF1 RNAi or OE on C-I30-Flag aggregation. (G) Immunoblots showing effect of PINK1 on ANKZF1 and NEMF interaction with ABCE1 in co-IP assays. Scale bar, 3µm in B, E, F. See also Figure S7.

    Journal: bioRxiv

    Article Title: MISTERMINATE Mechanistically Links Mitochondrial Dysfunction with Proteostasis Failure

    doi: 10.1101/554634

    Figure Lengend Snippet: Mechanism of C-I30-u Formation and Aggregation in Human Cells (A) Immunoblots showing CCCP-induced CTE forms (red *) of C-I30 and ATP5a in HeLa cells but not HeLa/GFP-Parkin cells. Blue * indicate preprotein of SDHA that accumulates in both HeLa and HeLa/GFP-Parkin cells upon CCCP treatment. (B) Immunostaining of HeLa cells transfected with C-I30-Flag and treated with CCCP or CCCP+anisomycin. Arrowheads: aggregates outside (white) or inside (yellow) mitochondria. (C) Immunoblots of showing effect of anisomycin treatment on the CTE forms of endogenous ATP5a and C-I30 or C-I30-Flag in HeLa cell. (D) Immunoblots showing effects of the various genetic manipulations or VCP inhibitor treatment on C-I30-Flag-u formation. (E) Immunostaining of HeLa cells co-transfected with C-I30-FLAG and various modifiers, showing effects on CCCP-induced FLAG+ aggregates. Arrowheads: aggregates. (F) Immunostaining of HeLa cells showing effects of ANKZF1 RNAi or OE on C-I30-Flag aggregation. (G) Immunoblots showing effect of PINK1 on ANKZF1 and NEMF interaction with ABCE1 in co-IP assays. Scale bar, 3µm in B, E, F. See also Figure S7.

    Article Snippet: Plasmids and Molecular Cloning The original human C-I30 CDS sequence was from the pPM-N-D-C-His (PV394217, abm) plasmid. pcDNA3.1(+)-C-I30-FLG and pcDNA3.1(+)-C-I30-FLG-TEV plasmids were generated by cloning the human C-I30 CDS with different tags into pcDNA3.1(+) vector via Kpn I and Xba I sites. pcDNA3.1(+)-C-I30-TEV-FLG ; pcDNA3.1(+)-C-I30-FLG-AT 5 ; pcDNA3.1(+)-C-I30-FLG-AT 23 ; pcDNA3.1(+)-C-I30-FLG-non-AT 25 ; pcDNA3.1(+)-C-I30(mMTS)-FLG (36R →A) ; pcDNA3.1(+)-C-I30-FLG-A stop ; pcDNA3.1(+)-C-I30-FLG-AK stop ; pcDNA3.1(+)-C-I30-FLG-AKK stop and pcDNA3.1(+)-HA-C-I30-FLG ; were modified based on the pcDNA3.1(+)-C-I30-FLG-TEV plasmid via the Q5 Site-Directed Mutagenesis Kit (cat#: E0554S, NEB). pCMV-FLAG-ABCE1 was a gift from Dr. Ramanujan Hegde. pCMV6-FLAG-NOT4 and pCMV6-ANKZF1 was obtained from OriGene Inc (cat#: RC217418 and RC201054 TrueORF). pCMV-SPORT6.1-eRF1 (cat#: MHS6278-202804766, Dharmacon™) and pCMV-SPORT6.1-VCP (cat#: MHS6278-202760239, Dharmacon™) were from GE healthcare.

    Techniques: Western Blot, Immunostaining, Transfection, Co-Immunoprecipitation Assay

    EZH2 increases the FAK upstream ITGB1 expression. a Screenshot of the RNA Pol II ChIP sequencing (ChIP-seq) signal (GSE188640) at the ITGB1 promoter locus in MDA-MB-231 (231_Pol _II.bw) and 231.KO#1 (KO_Pol_II.bw) cells. b qRT-PCR analysis of ITGB1 mRNA expression in the indicated cells. Data are presented as means ± S.E.M. t -test (two-sided). Three biologically independent experiments. c qRT-PCR analysis of ITGB1 mRNA expression in the indicated cells. Data are presented as means ± S.E.M. t -test (two-sided). Three biologically independent experiments. d ITGB1 promoter activity in MDA-MB-231, 231.sgCtrl, 231.KO#1, and 231.KO#2 cells as measured using a dual-luciferase reporter assay. ITGB1 firefly luciferase signal was divided by the control renilla luciferase signal, and the ratios of luciferase/renilla in four cell lines were normalized to that of MDA-MB-231 cells. n = 6 biologically independent experiments in 231.sgCtrl and 231.KO#1 cells; n = 8 biologically independent experiments in MDA-MB-231 cells, n = 9 biologically independent experiments in 231.KO#2 cells. Data are means ± SEM, t -test (two-sided). e Top: the locations of the primers at the ITGB1 gene promoter area for ChIP-qPCR. TSS, transcription start site. Bottom: EZH2 was immunoprecipitated from MDA-MB-231 and 231.KO#1 cells, and EZH2 binding to ITGB1 in the cells was detected using qPCR with the indicated primers. All fold-enrichment values were normalized according to IgG values. Three biologically independent experiments. Data are presented as means ± S.E.M. t -test (two-sided). f RNA Pol II was immunoprecipitated from MDA-MB-231 and 231.KO#1 cells, and RNA Pol II binding to ITGB1 in the cells was detected using qPCR with the indicated primers. All fold-enrichment values were normalized according to IgG values. Three biologically independent experiments. Data are presented as means ± S.E.M. t -test (two-sided). g RNA Pol II was immunoprecipitated from 231.shScr, 231.shEZH2#3, and 231.shEZH2#4 cells, and RNA Pol II binding to ITGB1 or HOXA9B in the cells was detected using qPCR with the indicated primers. HOXA9B was used as a negative control. All fold-enrichment values were normalized according to IgG values. Three biologically independent experiments using P1-P5 primers; six biologically independent experiments using HOXA9B primer. Data are presented as means ± S.E.M. t -test (two-sided). All P values are indicated in the figures.

    Journal: Nature Communications

    Article Title: EZH2 engages TGFβ signaling to promote breast cancer bone metastasis via integrin β1-FAK activation

    doi: 10.1038/s41467-022-30105-0

    Figure Lengend Snippet: EZH2 increases the FAK upstream ITGB1 expression. a Screenshot of the RNA Pol II ChIP sequencing (ChIP-seq) signal (GSE188640) at the ITGB1 promoter locus in MDA-MB-231 (231_Pol _II.bw) and 231.KO#1 (KO_Pol_II.bw) cells. b qRT-PCR analysis of ITGB1 mRNA expression in the indicated cells. Data are presented as means ± S.E.M. t -test (two-sided). Three biologically independent experiments. c qRT-PCR analysis of ITGB1 mRNA expression in the indicated cells. Data are presented as means ± S.E.M. t -test (two-sided). Three biologically independent experiments. d ITGB1 promoter activity in MDA-MB-231, 231.sgCtrl, 231.KO#1, and 231.KO#2 cells as measured using a dual-luciferase reporter assay. ITGB1 firefly luciferase signal was divided by the control renilla luciferase signal, and the ratios of luciferase/renilla in four cell lines were normalized to that of MDA-MB-231 cells. n = 6 biologically independent experiments in 231.sgCtrl and 231.KO#1 cells; n = 8 biologically independent experiments in MDA-MB-231 cells, n = 9 biologically independent experiments in 231.KO#2 cells. Data are means ± SEM, t -test (two-sided). e Top: the locations of the primers at the ITGB1 gene promoter area for ChIP-qPCR. TSS, transcription start site. Bottom: EZH2 was immunoprecipitated from MDA-MB-231 and 231.KO#1 cells, and EZH2 binding to ITGB1 in the cells was detected using qPCR with the indicated primers. All fold-enrichment values were normalized according to IgG values. Three biologically independent experiments. Data are presented as means ± S.E.M. t -test (two-sided). f RNA Pol II was immunoprecipitated from MDA-MB-231 and 231.KO#1 cells, and RNA Pol II binding to ITGB1 in the cells was detected using qPCR with the indicated primers. All fold-enrichment values were normalized according to IgG values. Three biologically independent experiments. Data are presented as means ± S.E.M. t -test (two-sided). g RNA Pol II was immunoprecipitated from 231.shScr, 231.shEZH2#3, and 231.shEZH2#4 cells, and RNA Pol II binding to ITGB1 or HOXA9B in the cells was detected using qPCR with the indicated primers. HOXA9B was used as a negative control. All fold-enrichment values were normalized according to IgG values. Three biologically independent experiments using P1-P5 primers; six biologically independent experiments using HOXA9B primer. Data are presented as means ± S.E.M. t -test (two-sided). All P values are indicated in the figures.

    Article Snippet: Site-specific mutation was performed using a Q5 Site-Directed Mutagenesis Kit (New England BioLabs; #E0554S).

    Techniques: Expressing, Chromatin Immunoprecipitation, Sequencing, Multiple Displacement Amplification, Quantitative RT-PCR, Activity Assay, Luciferase, Reporter Assay, Real-time Polymerase Chain Reaction, Immunoprecipitation, Binding Assay, Negative Control

    Cholesterol Depletion of Hepatocytes Decreases TAZ in a LATS2-Dependent Manner (A) LATS2 kinase assay, using rTAZ as substrate, of extracts of livers of mice fed the NAFLD diet containing 0.2%, 0.5%, or 1.25% cholesterol for 16 weeks. (B) Immunoblots of phospho- and total TAZ in TAZ immunoprecipitates from AML12 cells incubated as follows: left blot, 4 h with MG132 plus vehicle (Veh) or liposomes; right blot, 18 h with liposomes and then 4 h with MG132 plus vehicle or Lipo-Chol. (C) Immunoblots of phospho- and total TAZ in TAZ immunoprecipitates from primary human hepatocytes incubated for 8 h with vehicle or Lipo-Chol, with MG132 included during the last 4 h. (D) TAZ and YAP immunoblots of siScr-treated or siLats2-treated AML12 cells that were incubated for 24 h with vehicle or liposomes. (E) HA-TAZ immunoblot of HA-WT-human TAZ- or HA-S117A-human TAZ-transfected AML12 cells that were incubated for 24 h with vehicle, or liposomes for 16 h and then Lipo-Chol for 8 h. (F-J) The following parameters were measured in Wwtr1 fl/fl mice fed the NAFLD diet containing 0.2% cholesterol for 16 weeks, with AAV8-TBG-Cre plus either AAV8-TBG-HA-WT-hTAZ or AAV8-TBG-HA-S117A-hTAZ injected at the 8-week time point (n = 10 mice/group; means ± SEM; *p

    Journal: Cell metabolism

    Article Title: Cholesterol Stabilizes TAZ in Hepatocytes to Promote Experimental Nonalcoholic Steatohepatitis

    doi: 10.1016/j.cmet.2020.03.010

    Figure Lengend Snippet: Cholesterol Depletion of Hepatocytes Decreases TAZ in a LATS2-Dependent Manner (A) LATS2 kinase assay, using rTAZ as substrate, of extracts of livers of mice fed the NAFLD diet containing 0.2%, 0.5%, or 1.25% cholesterol for 16 weeks. (B) Immunoblots of phospho- and total TAZ in TAZ immunoprecipitates from AML12 cells incubated as follows: left blot, 4 h with MG132 plus vehicle (Veh) or liposomes; right blot, 18 h with liposomes and then 4 h with MG132 plus vehicle or Lipo-Chol. (C) Immunoblots of phospho- and total TAZ in TAZ immunoprecipitates from primary human hepatocytes incubated for 8 h with vehicle or Lipo-Chol, with MG132 included during the last 4 h. (D) TAZ and YAP immunoblots of siScr-treated or siLats2-treated AML12 cells that were incubated for 24 h with vehicle or liposomes. (E) HA-TAZ immunoblot of HA-WT-human TAZ- or HA-S117A-human TAZ-transfected AML12 cells that were incubated for 24 h with vehicle, or liposomes for 16 h and then Lipo-Chol for 8 h. (F-J) The following parameters were measured in Wwtr1 fl/fl mice fed the NAFLD diet containing 0.2% cholesterol for 16 weeks, with AAV8-TBG-Cre plus either AAV8-TBG-HA-WT-hTAZ or AAV8-TBG-HA-S117A-hTAZ injected at the 8-week time point (n = 10 mice/group; means ± SEM; *p

    Article Snippet: The kit to carry out mutagenesis of WWTR1 was from New England Biolabs (#E0554S), and the primers are listed in .

    Techniques: Kinase Assay, Mouse Assay, Western Blot, Incubation, Transfection, Injection

    Characterization of a mutant bovine ARGFX allele. ( A ) Structure of WT and mutant genes and deduced proteins. The WT gene comprises five coding exons, with the homeobox (pink) split across exons 4 and 5. The mutant allele has a 13 bp deletion in exon 2 causing a frameshift (gray) and an early stop codon truncating the protein in exon 3 before the homeodomain. Scale bar 1 kb. ( B ) Map showing the distribution of the mutant allele. Dark brown/black = deletion, pale brown = WT. Pie charts show allele frequency for each breed; pie size proportional to sample size. Pie charts are located at the approximate location of the breed’s origin. Map generated using Bovine Genome Variation Database ( Chen et al. 2020 ). ( Ci ) Agarose gel shows that individuals can be homozygous for the mutant ARGFX allele. PCR primers were used that anneal either side of the deletion site, producing a shorter amplicon in the deletion allele. Lane 1 = Bioline HyperLadder 50 bp. Lanes 2 and 16 = homozygous WT control. Lanes 3 and 15 = heterozygous control. Lanes 4 and 11 are homozygous WT Jersey individuals, lanes 5, 12, and 13 are homozygous mutant Jersey individuals and lanes 6, 7, 8, 9, and 14 are heterozygous Jersey individuals. Lane 10 is inconclusive, with a strong WT band and very weak mutant band. ( Cii ) Agarose gel showing that BFF cells have a WT ARGFX gene. Lane 1 = Bioline HyperLadder 50 bp. Lane 2 = BFF band. Lane 3 = heterozygote bands. ( D ) Volcano plot showing genes DE in response to ARGFX mutant ectopic expression. Each point represents a gene. Points in red are considered DE (adjusted P

    Journal: Molecular Biology and Evolution

    Article Title: PRD-Class Homeobox Genes in Bovine Early Embryos: Function, Evolution, and Overlapping Roles

    doi: 10.1093/molbev/msac098

    Figure Lengend Snippet: Characterization of a mutant bovine ARGFX allele. ( A ) Structure of WT and mutant genes and deduced proteins. The WT gene comprises five coding exons, with the homeobox (pink) split across exons 4 and 5. The mutant allele has a 13 bp deletion in exon 2 causing a frameshift (gray) and an early stop codon truncating the protein in exon 3 before the homeodomain. Scale bar 1 kb. ( B ) Map showing the distribution of the mutant allele. Dark brown/black = deletion, pale brown = WT. Pie charts show allele frequency for each breed; pie size proportional to sample size. Pie charts are located at the approximate location of the breed’s origin. Map generated using Bovine Genome Variation Database ( Chen et al. 2020 ). ( Ci ) Agarose gel shows that individuals can be homozygous for the mutant ARGFX allele. PCR primers were used that anneal either side of the deletion site, producing a shorter amplicon in the deletion allele. Lane 1 = Bioline HyperLadder 50 bp. Lanes 2 and 16 = homozygous WT control. Lanes 3 and 15 = heterozygous control. Lanes 4 and 11 are homozygous WT Jersey individuals, lanes 5, 12, and 13 are homozygous mutant Jersey individuals and lanes 6, 7, 8, 9, and 14 are heterozygous Jersey individuals. Lane 10 is inconclusive, with a strong WT band and very weak mutant band. ( Cii ) Agarose gel showing that BFF cells have a WT ARGFX gene. Lane 1 = Bioline HyperLadder 50 bp. Lane 2 = BFF band. Lane 3 = heterozygote bands. ( D ) Volcano plot showing genes DE in response to ARGFX mutant ectopic expression. Each point represents a gene. Points in red are considered DE (adjusted P

    Article Snippet: An ARGFX mutant containing the 13 bp deletion found in the ARS-UCD1.2 reference genome was produced using a Q5 Site-Directed Mutagenesis Kit (New England BioLabs #E0554S).

    Techniques: Mutagenesis, Generated, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Expressing