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  • 94
    Thermo Fisher pgk 1
    Functional interaction between UNC-45, CHN-1, and UFD-2 in vivo. a Comparison of UNC-45 protein levels when overexpressed in unc-45(OE) , ufd-2(tm1380), and chn-1(by155) adult worms. <t>PGK-1</t> was used as a loading control. b Western blot analysis showing the protein levels of endogenous UNC-45 at different stages of C. elegans development. c Western blot analysis showing protein levels of endogenous UNC-45 in wild-type, ufd-2(tm1380), and chn-1(by155) worms at the L3, L4, and adult developmental stages, respectively. d Average speed (mm s −1 ) of indicated worms as determined in motility assays. Error bars represent sample standard deviation. Uncropped images of all western blots are shown in Supplementary Fig. 7
    Pgk 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam phosphoglycerate pgk 1
    <t>PGK1</t> is a direct target gene of NFAT5. a Venn diagram showing the intersection of five gene sets. b The relevance of NFAT5 with PGK1 and MET is displayed in the Renji cohort and TCGA database. c , d Relative PGK1 and MET mRNA expression in the control and NFAT5 knockdown PDAC cell lines. e Heatmap showing the counted numbers of four levels of PGK1 expression and its correlation with NFAT5. f Standard immunohistochemical scoring pictures of PGK1 expression in 311 pancreatic cancer tumors and adjacent normal tissues. g Respective IHC staining of NFAT5 and PGK1 in orthotopic xenograft models. h Respective IHC staining of NFAT5 and PGK1 in KPC mice. Assays shown in Fig. 5c, d were performed in hypoxia condition. * P
    Phosphoglycerate Pgk 1, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc pgk1
    <t>Pgk1</t> as the target of TZ
    Pgk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addgene inc pgk 1 gene
    <t>Pgk1</t> as the target of TZ
    Pgk 1 Gene, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Santa Cruz Biotechnology anti pgk 1
    Figure 6. Inhibition of cell growth and invasion by miR-29a, miR-1256 and isoflavone through TRIM68/AR and <t>PGK-1</t> signaling. Transfection of miR-29a and miR-1256 mimic into PCa cells, and isoflavone treatment significantly inhibited cell growth (A) and invasion (B) of PCa cells (*: p
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    90
    WuXi AppTec anti pgk1
    Glucose availability alters 3BP sensitivity in CRC cells HCT116 and DLD-1 cells were treated with 3BP under different media glucose concentrations. HKII expression was assessed via western blot analysis. Representative blots from three biological replicates ( A ) and graphical outputs for densitometric analysis of HKII expression normalized to α-tubulin from three biological replicates ± S.E.M. are shown ( B ). Representative blots from three biological replicates of putative 3BP targets SDH, GAPDH and <t>PGK1</t> show no effects of altered glucose concentrations ( C ). Growth curves for CRC cells grown under different media glucose concentrations in the presence or absence of 3BP over 72 h ( D ). Dose-effects of 3BP on cell growth following 72 h treatment in CRC cells under different media glucose concentrations ( E ) (* P ≤0.05, ** P ≤0.01, *** P ≤0.001, **** P ≤0.0001 compared with control (0 μM 3BP)).
    Anti Pgk1, supplied by WuXi AppTec, used in various techniques. Bioz Stars score: 90/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti pgk1
    <t>PGK1</t> interacts directly with HSP90 and modulates the ATPase activity of HSP90 . a . Western blot was performed to assess the effects of PGK1 knockdown on expression levels of HSP90 in Ishikawa cancer cell line. β-actin was used as a loading control. b . Semi-quantitative analysis of protein level in A. c . Western blot was performed to assess the effects of PGK1 knockdown on expression levels of HSP90 in HEC1A cancer cell line. β-actin was used as a loading control. d . Semi-quantitative analysis of protein level in C. e . Effect of PGK1 overexpression and HSP90 inhibitor 17-AAG on cellular ATP level. Vector, empty vector; PGK1, vector for PGK1 overexpression; PGK1 + 17-AAG, vector for PGK1 overexpression supplemented with 17-AAG. f . Co-immunoprecipitation was used to detect binding between PGK1 and HSP90. Hemagglutinin (HA)-tagged PGK1 (PGK1-HA) was overexpressed in Ishikawa cells. Data were shown as mean ± SD based on three independent experiments. *, P
    Anti Pgk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Research Genetics Inc pgk 1
    In situ PCR in hybrid model and control. A: One or two <t>pgk-1</t> spot(s) were detected in the epithelial nuclei of cholesteatoma. B: On the other hand, one pgk-1 spot was detected in the cells of EAC. Original magnification, ×350. C: One pgk-1 spot
    Pgk 1, supplied by Research Genetics Inc, used in various techniques. Bioz Stars score: 88/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GenePharma Company pgk1
    <t>PGK1</t> interacts directly with HSP90 and modulates the ATPase activity of HSP90 . a . Western blot was performed to assess the effects of PGK1 knockdown on expression levels of HSP90 in Ishikawa cancer cell line. β-actin was used as a loading control. b . Semi-quantitative analysis of protein level in A. c . Western blot was performed to assess the effects of PGK1 knockdown on expression levels of HSP90 in HEC1A cancer cell line. β-actin was used as a loading control. d . Semi-quantitative analysis of protein level in C. e . Effect of PGK1 overexpression and HSP90 inhibitor 17-AAG on cellular ATP level. Vector, empty vector; PGK1, vector for PGK1 overexpression; PGK1 + 17-AAG, vector for PGK1 overexpression supplemented with 17-AAG. f . Co-immunoprecipitation was used to detect binding between PGK1 and HSP90. Hemagglutinin (HA)-tagged PGK1 (PGK1-HA) was overexpressed in Ishikawa cells. Data were shown as mean ± SD based on three independent experiments. *, P
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    pgk1  (Abcam)
    90
    Abcam pgk1
    Gankyrin potentiates mTORC1 signaling via <t>PGK1/AKT.</t> (a) Overexpressed gankyrin activates AKT/mTORC1 signaling in gastric cancer cell lines, MKN45, MKN74, and AGS. The protein levels of PGK1, pAKT, AKT, pS6 K1, S6 K1, p4E-BP1, and 4E-BP1 were analyzed by immunoblotting. (b) The correlation plot of gankyrin and PGK1 mRNA level in 414 gastric cancer samples was presented by analyzing the TCGA cancer genome database.
    Pgk1, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 141 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The Jackson Laboratory pgk1 cre
    Gankyrin potentiates mTORC1 signaling via <t>PGK1/AKT.</t> (a) Overexpressed gankyrin activates AKT/mTORC1 signaling in gastric cancer cell lines, MKN45, MKN74, and AGS. The protein levels of PGK1, pAKT, AKT, pS6 K1, S6 K1, p4E-BP1, and 4E-BP1 were analyzed by immunoblotting. (b) The correlation plot of gankyrin and PGK1 mRNA level in 414 gastric cancer samples was presented by analyzing the TCGA cancer genome database.
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    Genloci Biotechnologies Inc pgk1 1
    Gankyrin potentiates mTORC1 signaling via <t>PGK1/AKT.</t> (a) Overexpressed gankyrin activates AKT/mTORC1 signaling in gastric cancer cell lines, MKN45, MKN74, and AGS. The protein levels of PGK1, pAKT, AKT, pS6 K1, S6 K1, p4E-BP1, and 4E-BP1 were analyzed by immunoblotting. (b) The correlation plot of gankyrin and PGK1 mRNA level in 414 gastric cancer samples was presented by analyzing the TCGA cancer genome database.
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    91
    Abcam α pgk1
    Rrp12 is required for the export of pre-40S particles out of the nucleus. ( A to G ) Top panels, epifluorescence microscopy analysis of the subcellular distribution of GFP-tagged Enp1 (A), Dim1 (B), Tsr1 (C), Pno1 (D), Ltv1 (E), Nob1 (F) and Rio2 (G) in the indicated yeast strains and culture conditions (top). Bottom panels, differential interference contrast (DIC) images of the above preparations. ( H ) Western blot analysis showing the distribution of Rio2-GFP (top panel) and <t>Pgk1</t> (bottom panel) in whole cell lysates (W), cytosolic (C) and nuclear (F) fractions obtained from either control rio2-GFP cells (lanes 1 to 3) or GAL::HA-rrp12/rio2-GFP cells growing in galactose-containing medium (lanes 4 to 6) or upon a shift to glucose-containing medium for 9 hours (lanes 7 to 9).
    α Pgk1, supplied by Abcam, used in various techniques. Bioz Stars score: 91/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Abcam anti pgk1 22c5d8
    Rrp12 is required for the export of pre-40S particles out of the nucleus. ( A to G ) Top panels, epifluorescence microscopy analysis of the subcellular distribution of GFP-tagged Enp1 (A), Dim1 (B), Tsr1 (C), Pno1 (D), Ltv1 (E), Nob1 (F) and Rio2 (G) in the indicated yeast strains and culture conditions (top). Bottom panels, differential interference contrast (DIC) images of the above preparations. ( H ) Western blot analysis showing the distribution of Rio2-GFP (top panel) and <t>Pgk1</t> (bottom panel) in whole cell lysates (W), cytosolic (C) and nuclear (F) fractions obtained from either control rio2-GFP cells (lanes 1 to 3) or GAL::HA-rrp12/rio2-GFP cells growing in galactose-containing medium (lanes 4 to 6) or upon a shift to glucose-containing medium for 9 hours (lanes 7 to 9).
    Anti Pgk1 22c5d8, supplied by Abcam, used in various techniques. Bioz Stars score: 96/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Novus Biologicals anti pgk1 antibody
    Overexpression of RAD53 in HSP90 -overexpressing cell restores DNA damage hypersensitivity. (A) Percentage survivability of four different strains upon 0.03% MMS treatment plotted as indicated along the x -axis. Average viability ± SD for each strain from five independent experiments. (B) Percentage survivability to UV radiation for the same sets of strains as in A against different UV doses as indicated along the x -axis. (C) Representative Western blots, showing the restoration of Rad51p up-regulation upon treatment with MMS in the WT + p HSP90 + p RAD53 strain compared with the HSP90 -overexpressing strain. The increased level of Rad53p in the RAD53 -overexpressing strain is shown by probing with anti-Flag antibody. –, MMS-untreated fraction; +, fractions treated with 0.05% MMS for 2 h. <t>Pgk1</t> acts as loading control. Rad53p level was monitored using anti-Flag antibody. (D) Quantification of band intensities from two independent experiments showing that the extent of up-regulation of Rad51p in the wild-type strain (WT + pEMPTY(2)) is similar to that for the WT + p HSP90 + p RAD53 strain. Each lane was normalized against actin. Average band intensities ± SD. (E, F) Real time RT-PCR showing relative abundance of CLN1 and CLN2 transcripts, respectively, for the strains noted along the x -axis. There was no significant change between the levels of G1-cyclins from the 30-min, MMS-treated samples in G1-arrested cells and the 120-min post–MMS treatment samples. (G) Representative cell images for WT, the strain overexpressing RAD53 against the HSP90 -overexpression background (WT + p HSP90 + p RAD53 ), and the RAD53 -overexpressing (WT + p RAD53 ) strain. (H) Average cell size ( y -axis) for each strain type ( x -axis). Sample size > 100. Cell sizes all lie in the same range. Error bar indicates ±SD. *, p
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    Abcam pgk1 antibody
    Overexpression of RAD53 in HSP90 -overexpressing cell restores DNA damage hypersensitivity. (A) Percentage survivability of four different strains upon 0.03% MMS treatment plotted as indicated along the x -axis. Average viability ± SD for each strain from five independent experiments. (B) Percentage survivability to UV radiation for the same sets of strains as in A against different UV doses as indicated along the x -axis. (C) Representative Western blots, showing the restoration of Rad51p up-regulation upon treatment with MMS in the WT + p HSP90 + p RAD53 strain compared with the HSP90 -overexpressing strain. The increased level of Rad53p in the RAD53 -overexpressing strain is shown by probing with anti-Flag antibody. –, MMS-untreated fraction; +, fractions treated with 0.05% MMS for 2 h. <t>Pgk1</t> acts as loading control. Rad53p level was monitored using anti-Flag antibody. (D) Quantification of band intensities from two independent experiments showing that the extent of up-regulation of Rad51p in the wild-type strain (WT + pEMPTY(2)) is similar to that for the WT + p HSP90 + p RAD53 strain. Each lane was normalized against actin. Average band intensities ± SD. (E, F) Real time RT-PCR showing relative abundance of CLN1 and CLN2 transcripts, respectively, for the strains noted along the x -axis. There was no significant change between the levels of G1-cyclins from the 30-min, MMS-treated samples in G1-arrested cells and the 120-min post–MMS treatment samples. (G) Representative cell images for WT, the strain overexpressing RAD53 against the HSP90 -overexpression background (WT + p HSP90 + p RAD53 ), and the RAD53 -overexpressing (WT + p RAD53 ) strain. (H) Average cell size ( y -axis) for each strain type ( x -axis). Sample size > 100. Cell sizes all lie in the same range. Error bar indicates ±SD. *, p
    Pgk1 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher pgk1 polyclonal antibody
    Overexpression of RAD53 in HSP90 -overexpressing cell restores DNA damage hypersensitivity. (A) Percentage survivability of four different strains upon 0.03% MMS treatment plotted as indicated along the x -axis. Average viability ± SD for each strain from five independent experiments. (B) Percentage survivability to UV radiation for the same sets of strains as in A against different UV doses as indicated along the x -axis. (C) Representative Western blots, showing the restoration of Rad51p up-regulation upon treatment with MMS in the WT + p HSP90 + p RAD53 strain compared with the HSP90 -overexpressing strain. The increased level of Rad53p in the RAD53 -overexpressing strain is shown by probing with anti-Flag antibody. –, MMS-untreated fraction; +, fractions treated with 0.05% MMS for 2 h. <t>Pgk1</t> acts as loading control. Rad53p level was monitored using anti-Flag antibody. (D) Quantification of band intensities from two independent experiments showing that the extent of up-regulation of Rad51p in the wild-type strain (WT + pEMPTY(2)) is similar to that for the WT + p HSP90 + p RAD53 strain. Each lane was normalized against actin. Average band intensities ± SD. (E, F) Real time RT-PCR showing relative abundance of CLN1 and CLN2 transcripts, respectively, for the strains noted along the x -axis. There was no significant change between the levels of G1-cyclins from the 30-min, MMS-treated samples in G1-arrested cells and the 120-min post–MMS treatment samples. (G) Representative cell images for WT, the strain overexpressing RAD53 against the HSP90 -overexpression background (WT + p HSP90 + p RAD53 ), and the RAD53 -overexpressing (WT + p RAD53 ) strain. (H) Average cell size ( y -axis) for each strain type ( x -axis). Sample size > 100. Cell sizes all lie in the same range. Error bar indicates ±SD. *, p
    Pgk1 Polyclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Nordic-Mubio anti pgk1
    Rpd3 deacetylates Rad53 upon HO-induced DSB. (A) RPD3 deletion increases acetylation levels of Rad53 and Rfa1. Wild-type or rpd3 Δ cells expressing Rad53-FLAG, Rfa1-FLAG, or Cdk1-FLAG were lysed and subjected to antiacetyllysine immunoprecipitation and then anti-FLAG immunoblotting to detect protein acetylation (left) or to direct immunoblotting to verify protein expression (right). (B) Same as panel A except that cell lysates were subjected to anti-FLAG immunoprecipitation (IP) and then antiacetyllysine immunoblotting to detect protein acetylation. WB, Western blotting. (C) Rad53 is deacetylated by Rpd3 in vitro . Rad53-FLAG immunoprecipitated from rpd3 Δ cells expressing Rad53-FLAG was mixed with purified Rpd3-GST or deacetylase-deficient Rpd3((H150A; H151A))-GST to carry out in vitro deacetylation. Rad53 acetylation was assessed by antiacetyllysine immunoblotting (top). Anti-FLAG (middle) or anti-GST (bottom) immunoblotting was used to confirm Rad53 or Rpd3 protein levels. 2HA stands for the H150A and H151A double mutations. Ac-K, acetylated lysine. (D) Rpd3 interacts with Rad53. The extracts of untreated or MMS-treated cells expressing Rpd3-HA and/or Rad53-FLAG were subjected to anti-HA immunoprecipitation and then anti-FLAG immunoblotting (top) or to direct immunoblotting to verify protein expression (middle and bottom). (E) Rad53 is deacetylated after HO induction in a Rpd3-dependent manner. Wild-type or rpd3 Δ cells expressing Rad53-FLAG were grown on YEP-lactate medium to an OD 660 of 0.6 to 0.8, and HO endonuclease was induced by 2% galactose. Samples were taken at the indicated times and subjected to antiacetyllysine immunoprecipitation as described above for panel A. <t>Pgk1</t> is shown as a loading control. (F) Identification of Rad53 Lys22 acetylation by HPLC-MS/MS analysis. The MS/MS spectrum of a dual-charged ion at m/z 1,126.57 for MH22 + corresponding to the mass of the acetylated peptide FLIEK(Ac)FSQEQIGENIVC(carboxyamido)R is shown. All major fragment ions match the predicted b and y ions of the acetylated peptide. (G) Mutation of Lys22 and Lys213 reduces acetylation of Rad53. sml1 Δ rad53 Δ rpd3 Δ cells transfected with the indicated plasmids under the control of the Rad53 promoter were lysed and subjected to antiacetyllysine immunoprecipitation as described above for panel A. (H) K235 of human Chk2 is conserved with K213 of yeast Rad53. The conserved amino acids are highlighted in gray.
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    Image Search Results


    Functional interaction between UNC-45, CHN-1, and UFD-2 in vivo. a Comparison of UNC-45 protein levels when overexpressed in unc-45(OE) , ufd-2(tm1380), and chn-1(by155) adult worms. PGK-1 was used as a loading control. b Western blot analysis showing the protein levels of endogenous UNC-45 at different stages of C. elegans development. c Western blot analysis showing protein levels of endogenous UNC-45 in wild-type, ufd-2(tm1380), and chn-1(by155) worms at the L3, L4, and adult developmental stages, respectively. d Average speed (mm s −1 ) of indicated worms as determined in motility assays. Error bars represent sample standard deviation. Uncropped images of all western blots are shown in Supplementary Fig. 7

    Journal: Nature Communications

    Article Title: UFD-2 is an adaptor-assisted E3 ligase targeting unfolded proteins

    doi: 10.1038/s41467-018-02924-7

    Figure Lengend Snippet: Functional interaction between UNC-45, CHN-1, and UFD-2 in vivo. a Comparison of UNC-45 protein levels when overexpressed in unc-45(OE) , ufd-2(tm1380), and chn-1(by155) adult worms. PGK-1 was used as a loading control. b Western blot analysis showing the protein levels of endogenous UNC-45 at different stages of C. elegans development. c Western blot analysis showing protein levels of endogenous UNC-45 in wild-type, ufd-2(tm1380), and chn-1(by155) worms at the L3, L4, and adult developmental stages, respectively. d Average speed (mm s −1 ) of indicated worms as determined in motility assays. Error bars represent sample standard deviation. Uncropped images of all western blots are shown in Supplementary Fig. 7

    Article Snippet: Worm lysates were subjected to western blot analysis using UNC-45, UNC-54 (mAb 28.2) , and PGK-1 (Invitrogen)-specific antibodies.

    Techniques: Functional Assay, In Vivo, Western Blot, Standard Deviation

    Functional interaction of UFD-2, UNC-45, and myosin in vivo. a Summary of crawling assays performed with unc-45(m94) and unc-45(m94) ufd-2(tm1380) adult worms at the restrictive temperature of 23 °C for 60 min. Representative images show tracks generated by a single worm. For quantification, movement behavior was classified into three different categories - unc (uncoordinated phenotype), mobile (reduced motility), and wild-type-like motility. b Western blot analysis showing UNC-45 protein levels in wild-type, unc-45(m94) , and unc-45(m94) ufd-2(tm1380) adult worms. PGK-1 was used as a loading control. c Western blot analysis using an MHC-B specific antibody, showing levels of C. elegans muscle myosin in the indicated worm strains. d Quantification of the protein area of UNC-45 and UFD-2 in anti-UNC-45 and anti-UFD-2 IPs derived from parallel reaction monitoring (PRM). Two technical replicates are shown (two separate sets of IPs from the same lysate). No peptides were detected in control IPs

    Journal: Nature Communications

    Article Title: UFD-2 is an adaptor-assisted E3 ligase targeting unfolded proteins

    doi: 10.1038/s41467-018-02924-7

    Figure Lengend Snippet: Functional interaction of UFD-2, UNC-45, and myosin in vivo. a Summary of crawling assays performed with unc-45(m94) and unc-45(m94) ufd-2(tm1380) adult worms at the restrictive temperature of 23 °C for 60 min. Representative images show tracks generated by a single worm. For quantification, movement behavior was classified into three different categories - unc (uncoordinated phenotype), mobile (reduced motility), and wild-type-like motility. b Western blot analysis showing UNC-45 protein levels in wild-type, unc-45(m94) , and unc-45(m94) ufd-2(tm1380) adult worms. PGK-1 was used as a loading control. c Western blot analysis using an MHC-B specific antibody, showing levels of C. elegans muscle myosin in the indicated worm strains. d Quantification of the protein area of UNC-45 and UFD-2 in anti-UNC-45 and anti-UFD-2 IPs derived from parallel reaction monitoring (PRM). Two technical replicates are shown (two separate sets of IPs from the same lysate). No peptides were detected in control IPs

    Article Snippet: Worm lysates were subjected to western blot analysis using UNC-45, UNC-54 (mAb 28.2) , and PGK-1 (Invitrogen)-specific antibodies.

    Techniques: Functional Assay, In Vivo, Generated, Western Blot, Derivative Assay

    Phenotypic rescue by Tsa1 variants forming partial decamers is temperature-sensitive. (A) Yeast strains expressing partially functional Tsa1 variants were assayed for temperature sensitivity by plating serial dilutions on YPD or YPD + 4 mM H 2 O 2 and incubating at 30 or 37 °C for 48 h. (B) Temperature-sensitive variants of Tsa1 were expressed in tsa1 Δ tsa2 Δ cells were monitored for stability at 30 or 37 °C, following growth of cultures for 8 h at the indicated temperature. FLAG-tagged Tsa1 proteins were detected by immunoblot. Pgk1 levels were monitored as a loading control. Results are representative of three independent experiments.

    Journal: Chemical research in toxicology

    Article Title: Aromatic Residues at the Dimer–Dimer Interface in the Peroxiredoxin Tsa1 Facilitate Decamer Formation and Biological Function

    doi: 10.1021/acs.chemrestox.8b00346

    Figure Lengend Snippet: Phenotypic rescue by Tsa1 variants forming partial decamers is temperature-sensitive. (A) Yeast strains expressing partially functional Tsa1 variants were assayed for temperature sensitivity by plating serial dilutions on YPD or YPD + 4 mM H 2 O 2 and incubating at 30 or 37 °C for 48 h. (B) Temperature-sensitive variants of Tsa1 were expressed in tsa1 Δ tsa2 Δ cells were monitored for stability at 30 or 37 °C, following growth of cultures for 8 h at the indicated temperature. FLAG-tagged Tsa1 proteins were detected by immunoblot. Pgk1 levels were monitored as a loading control. Results are representative of three independent experiments.

    Article Snippet: Immunoblots were conducted using reducing SDS-PAGE prior to transfer onto PVDF and detection with antibodies against the FLAG epitope (Sigma), TAP tag (antiprotein A, Sigma), or Pgk1 (Invitrogen).

    Techniques: Expressing, Functional Assay

    Substitution of conserved aromatic residues at the Tsa1 decamer interface compromise the protein’s ability to protect against oxidants and genomic instability. (A) Expression levels of decamer interface variants were compared with that of wt Tsa1 via immunoblot. Pgk1 levels were monitored as a loading control. (B) Decamer interface variants of Tsa1 were tested for their ability to prevent peroxide-mediated toxicity. Strains were grown to stationary phase, diluted serially before plating on YPD medium or YPD medium containing 4 mM H 2 O 2 , and grown for 48 h at 30 °C. Results are representative of three independent experiments. (C) Strains expressing Tsa1 decamer interface variants were monitored for spontaneous mutation of the gene encoding the arginine transporter ( CAN1 section. Results shown are the median mutation rate ±95% confidence limit for two independent trials, each performed with nine individual isolates of the indicated strain.

    Journal: Chemical research in toxicology

    Article Title: Aromatic Residues at the Dimer–Dimer Interface in the Peroxiredoxin Tsa1 Facilitate Decamer Formation and Biological Function

    doi: 10.1021/acs.chemrestox.8b00346

    Figure Lengend Snippet: Substitution of conserved aromatic residues at the Tsa1 decamer interface compromise the protein’s ability to protect against oxidants and genomic instability. (A) Expression levels of decamer interface variants were compared with that of wt Tsa1 via immunoblot. Pgk1 levels were monitored as a loading control. (B) Decamer interface variants of Tsa1 were tested for their ability to prevent peroxide-mediated toxicity. Strains were grown to stationary phase, diluted serially before plating on YPD medium or YPD medium containing 4 mM H 2 O 2 , and grown for 48 h at 30 °C. Results are representative of three independent experiments. (C) Strains expressing Tsa1 decamer interface variants were monitored for spontaneous mutation of the gene encoding the arginine transporter ( CAN1 section. Results shown are the median mutation rate ±95% confidence limit for two independent trials, each performed with nine individual isolates of the indicated strain.

    Article Snippet: Immunoblots were conducted using reducing SDS-PAGE prior to transfer onto PVDF and detection with antibodies against the FLAG epitope (Sigma), TAP tag (antiprotein A, Sigma), or Pgk1 (Invitrogen).

    Techniques: Expressing, Mutagenesis

    Tsa1 decamer interface variants undergo less DVSF-mediated cross-linking to thioredoxin. (A) Possible cross-linking reactions that Tsa1 undergoes in cells treated with the bifunctional electrophile DVSF. (B) Wild-type cells (BY4741) or cells lacking Trx1 and Trx2 ( trx Δ) expressing FLAG-tagged Tsa1 variants were treated with 1 mM DVSF for 1 h at 30 °C. FLAG-tagged Tsa1 and its cross-linked species were detected via immunoblot. Pgk1 levels were monitored as a loading control. (C) FLAG-tagged Tsa1 variants were expressed in cells containing a TAP-tagged Trx2 and were subsequently immunoprecipitated from cell lysates treated with DVSF. Immunoprecipitates were probed with antibodies against the FLAG or TAP tags via immunoblot. Results are representative of three independent experiments.

    Journal: Chemical research in toxicology

    Article Title: Aromatic Residues at the Dimer–Dimer Interface in the Peroxiredoxin Tsa1 Facilitate Decamer Formation and Biological Function

    doi: 10.1021/acs.chemrestox.8b00346

    Figure Lengend Snippet: Tsa1 decamer interface variants undergo less DVSF-mediated cross-linking to thioredoxin. (A) Possible cross-linking reactions that Tsa1 undergoes in cells treated with the bifunctional electrophile DVSF. (B) Wild-type cells (BY4741) or cells lacking Trx1 and Trx2 ( trx Δ) expressing FLAG-tagged Tsa1 variants were treated with 1 mM DVSF for 1 h at 30 °C. FLAG-tagged Tsa1 and its cross-linked species were detected via immunoblot. Pgk1 levels were monitored as a loading control. (C) FLAG-tagged Tsa1 variants were expressed in cells containing a TAP-tagged Trx2 and were subsequently immunoprecipitated from cell lysates treated with DVSF. Immunoprecipitates were probed with antibodies against the FLAG or TAP tags via immunoblot. Results are representative of three independent experiments.

    Article Snippet: Immunoblots were conducted using reducing SDS-PAGE prior to transfer onto PVDF and detection with antibodies against the FLAG epitope (Sigma), TAP tag (antiprotein A, Sigma), or Pgk1 (Invitrogen).

    Techniques: Expressing, Immunoprecipitation

    Pch2 and Rad17 promote Hop1 and Mek1 activation. (A) Western blot analysis of WT, rad17Δ , pch2Δ and pch2Δ rad17Δ at indicated time points after transfer to SPM using α-Hop1 antibody. Pgk1 Western blot was used as the loading control. The phosphorylated isoforms of Hop1 are detectable as slow-moving species. (B) Mek1–3HA immunoprecipitates from WT, rad17Δ , pch2Δ and pch2Δ rad17Δ at indicated time points were analyzed by Western blot using α-phospho-Akt substrate (recognizing pT327 of Mek1) and α-HA antibodies. *IgG heavy chain. Cell lysates were analyzed by Western blot using α-HA and α-Pgk1 antibodies.

    Journal: PLoS Genetics

    Article Title: Pch2 Acts through Xrs2 and Tel1/ATM to Modulate Interhomolog Bias and Checkpoint Function during Meiosis

    doi: 10.1371/journal.pgen.1002351

    Figure Lengend Snippet: Pch2 and Rad17 promote Hop1 and Mek1 activation. (A) Western blot analysis of WT, rad17Δ , pch2Δ and pch2Δ rad17Δ at indicated time points after transfer to SPM using α-Hop1 antibody. Pgk1 Western blot was used as the loading control. The phosphorylated isoforms of Hop1 are detectable as slow-moving species. (B) Mek1–3HA immunoprecipitates from WT, rad17Δ , pch2Δ and pch2Δ rad17Δ at indicated time points were analyzed by Western blot using α-phospho-Akt substrate (recognizing pT327 of Mek1) and α-HA antibodies. *IgG heavy chain. Cell lysates were analyzed by Western blot using α-HA and α-Pgk1 antibodies.

    Article Snippet: Proteins from denaturing whole-cell extracts were detected by Western blotting using α-Hop1 (S. Roeder), α-HA (Santa Cruz, sc-7392), α-phospho-Akt substrate (Cell signaling, #9614), and α-Pgk1 (Invitrogen, A-6457).

    Techniques: Activation Assay, Western Blot

    PGK1 is a direct target gene of NFAT5. a Venn diagram showing the intersection of five gene sets. b The relevance of NFAT5 with PGK1 and MET is displayed in the Renji cohort and TCGA database. c , d Relative PGK1 and MET mRNA expression in the control and NFAT5 knockdown PDAC cell lines. e Heatmap showing the counted numbers of four levels of PGK1 expression and its correlation with NFAT5. f Standard immunohistochemical scoring pictures of PGK1 expression in 311 pancreatic cancer tumors and adjacent normal tissues. g Respective IHC staining of NFAT5 and PGK1 in orthotopic xenograft models. h Respective IHC staining of NFAT5 and PGK1 in KPC mice. Assays shown in Fig. 5c, d were performed in hypoxia condition. * P

    Journal: Cell Death & Disease

    Article Title: Transcription factor NFAT5 contributes to the glycolytic phenotype rewiring and pancreatic cancer progression via transcription of PGK1

    doi: 10.1038/s41419-019-2072-5

    Figure Lengend Snippet: PGK1 is a direct target gene of NFAT5. a Venn diagram showing the intersection of five gene sets. b The relevance of NFAT5 with PGK1 and MET is displayed in the Renji cohort and TCGA database. c , d Relative PGK1 and MET mRNA expression in the control and NFAT5 knockdown PDAC cell lines. e Heatmap showing the counted numbers of four levels of PGK1 expression and its correlation with NFAT5. f Standard immunohistochemical scoring pictures of PGK1 expression in 311 pancreatic cancer tumors and adjacent normal tissues. g Respective IHC staining of NFAT5 and PGK1 in orthotopic xenograft models. h Respective IHC staining of NFAT5 and PGK1 in KPC mice. Assays shown in Fig. 5c, d were performed in hypoxia condition. * P

    Article Snippet: Antibodies used were mouse anti-GAPDH (1:10,000, Abcam, ab8245), rabbit anti-NFAT5 (1:1000, Abcam, ab226308), and rabbit anti-PGK1 (1:500, Abcam, ab38007).

    Techniques: Expressing, Immunohistochemistry, Staining, Mouse Assay

    PGK1 is a direct target gene of NFAT5. a The sequence logo graph manifested the canonical binding site of NFAT5 predicted by JASPAR. b Four potential binding sequences were found in the PGK1 promoter region. c Mutated binding sites of NFAT5 lead to impaired luciferase activity in two cell lines. * P

    Journal: Cell Death & Disease

    Article Title: Transcription factor NFAT5 contributes to the glycolytic phenotype rewiring and pancreatic cancer progression via transcription of PGK1

    doi: 10.1038/s41419-019-2072-5

    Figure Lengend Snippet: PGK1 is a direct target gene of NFAT5. a The sequence logo graph manifested the canonical binding site of NFAT5 predicted by JASPAR. b Four potential binding sequences were found in the PGK1 promoter region. c Mutated binding sites of NFAT5 lead to impaired luciferase activity in two cell lines. * P

    Article Snippet: Antibodies used were mouse anti-GAPDH (1:10,000, Abcam, ab8245), rabbit anti-NFAT5 (1:1000, Abcam, ab226308), and rabbit anti-PGK1 (1:500, Abcam, ab38007).

    Techniques: Sequencing, Binding Assay, Luciferase, Activity Assay

    NFAT5 boosts proliferation and the Warburg effect partially via PGK1. a Overexpression efficacy of PGK1 in sh-NFAT5-treated AsPC-1 and BxPC-3 cells was determined by western blotting. b , c Altered level of ECAR and OCR in AsPC-1 and BxPC-3 cells in three different groups (control, shRNA, and PGK1 over-expression). Values are means ± SD. d Relative glucose consumption and lactate production in AsPC-1 and BxPC-3 cell lines in three different groups (control, shRNA, and PGK1 over-expression). e PGK1-overexpression reversed the inhibitory effects of the knockdown of NFAT5 on the CCK8 assay of PDAC cells, and values are means ± SD. f PGK1-overexpression reversed the inhibitory effects of the knockdown of NFAT5 on the colony formation properties of PDAC cells. g Representative bioluminescence photograph of mice orthotopically implanted with luciferase-expressing AsPC-1 cells. h IHC staining of PCNA in mice orthotopically implanted tissue. Assays shown in Fig. 7b–f were performed in hypoxia condition. Values are means ± SD. * P

    Journal: Cell Death & Disease

    Article Title: Transcription factor NFAT5 contributes to the glycolytic phenotype rewiring and pancreatic cancer progression via transcription of PGK1

    doi: 10.1038/s41419-019-2072-5

    Figure Lengend Snippet: NFAT5 boosts proliferation and the Warburg effect partially via PGK1. a Overexpression efficacy of PGK1 in sh-NFAT5-treated AsPC-1 and BxPC-3 cells was determined by western blotting. b , c Altered level of ECAR and OCR in AsPC-1 and BxPC-3 cells in three different groups (control, shRNA, and PGK1 over-expression). Values are means ± SD. d Relative glucose consumption and lactate production in AsPC-1 and BxPC-3 cell lines in three different groups (control, shRNA, and PGK1 over-expression). e PGK1-overexpression reversed the inhibitory effects of the knockdown of NFAT5 on the CCK8 assay of PDAC cells, and values are means ± SD. f PGK1-overexpression reversed the inhibitory effects of the knockdown of NFAT5 on the colony formation properties of PDAC cells. g Representative bioluminescence photograph of mice orthotopically implanted with luciferase-expressing AsPC-1 cells. h IHC staining of PCNA in mice orthotopically implanted tissue. Assays shown in Fig. 7b–f were performed in hypoxia condition. Values are means ± SD. * P

    Article Snippet: Antibodies used were mouse anti-GAPDH (1:10,000, Abcam, ab8245), rabbit anti-NFAT5 (1:1000, Abcam, ab226308), and rabbit anti-PGK1 (1:500, Abcam, ab38007).

    Techniques: Over Expression, Western Blot, shRNA, CCK-8 Assay, Mouse Assay, Luciferase, Expressing, Immunohistochemistry, Staining

    Pgk1 as the target of TZ

    Journal: Nature chemical biology

    Article Title: Terazosin activated Pgk1 and Hsp90 to promote stress resistance

    doi: 10.1038/nchembio.1657

    Figure Lengend Snippet: Pgk1 as the target of TZ

    Article Snippet: Caspase 3 (9662, Cell Signaling); PARP1 (9532, Cell Signaling); HSP70 (4872, Cell Signaling); HSP90 for IP (ab1429, Abcam); β-actin ( , Abmart); HA (H6908, sigma); Hsp90 for WB (4874, Cell Signaling); Pgk1 for IP (sc-17943, Santa Cruz); P23 (sc-376725, Santa Cruz); GFP (BE2001, Easybio); Pgk1 for WB (sc-48342, Santa Cruz); HSF-1 (12972, Cell Signaling); IgG (invitrogen).

    Techniques:

    Crystal structure of Pgk1 and TZ binding

    Journal: Nature chemical biology

    Article Title: Terazosin activated Pgk1 and Hsp90 to promote stress resistance

    doi: 10.1038/nchembio.1657

    Figure Lengend Snippet: Crystal structure of Pgk1 and TZ binding

    Article Snippet: Caspase 3 (9662, Cell Signaling); PARP1 (9532, Cell Signaling); HSP70 (4872, Cell Signaling); HSP90 for IP (ab1429, Abcam); β-actin ( , Abmart); HA (H6908, sigma); Hsp90 for WB (4874, Cell Signaling); Pgk1 for IP (sc-17943, Santa Cruz); P23 (sc-376725, Santa Cruz); GFP (BE2001, Easybio); Pgk1 for WB (sc-48342, Santa Cruz); HSF-1 (12972, Cell Signaling); IgG (invitrogen).

    Techniques: Binding Assay

    Figure 6. Inhibition of cell growth and invasion by miR-29a, miR-1256 and isoflavone through TRIM68/AR and PGK-1 signaling. Transfection of miR-29a and miR-1256 mimic into PCa cells, and isoflavone treatment significantly inhibited cell growth (A) and invasion (B) of PCa cells (*: p

    Journal: Epigenetics

    Article Title: Epigenetic deregulation of miR-29a and miR-1256 by isoflavone contributes to the inhibition of prostate cancer cell growth and invasion

    doi: 10.4161/epi.21236

    Figure Lengend Snippet: Figure 6. Inhibition of cell growth and invasion by miR-29a, miR-1256 and isoflavone through TRIM68/AR and PGK-1 signaling. Transfection of miR-29a and miR-1256 mimic into PCa cells, and isoflavone treatment significantly inhibited cell growth (A) and invasion (B) of PCa cells (*: p

    Article Snippet: Anti-TRIM68 (Santa Cruz), anti-PGK-1 (Santa Cruz), anti-β-actin (Sigma) and anti-GAPDH (Sigma) primary antibodies were used for Western Blot analysis.

    Techniques: Inhibition, Transfection

    Figure 4. The expression of TRIM68 and PGK-1. A significantly higher mRNA expression of TRIM68 and PGK-1 was observed in PCa cells [(A) *: p

    Journal: Epigenetics

    Article Title: Epigenetic deregulation of miR-29a and miR-1256 by isoflavone contributes to the inhibition of prostate cancer cell growth and invasion

    doi: 10.4161/epi.21236

    Figure Lengend Snippet: Figure 4. The expression of TRIM68 and PGK-1. A significantly higher mRNA expression of TRIM68 and PGK-1 was observed in PCa cells [(A) *: p

    Article Snippet: Anti-TRIM68 (Santa Cruz), anti-PGK-1 (Santa Cruz), anti-β-actin (Sigma) and anti-GAPDH (Sigma) primary antibodies were used for Western Blot analysis.

    Techniques: Expressing

    Figure 3. Targeting TRIM68 by miR-29a and miR-1256. Computerized analysis showed miR-29a and miR-1256 sequence alignment to the sequence of TRIM68 3′UTR (A). Co-transfection of TRIM68 3′UTR-Luc and miR-29a, miR-1256 mimic, or negative control miRNA showed that miR-29a and miR-1256 directly bond to the 3′UTR of TRIM68 (B). Transfection of miR-29a, miR-1256 mimic, or negative control miRNA into PCa cells caused downregulation of TRIM68 and PGK-1 at mRNA (C) and protein (D) levels. (Neg: negative control miRNA; 29a: miR-29a; 1256: miR-1256; *: p

    Journal: Epigenetics

    Article Title: Epigenetic deregulation of miR-29a and miR-1256 by isoflavone contributes to the inhibition of prostate cancer cell growth and invasion

    doi: 10.4161/epi.21236

    Figure Lengend Snippet: Figure 3. Targeting TRIM68 by miR-29a and miR-1256. Computerized analysis showed miR-29a and miR-1256 sequence alignment to the sequence of TRIM68 3′UTR (A). Co-transfection of TRIM68 3′UTR-Luc and miR-29a, miR-1256 mimic, or negative control miRNA showed that miR-29a and miR-1256 directly bond to the 3′UTR of TRIM68 (B). Transfection of miR-29a, miR-1256 mimic, or negative control miRNA into PCa cells caused downregulation of TRIM68 and PGK-1 at mRNA (C) and protein (D) levels. (Neg: negative control miRNA; 29a: miR-29a; 1256: miR-1256; *: p

    Article Snippet: Anti-TRIM68 (Santa Cruz), anti-PGK-1 (Santa Cruz), anti-β-actin (Sigma) and anti-GAPDH (Sigma) primary antibodies were used for Western Blot analysis.

    Techniques: Sequencing, Cotransfection, Negative Control, Transfection

    Glucose availability alters 3BP sensitivity in CRC cells HCT116 and DLD-1 cells were treated with 3BP under different media glucose concentrations. HKII expression was assessed via western blot analysis. Representative blots from three biological replicates ( A ) and graphical outputs for densitometric analysis of HKII expression normalized to α-tubulin from three biological replicates ± S.E.M. are shown ( B ). Representative blots from three biological replicates of putative 3BP targets SDH, GAPDH and PGK1 show no effects of altered glucose concentrations ( C ). Growth curves for CRC cells grown under different media glucose concentrations in the presence or absence of 3BP over 72 h ( D ). Dose-effects of 3BP on cell growth following 72 h treatment in CRC cells under different media glucose concentrations ( E ) (* P ≤0.05, ** P ≤0.01, *** P ≤0.001, **** P ≤0.0001 compared with control (0 μM 3BP)).

    Journal: Bioscience Reports

    Article Title: The effect of 3-bromopyruvate on human colorectal cancer cells is dependent on glucose concentration but not hexokinase II expression

    doi: 10.1042/BSR20150267

    Figure Lengend Snippet: Glucose availability alters 3BP sensitivity in CRC cells HCT116 and DLD-1 cells were treated with 3BP under different media glucose concentrations. HKII expression was assessed via western blot analysis. Representative blots from three biological replicates ( A ) and graphical outputs for densitometric analysis of HKII expression normalized to α-tubulin from three biological replicates ± S.E.M. are shown ( B ). Representative blots from three biological replicates of putative 3BP targets SDH, GAPDH and PGK1 show no effects of altered glucose concentrations ( C ). Growth curves for CRC cells grown under different media glucose concentrations in the presence or absence of 3BP over 72 h ( D ). Dose-effects of 3BP on cell growth following 72 h treatment in CRC cells under different media glucose concentrations ( E ) (* P ≤0.05, ** P ≤0.01, *** P ≤0.001, **** P ≤0.0001 compared with control (0 μM 3BP)).

    Article Snippet: Primary antibodies used included rabbit anti-pAkt Ser-473 (9271; 1:1000; Cell Signaling), anti-pAkt Thr-308 (4056; 1:1000; Cell Signaling), anti-pan-Akt (4691; 1:1000; Cell Signaling), anti-caspase 3 (9662S; 1:1000; Cell Signaling), anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; AP7873a; 1:1000; Abgent), anti-HKII (2867; 1:5000; Cell Signaling), anti-pPDK1 Ser-241 (3438; 1:1000; Cell Signaling), anti-PDK1 (3062; 1:1000; Cell Signaling), anti-PGK1 (AP7094b; 1:1000; Abgent), anti-PTEN (9188; 1:1000; Cell Signaling), anti-SDH (AP19974b; 1:1000; Abgent), mouse anti-α-tubulin (T5168; 1:200000; Sigma–Aldrich) and rabbit or mouse HRP-labelled secondary antibodies (all 1:20000; Sigma–Aldrich).

    Techniques: Expressing, Western Blot

    PGK1 interacts directly with HSP90 and modulates the ATPase activity of HSP90 . a . Western blot was performed to assess the effects of PGK1 knockdown on expression levels of HSP90 in Ishikawa cancer cell line. β-actin was used as a loading control. b . Semi-quantitative analysis of protein level in A. c . Western blot was performed to assess the effects of PGK1 knockdown on expression levels of HSP90 in HEC1A cancer cell line. β-actin was used as a loading control. d . Semi-quantitative analysis of protein level in C. e . Effect of PGK1 overexpression and HSP90 inhibitor 17-AAG on cellular ATP level. Vector, empty vector; PGK1, vector for PGK1 overexpression; PGK1 + 17-AAG, vector for PGK1 overexpression supplemented with 17-AAG. f . Co-immunoprecipitation was used to detect binding between PGK1 and HSP90. Hemagglutinin (HA)-tagged PGK1 (PGK1-HA) was overexpressed in Ishikawa cells. Data were shown as mean ± SD based on three independent experiments. *, P

    Journal: Molecular Medicine

    Article Title: PGK1 facilities cisplatin chemoresistance by triggering HSP90/ERK pathway mediated DNA repair and methylation in endometrial endometrioid adenocarcinoma

    doi: 10.1186/s10020-019-0079-0

    Figure Lengend Snippet: PGK1 interacts directly with HSP90 and modulates the ATPase activity of HSP90 . a . Western blot was performed to assess the effects of PGK1 knockdown on expression levels of HSP90 in Ishikawa cancer cell line. β-actin was used as a loading control. b . Semi-quantitative analysis of protein level in A. c . Western blot was performed to assess the effects of PGK1 knockdown on expression levels of HSP90 in HEC1A cancer cell line. β-actin was used as a loading control. d . Semi-quantitative analysis of protein level in C. e . Effect of PGK1 overexpression and HSP90 inhibitor 17-AAG on cellular ATP level. Vector, empty vector; PGK1, vector for PGK1 overexpression; PGK1 + 17-AAG, vector for PGK1 overexpression supplemented with 17-AAG. f . Co-immunoprecipitation was used to detect binding between PGK1 and HSP90. Hemagglutinin (HA)-tagged PGK1 (PGK1-HA) was overexpressed in Ishikawa cells. Data were shown as mean ± SD based on three independent experiments. *, P

    Article Snippet: Primary antibodies against POLD1, JNK, p-JNK, c-JUN, and FOSL1 were purchased from Santa Cruz Biotech (Dallas, USA); primary antibodies against PGK1, HSP90, ERK, p-ERK, AKT, p-AKT, DNMT1, DNMT3A, DNMT3B, and SPARC were purchased from Cell Signaling Technology (Boston, USA); primary antibodies against Bcl-xL, Mcl-1, and Bax were purchased from Abcam (Cambridge, UK).

    Techniques: Activity Assay, Western Blot, Expressing, Over Expression, Plasmid Preparation, Immunoprecipitation, Binding Assay

    PGK1 facilitates chemoresistance to cisplatin through HSP90/ERK pathway. a - b . MTT assay was used to evaluate the effects of PGK1 overexpression or HSP90 ATPase inhibitor 17-AAG on chemoresistance to cisplatin in Ishikawa ( a ) and HEC1A ( b ) cell lines. Vector, empty vector; PGK1, plasmid for PGK1 overexpression; PGK1 + 17-AAG, plasmid for PGK1 overexpression supplemented with 17-AAG. c - d . MTT assay was used to evaluate the effects of PGK1 overexpression or ERK pathway inhibitor PD98059 on chemoresistance to cisplatin in Ishikawa ( c ) and HEC1A (D) cell lines. Vector, empty vector; PGK1, plasmid for PGK1 overexpression; PGK1+ PD98059, plasmid for PGK1 overexpression supplemented with PD98059. Data were shown as mean ± SD based on three independent experiments. *, P

    Journal: Molecular Medicine

    Article Title: PGK1 facilities cisplatin chemoresistance by triggering HSP90/ERK pathway mediated DNA repair and methylation in endometrial endometrioid adenocarcinoma

    doi: 10.1186/s10020-019-0079-0

    Figure Lengend Snippet: PGK1 facilitates chemoresistance to cisplatin through HSP90/ERK pathway. a - b . MTT assay was used to evaluate the effects of PGK1 overexpression or HSP90 ATPase inhibitor 17-AAG on chemoresistance to cisplatin in Ishikawa ( a ) and HEC1A ( b ) cell lines. Vector, empty vector; PGK1, plasmid for PGK1 overexpression; PGK1 + 17-AAG, plasmid for PGK1 overexpression supplemented with 17-AAG. c - d . MTT assay was used to evaluate the effects of PGK1 overexpression or ERK pathway inhibitor PD98059 on chemoresistance to cisplatin in Ishikawa ( c ) and HEC1A (D) cell lines. Vector, empty vector; PGK1, plasmid for PGK1 overexpression; PGK1+ PD98059, plasmid for PGK1 overexpression supplemented with PD98059. Data were shown as mean ± SD based on three independent experiments. *, P

    Article Snippet: Primary antibodies against POLD1, JNK, p-JNK, c-JUN, and FOSL1 were purchased from Santa Cruz Biotech (Dallas, USA); primary antibodies against PGK1, HSP90, ERK, p-ERK, AKT, p-AKT, DNMT1, DNMT3A, DNMT3B, and SPARC were purchased from Cell Signaling Technology (Boston, USA); primary antibodies against Bcl-xL, Mcl-1, and Bax were purchased from Abcam (Cambridge, UK).

    Techniques: MTT Assay, Over Expression, Plasmid Preparation

    HSP90 inhibitor 17-AAG exhibits similar effects as knockdown of PGK1. a - b . MTT assay was used to evaluate the effects of HSP90 inhibitor 17-AAG on cell viability in endometrial cell lines Ishikawa ( a ) and HEC1A ( b ). DMSO was used as negative control. c . Western blot was used to detect the expression of DNA repair-related proteins c-JUN, FOSL1, and POLD1 in the presence of HSP90 inhibitor 17-AAG in Ishikawa cancer cell line. β-actin was used as a loading control. d . Semi-quantitative analysis of protein level in C. e . Western blot was used to detect the expression of DNA repair-related proteins c-JUN, FOSL1, and POLD1 in the presence of HSP90 inhibitor 17-AAG in HEC1A cancer cell line. β-actin was used as a loading control. f . Semi-quantitative analysis of protein level in E. g . Western blot was performed to examine the effect of HSP90 inhibitor 17-AAG on the expression of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) and SPARC in Ishikawa cancer cell line. β-actin was used as a loading control. h . Semi-quantitative analysis of protein level in G. i . Western blot was performed to examine the effect of HSP90 inhibitor 17-AAG on the expression of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) and SPARC in HEC1A cancer cell line. β-actin was used as a loading control. j . Semi-quantitative analysis of protein level in I. Data were shown as mean ± SD based on three independent experiments. *, P

    Journal: Molecular Medicine

    Article Title: PGK1 facilities cisplatin chemoresistance by triggering HSP90/ERK pathway mediated DNA repair and methylation in endometrial endometrioid adenocarcinoma

    doi: 10.1186/s10020-019-0079-0

    Figure Lengend Snippet: HSP90 inhibitor 17-AAG exhibits similar effects as knockdown of PGK1. a - b . MTT assay was used to evaluate the effects of HSP90 inhibitor 17-AAG on cell viability in endometrial cell lines Ishikawa ( a ) and HEC1A ( b ). DMSO was used as negative control. c . Western blot was used to detect the expression of DNA repair-related proteins c-JUN, FOSL1, and POLD1 in the presence of HSP90 inhibitor 17-AAG in Ishikawa cancer cell line. β-actin was used as a loading control. d . Semi-quantitative analysis of protein level in C. e . Western blot was used to detect the expression of DNA repair-related proteins c-JUN, FOSL1, and POLD1 in the presence of HSP90 inhibitor 17-AAG in HEC1A cancer cell line. β-actin was used as a loading control. f . Semi-quantitative analysis of protein level in E. g . Western blot was performed to examine the effect of HSP90 inhibitor 17-AAG on the expression of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) and SPARC in Ishikawa cancer cell line. β-actin was used as a loading control. h . Semi-quantitative analysis of protein level in G. i . Western blot was performed to examine the effect of HSP90 inhibitor 17-AAG on the expression of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) and SPARC in HEC1A cancer cell line. β-actin was used as a loading control. j . Semi-quantitative analysis of protein level in I. Data were shown as mean ± SD based on three independent experiments. *, P

    Article Snippet: Primary antibodies against POLD1, JNK, p-JNK, c-JUN, and FOSL1 were purchased from Santa Cruz Biotech (Dallas, USA); primary antibodies against PGK1, HSP90, ERK, p-ERK, AKT, p-AKT, DNMT1, DNMT3A, DNMT3B, and SPARC were purchased from Cell Signaling Technology (Boston, USA); primary antibodies against Bcl-xL, Mcl-1, and Bax were purchased from Abcam (Cambridge, UK).

    Techniques: MTT Assay, Negative Control, Western Blot, Expressing

    PGK1 expression is elevated in endometrial cancer tissues and is associated with FIGO stages and lymphatic metastasis. a . qPCR was used to detect the expression levels of PGK1 in endometrial carcinoma tissues ( n = 56) and in normal human endometrial tissues ( n = 20). b . qPCR was used to compare the expression levels of PGK1 in stage I/II endometrial carcinoma tissues ( n = 37) and in stage III/IV endometrial carcinoma tissues ( n = 19). c . qPCR was performed to examine the expression levels of PGK1 in endometrial carcinoma tissues with positive lymph node metastasis ( n = 14) and with negative lymph node metastasis ( n = 42). Data were shown as mean ± SD based on three independent experiments. ***, P

    Journal: Molecular Medicine

    Article Title: PGK1 facilities cisplatin chemoresistance by triggering HSP90/ERK pathway mediated DNA repair and methylation in endometrial endometrioid adenocarcinoma

    doi: 10.1186/s10020-019-0079-0

    Figure Lengend Snippet: PGK1 expression is elevated in endometrial cancer tissues and is associated with FIGO stages and lymphatic metastasis. a . qPCR was used to detect the expression levels of PGK1 in endometrial carcinoma tissues ( n = 56) and in normal human endometrial tissues ( n = 20). b . qPCR was used to compare the expression levels of PGK1 in stage I/II endometrial carcinoma tissues ( n = 37) and in stage III/IV endometrial carcinoma tissues ( n = 19). c . qPCR was performed to examine the expression levels of PGK1 in endometrial carcinoma tissues with positive lymph node metastasis ( n = 14) and with negative lymph node metastasis ( n = 42). Data were shown as mean ± SD based on three independent experiments. ***, P

    Article Snippet: Primary antibodies against POLD1, JNK, p-JNK, c-JUN, and FOSL1 were purchased from Santa Cruz Biotech (Dallas, USA); primary antibodies against PGK1, HSP90, ERK, p-ERK, AKT, p-AKT, DNMT1, DNMT3A, DNMT3B, and SPARC were purchased from Cell Signaling Technology (Boston, USA); primary antibodies against Bcl-xL, Mcl-1, and Bax were purchased from Abcam (Cambridge, UK).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    PGK1 knockdown inhibits proliferation and enhances cisplatin sensitivity in endometrial cancer cell lines. a . Western blot was performed to confirm knockdown of PGK1 in endometrial cancer cell lines (Ishikawa and HEC1A). α-tubulin was used as a loading control. b . MTT assay was used to evaluate the effects of PGK1 knockdown on cell viability in endometrial cancer cell lines (Ishikawa and HEC1A). c - d . Flow cytometry was used to assess the effect of PGK1 knockdown on apoptosis of endometrial cancer cells (Ishikawa and HEC1A). e - f . Effect of PGK1 knockdown on tumor growth in a Ishikawa cell-induced xenograft endometrial cancer model. g . MTT assay was used to evaluate the effect of PGK1 knockdown on the sensitivity of Ishikawa endometrial cancer cell line to cisplatin and the IC 50 value was calculated using GraphPad Prism 6. h . MTT assay was used to test the effect of PGK1 knockdown on the sensitivity of HEC1A endometrial cancer cell line to cisplatin and the IC 50 value was calculated using GraphPad Prism 6. i . Western blot was performed to confirm knockdown of PGK1 in cisplatin-resistant endometrial cancer cell line (Ishikawa/DDP). j . MTT assay was used to evaluate the effect of PGK1 knockdown on the sensitivity of Ishikawa/DDP cell line to cisplatin and the IC 50 value was calculated using GraphPad Prism 6. Data were shown as mean ± SD based on three independent experiments. *, P

    Journal: Molecular Medicine

    Article Title: PGK1 facilities cisplatin chemoresistance by triggering HSP90/ERK pathway mediated DNA repair and methylation in endometrial endometrioid adenocarcinoma

    doi: 10.1186/s10020-019-0079-0

    Figure Lengend Snippet: PGK1 knockdown inhibits proliferation and enhances cisplatin sensitivity in endometrial cancer cell lines. a . Western blot was performed to confirm knockdown of PGK1 in endometrial cancer cell lines (Ishikawa and HEC1A). α-tubulin was used as a loading control. b . MTT assay was used to evaluate the effects of PGK1 knockdown on cell viability in endometrial cancer cell lines (Ishikawa and HEC1A). c - d . Flow cytometry was used to assess the effect of PGK1 knockdown on apoptosis of endometrial cancer cells (Ishikawa and HEC1A). e - f . Effect of PGK1 knockdown on tumor growth in a Ishikawa cell-induced xenograft endometrial cancer model. g . MTT assay was used to evaluate the effect of PGK1 knockdown on the sensitivity of Ishikawa endometrial cancer cell line to cisplatin and the IC 50 value was calculated using GraphPad Prism 6. h . MTT assay was used to test the effect of PGK1 knockdown on the sensitivity of HEC1A endometrial cancer cell line to cisplatin and the IC 50 value was calculated using GraphPad Prism 6. i . Western blot was performed to confirm knockdown of PGK1 in cisplatin-resistant endometrial cancer cell line (Ishikawa/DDP). j . MTT assay was used to evaluate the effect of PGK1 knockdown on the sensitivity of Ishikawa/DDP cell line to cisplatin and the IC 50 value was calculated using GraphPad Prism 6. Data were shown as mean ± SD based on three independent experiments. *, P

    Article Snippet: Primary antibodies against POLD1, JNK, p-JNK, c-JUN, and FOSL1 were purchased from Santa Cruz Biotech (Dallas, USA); primary antibodies against PGK1, HSP90, ERK, p-ERK, AKT, p-AKT, DNMT1, DNMT3A, DNMT3B, and SPARC were purchased from Cell Signaling Technology (Boston, USA); primary antibodies against Bcl-xL, Mcl-1, and Bax were purchased from Abcam (Cambridge, UK).

    Techniques: Western Blot, MTT Assay, Flow Cytometry, Cytometry

    PGK1 knockdown inhibits phosphorylation of JNK, ERK, and AKT pathway. a . Western blot was performed to evaluate the effects of PGK1 knockdown on phosphorylation levels of JNK, ERK, and AKT in Ishikawa cancer cell line. β-actin was used as a loading control. b . Semi-quantitative analysis of protein level in A. c . Western blot was performed to evaluate the effects of PGK1 knockdown on phosphorylation levels of JNK, ERK, and AKT pathway in HEC1A cancer cell line. β-actin was used as a loading control. d . Semi-quantitative analysis of protein level in C. Data were shown as mean ± SD based on three independent experiments. *, P

    Journal: Molecular Medicine

    Article Title: PGK1 facilities cisplatin chemoresistance by triggering HSP90/ERK pathway mediated DNA repair and methylation in endometrial endometrioid adenocarcinoma

    doi: 10.1186/s10020-019-0079-0

    Figure Lengend Snippet: PGK1 knockdown inhibits phosphorylation of JNK, ERK, and AKT pathway. a . Western blot was performed to evaluate the effects of PGK1 knockdown on phosphorylation levels of JNK, ERK, and AKT in Ishikawa cancer cell line. β-actin was used as a loading control. b . Semi-quantitative analysis of protein level in A. c . Western blot was performed to evaluate the effects of PGK1 knockdown on phosphorylation levels of JNK, ERK, and AKT pathway in HEC1A cancer cell line. β-actin was used as a loading control. d . Semi-quantitative analysis of protein level in C. Data were shown as mean ± SD based on three independent experiments. *, P

    Article Snippet: Primary antibodies against POLD1, JNK, p-JNK, c-JUN, and FOSL1 were purchased from Santa Cruz Biotech (Dallas, USA); primary antibodies against PGK1, HSP90, ERK, p-ERK, AKT, p-AKT, DNMT1, DNMT3A, DNMT3B, and SPARC were purchased from Cell Signaling Technology (Boston, USA); primary antibodies against Bcl-xL, Mcl-1, and Bax were purchased from Abcam (Cambridge, UK).

    Techniques: Western Blot

    PGK1 knockdown reduces the expression of DNA repair-related proteins, methylation-related enzymes, and total cellular methylation level. a . Western blot was used to detect the expression of pro-survival proteins (Bcl-xL, Mcl-1) and pro-apoptotic protein (Bax) in Ishikawa endometrial cancer cell line. β-actin was used as a loading control. b . Semi-quantitative analysis of protein level in A. c . Western blot was used to detect the expression of pro-survival proteins (Bcl-xL, Mcl-1) and pro-apoptotic protein (Bax) in HEC1A endometrial cancer cell line. β-actin was used as a loading control. d . Semi-quantitative analysis of protein level in C. e . Western blot was used to detect the expression of DNA repair-related proteins c-JUN, FOSL1, and POLD1 in Ishikawa endometrial cancer cell line. β-actin was used as a loading control. f . Semi-quantitative analysis of protein level in A. g . Western blot was used to detect the expression of DNA repair-related proteins c-JUN, FOSL1, and POLD1 in HEC1A endometrial cancer cell line. β-actin was used as a loading control. h . Semi-quantitative analysis of protein level in C. i . Western blot was performed to determine the effect of PGK1 knockdown on the total intracellular methylation levels (amount of average methylated cytosine). j . Western blot was used to examine the effect of PGK1 knockdown on the expression of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) and SPARC in Ishikawa cancer cell line. β-actin was used as a loading control. k . Semi-quantitative analysis of protein level in F. l . Western blot was used to examine the effect of PGK1 knockdown on the expression of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) and SPARC in HEC1A endometrial cancer cell line. β-actin was used as a loading control. m . Semi-quantitative analysis of protein level in H. Data were shown as mean ± SD based on three independent experiments. *, P

    Journal: Molecular Medicine

    Article Title: PGK1 facilities cisplatin chemoresistance by triggering HSP90/ERK pathway mediated DNA repair and methylation in endometrial endometrioid adenocarcinoma

    doi: 10.1186/s10020-019-0079-0

    Figure Lengend Snippet: PGK1 knockdown reduces the expression of DNA repair-related proteins, methylation-related enzymes, and total cellular methylation level. a . Western blot was used to detect the expression of pro-survival proteins (Bcl-xL, Mcl-1) and pro-apoptotic protein (Bax) in Ishikawa endometrial cancer cell line. β-actin was used as a loading control. b . Semi-quantitative analysis of protein level in A. c . Western blot was used to detect the expression of pro-survival proteins (Bcl-xL, Mcl-1) and pro-apoptotic protein (Bax) in HEC1A endometrial cancer cell line. β-actin was used as a loading control. d . Semi-quantitative analysis of protein level in C. e . Western blot was used to detect the expression of DNA repair-related proteins c-JUN, FOSL1, and POLD1 in Ishikawa endometrial cancer cell line. β-actin was used as a loading control. f . Semi-quantitative analysis of protein level in A. g . Western blot was used to detect the expression of DNA repair-related proteins c-JUN, FOSL1, and POLD1 in HEC1A endometrial cancer cell line. β-actin was used as a loading control. h . Semi-quantitative analysis of protein level in C. i . Western blot was performed to determine the effect of PGK1 knockdown on the total intracellular methylation levels (amount of average methylated cytosine). j . Western blot was used to examine the effect of PGK1 knockdown on the expression of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) and SPARC in Ishikawa cancer cell line. β-actin was used as a loading control. k . Semi-quantitative analysis of protein level in F. l . Western blot was used to examine the effect of PGK1 knockdown on the expression of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) and SPARC in HEC1A endometrial cancer cell line. β-actin was used as a loading control. m . Semi-quantitative analysis of protein level in H. Data were shown as mean ± SD based on three independent experiments. *, P

    Article Snippet: Primary antibodies against POLD1, JNK, p-JNK, c-JUN, and FOSL1 were purchased from Santa Cruz Biotech (Dallas, USA); primary antibodies against PGK1, HSP90, ERK, p-ERK, AKT, p-AKT, DNMT1, DNMT3A, DNMT3B, and SPARC were purchased from Cell Signaling Technology (Boston, USA); primary antibodies against Bcl-xL, Mcl-1, and Bax were purchased from Abcam (Cambridge, UK).

    Techniques: Expressing, Methylation, Western Blot

    BTN2 expression under mild ethanol stress. Cells carrying a FLAG-tagged chromosomal copy of BTN2 gene in the exponential growth phase were treated with the indicated concentrations of ethanol for 60 min. (A) BTN2 mRNA level was determined by performing qRT-PCR and was normalized to that of ACT1 . (B) Btn2 protein level was determined by performing Western blotting with anti-FLAG antibody. Pgk1 was used as the loading control. Btn2 protein level was normalized to that of Pgk1, and the intensity of the Pgk1 band in each lane was set at 100%. Data are expressed as mean ± SD ( n = 3).

    Journal: Frontiers in Microbiology

    Article Title: Prioritized Expression of BTN2 of Saccharomyces cerevisiae under Pronounced Translation Repression Induced by Severe Ethanol Stress

    doi: 10.3389/fmicb.2016.01319

    Figure Lengend Snippet: BTN2 expression under mild ethanol stress. Cells carrying a FLAG-tagged chromosomal copy of BTN2 gene in the exponential growth phase were treated with the indicated concentrations of ethanol for 60 min. (A) BTN2 mRNA level was determined by performing qRT-PCR and was normalized to that of ACT1 . (B) Btn2 protein level was determined by performing Western blotting with anti-FLAG antibody. Pgk1 was used as the loading control. Btn2 protein level was normalized to that of Pgk1, and the intensity of the Pgk1 band in each lane was set at 100%. Data are expressed as mean ± SD ( n = 3).

    Article Snippet: The anti-FLAG and anti-Pgk1 primary antibodies were detected using HRP-conjugated anti-mouse secondary antibody (dilution, 1:1,000; Cell Signaling Technology, Beverly, MA, USA).

    Techniques: Expressing, Quantitative RT-PCR, Western Blot

    BTN2 was preferentially expressed under severe ethanol stress. Yeast cells carrying a FLAG-tagged chromosomal copy of BTN2 in the exponential growth phase were treated with 9 or 10% (v/v) ethanol (severe ethanol stress) for the indicated time (A–D) or treated with 9 or 10% (v/v) ethanol and 0.1 mg/ml cycloheximide (CHX) for 60 min (E) . (A,C) BTN2 mRNA level was determined by performing qRT-PCR and was normalized to that of ACT1 . (B,D,E) Btn2 protein level was determined by performing Western blotting with anti-FLAG antibody. Pgk1 was used as the loading control. Btn2 protein level was normalized to that of Pgk1, and the intensity of the Pgk1 band in each lane was set at 100%. Data are expressed as mean ± SD ( n = 3). ND, not detected.

    Journal: Frontiers in Microbiology

    Article Title: Prioritized Expression of BTN2 of Saccharomyces cerevisiae under Pronounced Translation Repression Induced by Severe Ethanol Stress

    doi: 10.3389/fmicb.2016.01319

    Figure Lengend Snippet: BTN2 was preferentially expressed under severe ethanol stress. Yeast cells carrying a FLAG-tagged chromosomal copy of BTN2 in the exponential growth phase were treated with 9 or 10% (v/v) ethanol (severe ethanol stress) for the indicated time (A–D) or treated with 9 or 10% (v/v) ethanol and 0.1 mg/ml cycloheximide (CHX) for 60 min (E) . (A,C) BTN2 mRNA level was determined by performing qRT-PCR and was normalized to that of ACT1 . (B,D,E) Btn2 protein level was determined by performing Western blotting with anti-FLAG antibody. Pgk1 was used as the loading control. Btn2 protein level was normalized to that of Pgk1, and the intensity of the Pgk1 band in each lane was set at 100%. Data are expressed as mean ± SD ( n = 3). ND, not detected.

    Article Snippet: The anti-FLAG and anti-Pgk1 primary antibodies were detected using HRP-conjugated anti-mouse secondary antibody (dilution, 1:1,000; Cell Signaling Technology, Beverly, MA, USA).

    Techniques: Quantitative RT-PCR, Western Blot

    Btn2 protein level decreased during recovery from severe ethanol stress. Cells carrying a FLAG-tagged chromosomal copy of BTN2 gene in the exponential growth phase were treated with severe ethanol stress (10% v/v) for 60 min. After the treatment with ethanol stress, cells were transferred to a fresh SD medium lacking ethanol (A,B) or containing 5% ethanol (C,D) and were incubated further at 28°C for the indicated time. (A,C) BTN2 mRNA level was determined by performing qRT-PCR and was normalized to that of ACT1 . (B,D) Btn2 protein level was determined by performing Western blotting with anti-FLAG antibody. Pgk1 was used as the loading control. Btn2 protein level was normalized to that of Pgk1, and the intensity of the Pgk1 band in each lane was set at 100%. Data are expressed as mean ± SD ( n = 3).

    Journal: Frontiers in Microbiology

    Article Title: Prioritized Expression of BTN2 of Saccharomyces cerevisiae under Pronounced Translation Repression Induced by Severe Ethanol Stress

    doi: 10.3389/fmicb.2016.01319

    Figure Lengend Snippet: Btn2 protein level decreased during recovery from severe ethanol stress. Cells carrying a FLAG-tagged chromosomal copy of BTN2 gene in the exponential growth phase were treated with severe ethanol stress (10% v/v) for 60 min. After the treatment with ethanol stress, cells were transferred to a fresh SD medium lacking ethanol (A,B) or containing 5% ethanol (C,D) and were incubated further at 28°C for the indicated time. (A,C) BTN2 mRNA level was determined by performing qRT-PCR and was normalized to that of ACT1 . (B,D) Btn2 protein level was determined by performing Western blotting with anti-FLAG antibody. Pgk1 was used as the loading control. Btn2 protein level was normalized to that of Pgk1, and the intensity of the Pgk1 band in each lane was set at 100%. Data are expressed as mean ± SD ( n = 3).

    Article Snippet: The anti-FLAG and anti-Pgk1 primary antibodies were detected using HRP-conjugated anti-mouse secondary antibody (dilution, 1:1,000; Cell Signaling Technology, Beverly, MA, USA).

    Techniques: Incubation, Quantitative RT-PCR, Western Blot

    The BTN2 promoter region induced the protein synthesis of non-native genes under severe ethanol stress. (A,B) Cells harboring the pYY plasmid series ( BTN2 promoter-driven expression system) in the exponential growth phase were treated with 9 or 10% ethanol for the indicated time. (A) The mRNA levels of each FLAG-tagged gene were analyzed by performing qRT-PCR and were normalized to that of ACT1 . (B) Protein levels of Cur1-FLAG, Gic2-FLAG, and Yur1-FLAG were determined by performing Western blotting with anti-FLAG antibody. (C) The BTN2 terminator region had a negligible effect on preferential translation upon severe ethanol stress. Cells carrying YIp- BTN2-FLAG-ADH6 Ter or YIp- BTN2-FLAG-TEF1 Ter in the exponential phase of growth were treated with 9 or 10% ethanol for 1 h. Protein levels of Btn2-FLAG were determined by Western blot analysis using an anti-FLAG antibody. Pgk1 was used as the loading control. Their protein levels were normalized to that of Pgk1, and the intensity of the Pgk1 band in each lane was set at 100%. Data are shown as mean ± SD ( n = 3). Ctrl, control cells not treated with ethanol stress.

    Journal: Frontiers in Microbiology

    Article Title: Prioritized Expression of BTN2 of Saccharomyces cerevisiae under Pronounced Translation Repression Induced by Severe Ethanol Stress

    doi: 10.3389/fmicb.2016.01319

    Figure Lengend Snippet: The BTN2 promoter region induced the protein synthesis of non-native genes under severe ethanol stress. (A,B) Cells harboring the pYY plasmid series ( BTN2 promoter-driven expression system) in the exponential growth phase were treated with 9 or 10% ethanol for the indicated time. (A) The mRNA levels of each FLAG-tagged gene were analyzed by performing qRT-PCR and were normalized to that of ACT1 . (B) Protein levels of Cur1-FLAG, Gic2-FLAG, and Yur1-FLAG were determined by performing Western blotting with anti-FLAG antibody. (C) The BTN2 terminator region had a negligible effect on preferential translation upon severe ethanol stress. Cells carrying YIp- BTN2-FLAG-ADH6 Ter or YIp- BTN2-FLAG-TEF1 Ter in the exponential phase of growth were treated with 9 or 10% ethanol for 1 h. Protein levels of Btn2-FLAG were determined by Western blot analysis using an anti-FLAG antibody. Pgk1 was used as the loading control. Their protein levels were normalized to that of Pgk1, and the intensity of the Pgk1 band in each lane was set at 100%. Data are shown as mean ± SD ( n = 3). Ctrl, control cells not treated with ethanol stress.

    Article Snippet: The anti-FLAG and anti-Pgk1 primary antibodies were detected using HRP-conjugated anti-mouse secondary antibody (dilution, 1:1,000; Cell Signaling Technology, Beverly, MA, USA).

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

    In situ PCR in hybrid model and control. A: One or two pgk-1 spot(s) were detected in the epithelial nuclei of cholesteatoma. B: On the other hand, one pgk-1 spot was detected in the cells of EAC. Original magnification, ×350. C: One pgk-1 spot

    Journal: The American Journal of Pathology

    Article Title: Pathogenesis of Middle Ear Cholesteatoma

    doi: 10.2353/ajpath.2010.091182

    Figure Lengend Snippet: In situ PCR in hybrid model and control. A: One or two pgk-1 spot(s) were detected in the epithelial nuclei of cholesteatoma. B: On the other hand, one pgk-1 spot was detected in the cells of EAC. Original magnification, ×350. C: One pgk-1 spot

    Article Snippet: Initial detection of the X chromosomes was achieved by simultaneous amplification of X-linked phosphoglycerate kinase locus ( pgk-1 )., All primer sequences were synthesized commercially and, apart from pgk-1 , sequences were obtained from Research Genetics Inc. (Huntsville, AL).

    Techniques: In Situ, Polymerase Chain Reaction

    PGK1 interacts directly with HSP90 and modulates the ATPase activity of HSP90 . a . Western blot was performed to assess the effects of PGK1 knockdown on expression levels of HSP90 in Ishikawa cancer cell line. β-actin was used as a loading control. b . Semi-quantitative analysis of protein level in A. c . Western blot was performed to assess the effects of PGK1 knockdown on expression levels of HSP90 in HEC1A cancer cell line. β-actin was used as a loading control. d . Semi-quantitative analysis of protein level in C. e . Effect of PGK1 overexpression and HSP90 inhibitor 17-AAG on cellular ATP level. Vector, empty vector; PGK1, vector for PGK1 overexpression; PGK1 + 17-AAG, vector for PGK1 overexpression supplemented with 17-AAG. f . Co-immunoprecipitation was used to detect binding between PGK1 and HSP90. Hemagglutinin (HA)-tagged PGK1 (PGK1-HA) was overexpressed in Ishikawa cells. Data were shown as mean ± SD based on three independent experiments. *, P

    Journal: Molecular Medicine

    Article Title: PGK1 facilities cisplatin chemoresistance by triggering HSP90/ERK pathway mediated DNA repair and methylation in endometrial endometrioid adenocarcinoma

    doi: 10.1186/s10020-019-0079-0

    Figure Lengend Snippet: PGK1 interacts directly with HSP90 and modulates the ATPase activity of HSP90 . a . Western blot was performed to assess the effects of PGK1 knockdown on expression levels of HSP90 in Ishikawa cancer cell line. β-actin was used as a loading control. b . Semi-quantitative analysis of protein level in A. c . Western blot was performed to assess the effects of PGK1 knockdown on expression levels of HSP90 in HEC1A cancer cell line. β-actin was used as a loading control. d . Semi-quantitative analysis of protein level in C. e . Effect of PGK1 overexpression and HSP90 inhibitor 17-AAG on cellular ATP level. Vector, empty vector; PGK1, vector for PGK1 overexpression; PGK1 + 17-AAG, vector for PGK1 overexpression supplemented with 17-AAG. f . Co-immunoprecipitation was used to detect binding between PGK1 and HSP90. Hemagglutinin (HA)-tagged PGK1 (PGK1-HA) was overexpressed in Ishikawa cells. Data were shown as mean ± SD based on three independent experiments. *, P

    Article Snippet: Cell transfection shRNA sequence targeting PGK1 and overexpressing plasmid of PGK1 were purchased from GenePharma (Shanghai, China).

    Techniques: Activity Assay, Western Blot, Expressing, Over Expression, Plasmid Preparation, Immunoprecipitation, Binding Assay

    PGK1 facilitates chemoresistance to cisplatin through HSP90/ERK pathway. a - b . MTT assay was used to evaluate the effects of PGK1 overexpression or HSP90 ATPase inhibitor 17-AAG on chemoresistance to cisplatin in Ishikawa ( a ) and HEC1A ( b ) cell lines. Vector, empty vector; PGK1, plasmid for PGK1 overexpression; PGK1 + 17-AAG, plasmid for PGK1 overexpression supplemented with 17-AAG. c - d . MTT assay was used to evaluate the effects of PGK1 overexpression or ERK pathway inhibitor PD98059 on chemoresistance to cisplatin in Ishikawa ( c ) and HEC1A (D) cell lines. Vector, empty vector; PGK1, plasmid for PGK1 overexpression; PGK1+ PD98059, plasmid for PGK1 overexpression supplemented with PD98059. Data were shown as mean ± SD based on three independent experiments. *, P

    Journal: Molecular Medicine

    Article Title: PGK1 facilities cisplatin chemoresistance by triggering HSP90/ERK pathway mediated DNA repair and methylation in endometrial endometrioid adenocarcinoma

    doi: 10.1186/s10020-019-0079-0

    Figure Lengend Snippet: PGK1 facilitates chemoresistance to cisplatin through HSP90/ERK pathway. a - b . MTT assay was used to evaluate the effects of PGK1 overexpression or HSP90 ATPase inhibitor 17-AAG on chemoresistance to cisplatin in Ishikawa ( a ) and HEC1A ( b ) cell lines. Vector, empty vector; PGK1, plasmid for PGK1 overexpression; PGK1 + 17-AAG, plasmid for PGK1 overexpression supplemented with 17-AAG. c - d . MTT assay was used to evaluate the effects of PGK1 overexpression or ERK pathway inhibitor PD98059 on chemoresistance to cisplatin in Ishikawa ( c ) and HEC1A (D) cell lines. Vector, empty vector; PGK1, plasmid for PGK1 overexpression; PGK1+ PD98059, plasmid for PGK1 overexpression supplemented with PD98059. Data were shown as mean ± SD based on three independent experiments. *, P

    Article Snippet: Cell transfection shRNA sequence targeting PGK1 and overexpressing plasmid of PGK1 were purchased from GenePharma (Shanghai, China).

    Techniques: MTT Assay, Over Expression, Plasmid Preparation

    HSP90 inhibitor 17-AAG exhibits similar effects as knockdown of PGK1. a - b . MTT assay was used to evaluate the effects of HSP90 inhibitor 17-AAG on cell viability in endometrial cell lines Ishikawa ( a ) and HEC1A ( b ). DMSO was used as negative control. c . Western blot was used to detect the expression of DNA repair-related proteins c-JUN, FOSL1, and POLD1 in the presence of HSP90 inhibitor 17-AAG in Ishikawa cancer cell line. β-actin was used as a loading control. d . Semi-quantitative analysis of protein level in C. e . Western blot was used to detect the expression of DNA repair-related proteins c-JUN, FOSL1, and POLD1 in the presence of HSP90 inhibitor 17-AAG in HEC1A cancer cell line. β-actin was used as a loading control. f . Semi-quantitative analysis of protein level in E. g . Western blot was performed to examine the effect of HSP90 inhibitor 17-AAG on the expression of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) and SPARC in Ishikawa cancer cell line. β-actin was used as a loading control. h . Semi-quantitative analysis of protein level in G. i . Western blot was performed to examine the effect of HSP90 inhibitor 17-AAG on the expression of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) and SPARC in HEC1A cancer cell line. β-actin was used as a loading control. j . Semi-quantitative analysis of protein level in I. Data were shown as mean ± SD based on three independent experiments. *, P

    Journal: Molecular Medicine

    Article Title: PGK1 facilities cisplatin chemoresistance by triggering HSP90/ERK pathway mediated DNA repair and methylation in endometrial endometrioid adenocarcinoma

    doi: 10.1186/s10020-019-0079-0

    Figure Lengend Snippet: HSP90 inhibitor 17-AAG exhibits similar effects as knockdown of PGK1. a - b . MTT assay was used to evaluate the effects of HSP90 inhibitor 17-AAG on cell viability in endometrial cell lines Ishikawa ( a ) and HEC1A ( b ). DMSO was used as negative control. c . Western blot was used to detect the expression of DNA repair-related proteins c-JUN, FOSL1, and POLD1 in the presence of HSP90 inhibitor 17-AAG in Ishikawa cancer cell line. β-actin was used as a loading control. d . Semi-quantitative analysis of protein level in C. e . Western blot was used to detect the expression of DNA repair-related proteins c-JUN, FOSL1, and POLD1 in the presence of HSP90 inhibitor 17-AAG in HEC1A cancer cell line. β-actin was used as a loading control. f . Semi-quantitative analysis of protein level in E. g . Western blot was performed to examine the effect of HSP90 inhibitor 17-AAG on the expression of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) and SPARC in Ishikawa cancer cell line. β-actin was used as a loading control. h . Semi-quantitative analysis of protein level in G. i . Western blot was performed to examine the effect of HSP90 inhibitor 17-AAG on the expression of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) and SPARC in HEC1A cancer cell line. β-actin was used as a loading control. j . Semi-quantitative analysis of protein level in I. Data were shown as mean ± SD based on three independent experiments. *, P

    Article Snippet: Cell transfection shRNA sequence targeting PGK1 and overexpressing plasmid of PGK1 were purchased from GenePharma (Shanghai, China).

    Techniques: MTT Assay, Negative Control, Western Blot, Expressing

    PGK1 expression is elevated in endometrial cancer tissues and is associated with FIGO stages and lymphatic metastasis. a . qPCR was used to detect the expression levels of PGK1 in endometrial carcinoma tissues ( n = 56) and in normal human endometrial tissues ( n = 20). b . qPCR was used to compare the expression levels of PGK1 in stage I/II endometrial carcinoma tissues ( n = 37) and in stage III/IV endometrial carcinoma tissues ( n = 19). c . qPCR was performed to examine the expression levels of PGK1 in endometrial carcinoma tissues with positive lymph node metastasis ( n = 14) and with negative lymph node metastasis ( n = 42). Data were shown as mean ± SD based on three independent experiments. ***, P

    Journal: Molecular Medicine

    Article Title: PGK1 facilities cisplatin chemoresistance by triggering HSP90/ERK pathway mediated DNA repair and methylation in endometrial endometrioid adenocarcinoma

    doi: 10.1186/s10020-019-0079-0

    Figure Lengend Snippet: PGK1 expression is elevated in endometrial cancer tissues and is associated with FIGO stages and lymphatic metastasis. a . qPCR was used to detect the expression levels of PGK1 in endometrial carcinoma tissues ( n = 56) and in normal human endometrial tissues ( n = 20). b . qPCR was used to compare the expression levels of PGK1 in stage I/II endometrial carcinoma tissues ( n = 37) and in stage III/IV endometrial carcinoma tissues ( n = 19). c . qPCR was performed to examine the expression levels of PGK1 in endometrial carcinoma tissues with positive lymph node metastasis ( n = 14) and with negative lymph node metastasis ( n = 42). Data were shown as mean ± SD based on three independent experiments. ***, P

    Article Snippet: Cell transfection shRNA sequence targeting PGK1 and overexpressing plasmid of PGK1 were purchased from GenePharma (Shanghai, China).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    PGK1 knockdown inhibits proliferation and enhances cisplatin sensitivity in endometrial cancer cell lines. a . Western blot was performed to confirm knockdown of PGK1 in endometrial cancer cell lines (Ishikawa and HEC1A). α-tubulin was used as a loading control. b . MTT assay was used to evaluate the effects of PGK1 knockdown on cell viability in endometrial cancer cell lines (Ishikawa and HEC1A). c - d . Flow cytometry was used to assess the effect of PGK1 knockdown on apoptosis of endometrial cancer cells (Ishikawa and HEC1A). e - f . Effect of PGK1 knockdown on tumor growth in a Ishikawa cell-induced xenograft endometrial cancer model. g . MTT assay was used to evaluate the effect of PGK1 knockdown on the sensitivity of Ishikawa endometrial cancer cell line to cisplatin and the IC 50 value was calculated using GraphPad Prism 6. h . MTT assay was used to test the effect of PGK1 knockdown on the sensitivity of HEC1A endometrial cancer cell line to cisplatin and the IC 50 value was calculated using GraphPad Prism 6. i . Western blot was performed to confirm knockdown of PGK1 in cisplatin-resistant endometrial cancer cell line (Ishikawa/DDP). j . MTT assay was used to evaluate the effect of PGK1 knockdown on the sensitivity of Ishikawa/DDP cell line to cisplatin and the IC 50 value was calculated using GraphPad Prism 6. Data were shown as mean ± SD based on three independent experiments. *, P

    Journal: Molecular Medicine

    Article Title: PGK1 facilities cisplatin chemoresistance by triggering HSP90/ERK pathway mediated DNA repair and methylation in endometrial endometrioid adenocarcinoma

    doi: 10.1186/s10020-019-0079-0

    Figure Lengend Snippet: PGK1 knockdown inhibits proliferation and enhances cisplatin sensitivity in endometrial cancer cell lines. a . Western blot was performed to confirm knockdown of PGK1 in endometrial cancer cell lines (Ishikawa and HEC1A). α-tubulin was used as a loading control. b . MTT assay was used to evaluate the effects of PGK1 knockdown on cell viability in endometrial cancer cell lines (Ishikawa and HEC1A). c - d . Flow cytometry was used to assess the effect of PGK1 knockdown on apoptosis of endometrial cancer cells (Ishikawa and HEC1A). e - f . Effect of PGK1 knockdown on tumor growth in a Ishikawa cell-induced xenograft endometrial cancer model. g . MTT assay was used to evaluate the effect of PGK1 knockdown on the sensitivity of Ishikawa endometrial cancer cell line to cisplatin and the IC 50 value was calculated using GraphPad Prism 6. h . MTT assay was used to test the effect of PGK1 knockdown on the sensitivity of HEC1A endometrial cancer cell line to cisplatin and the IC 50 value was calculated using GraphPad Prism 6. i . Western blot was performed to confirm knockdown of PGK1 in cisplatin-resistant endometrial cancer cell line (Ishikawa/DDP). j . MTT assay was used to evaluate the effect of PGK1 knockdown on the sensitivity of Ishikawa/DDP cell line to cisplatin and the IC 50 value was calculated using GraphPad Prism 6. Data were shown as mean ± SD based on three independent experiments. *, P

    Article Snippet: Cell transfection shRNA sequence targeting PGK1 and overexpressing plasmid of PGK1 were purchased from GenePharma (Shanghai, China).

    Techniques: Western Blot, MTT Assay, Flow Cytometry, Cytometry

    PGK1 knockdown inhibits phosphorylation of JNK, ERK, and AKT pathway. a . Western blot was performed to evaluate the effects of PGK1 knockdown on phosphorylation levels of JNK, ERK, and AKT in Ishikawa cancer cell line. β-actin was used as a loading control. b . Semi-quantitative analysis of protein level in A. c . Western blot was performed to evaluate the effects of PGK1 knockdown on phosphorylation levels of JNK, ERK, and AKT pathway in HEC1A cancer cell line. β-actin was used as a loading control. d . Semi-quantitative analysis of protein level in C. Data were shown as mean ± SD based on three independent experiments. *, P

    Journal: Molecular Medicine

    Article Title: PGK1 facilities cisplatin chemoresistance by triggering HSP90/ERK pathway mediated DNA repair and methylation in endometrial endometrioid adenocarcinoma

    doi: 10.1186/s10020-019-0079-0

    Figure Lengend Snippet: PGK1 knockdown inhibits phosphorylation of JNK, ERK, and AKT pathway. a . Western blot was performed to evaluate the effects of PGK1 knockdown on phosphorylation levels of JNK, ERK, and AKT in Ishikawa cancer cell line. β-actin was used as a loading control. b . Semi-quantitative analysis of protein level in A. c . Western blot was performed to evaluate the effects of PGK1 knockdown on phosphorylation levels of JNK, ERK, and AKT pathway in HEC1A cancer cell line. β-actin was used as a loading control. d . Semi-quantitative analysis of protein level in C. Data were shown as mean ± SD based on three independent experiments. *, P

    Article Snippet: Cell transfection shRNA sequence targeting PGK1 and overexpressing plasmid of PGK1 were purchased from GenePharma (Shanghai, China).

    Techniques: Western Blot

    PGK1 knockdown reduces the expression of DNA repair-related proteins, methylation-related enzymes, and total cellular methylation level. a . Western blot was used to detect the expression of pro-survival proteins (Bcl-xL, Mcl-1) and pro-apoptotic protein (Bax) in Ishikawa endometrial cancer cell line. β-actin was used as a loading control. b . Semi-quantitative analysis of protein level in A. c . Western blot was used to detect the expression of pro-survival proteins (Bcl-xL, Mcl-1) and pro-apoptotic protein (Bax) in HEC1A endometrial cancer cell line. β-actin was used as a loading control. d . Semi-quantitative analysis of protein level in C. e . Western blot was used to detect the expression of DNA repair-related proteins c-JUN, FOSL1, and POLD1 in Ishikawa endometrial cancer cell line. β-actin was used as a loading control. f . Semi-quantitative analysis of protein level in A. g . Western blot was used to detect the expression of DNA repair-related proteins c-JUN, FOSL1, and POLD1 in HEC1A endometrial cancer cell line. β-actin was used as a loading control. h . Semi-quantitative analysis of protein level in C. i . Western blot was performed to determine the effect of PGK1 knockdown on the total intracellular methylation levels (amount of average methylated cytosine). j . Western blot was used to examine the effect of PGK1 knockdown on the expression of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) and SPARC in Ishikawa cancer cell line. β-actin was used as a loading control. k . Semi-quantitative analysis of protein level in F. l . Western blot was used to examine the effect of PGK1 knockdown on the expression of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) and SPARC in HEC1A endometrial cancer cell line. β-actin was used as a loading control. m . Semi-quantitative analysis of protein level in H. Data were shown as mean ± SD based on three independent experiments. *, P

    Journal: Molecular Medicine

    Article Title: PGK1 facilities cisplatin chemoresistance by triggering HSP90/ERK pathway mediated DNA repair and methylation in endometrial endometrioid adenocarcinoma

    doi: 10.1186/s10020-019-0079-0

    Figure Lengend Snippet: PGK1 knockdown reduces the expression of DNA repair-related proteins, methylation-related enzymes, and total cellular methylation level. a . Western blot was used to detect the expression of pro-survival proteins (Bcl-xL, Mcl-1) and pro-apoptotic protein (Bax) in Ishikawa endometrial cancer cell line. β-actin was used as a loading control. b . Semi-quantitative analysis of protein level in A. c . Western blot was used to detect the expression of pro-survival proteins (Bcl-xL, Mcl-1) and pro-apoptotic protein (Bax) in HEC1A endometrial cancer cell line. β-actin was used as a loading control. d . Semi-quantitative analysis of protein level in C. e . Western blot was used to detect the expression of DNA repair-related proteins c-JUN, FOSL1, and POLD1 in Ishikawa endometrial cancer cell line. β-actin was used as a loading control. f . Semi-quantitative analysis of protein level in A. g . Western blot was used to detect the expression of DNA repair-related proteins c-JUN, FOSL1, and POLD1 in HEC1A endometrial cancer cell line. β-actin was used as a loading control. h . Semi-quantitative analysis of protein level in C. i . Western blot was performed to determine the effect of PGK1 knockdown on the total intracellular methylation levels (amount of average methylated cytosine). j . Western blot was used to examine the effect of PGK1 knockdown on the expression of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) and SPARC in Ishikawa cancer cell line. β-actin was used as a loading control. k . Semi-quantitative analysis of protein level in F. l . Western blot was used to examine the effect of PGK1 knockdown on the expression of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) and SPARC in HEC1A endometrial cancer cell line. β-actin was used as a loading control. m . Semi-quantitative analysis of protein level in H. Data were shown as mean ± SD based on three independent experiments. *, P

    Article Snippet: Cell transfection shRNA sequence targeting PGK1 and overexpressing plasmid of PGK1 were purchased from GenePharma (Shanghai, China).

    Techniques: Expressing, Methylation, Western Blot

    Gankyrin potentiates mTORC1 signaling via PGK1/AKT. (a) Overexpressed gankyrin activates AKT/mTORC1 signaling in gastric cancer cell lines, MKN45, MKN74, and AGS. The protein levels of PGK1, pAKT, AKT, pS6 K1, S6 K1, p4E-BP1, and 4E-BP1 were analyzed by immunoblotting. (b) The correlation plot of gankyrin and PGK1 mRNA level in 414 gastric cancer samples was presented by analyzing the TCGA cancer genome database.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Gankyrin Drives Malignant Transformation of Gastric Cancer and Alleviates Oxidative Stress via mTORC1 Activation

    doi: 10.1155/2018/9480316

    Figure Lengend Snippet: Gankyrin potentiates mTORC1 signaling via PGK1/AKT. (a) Overexpressed gankyrin activates AKT/mTORC1 signaling in gastric cancer cell lines, MKN45, MKN74, and AGS. The protein levels of PGK1, pAKT, AKT, pS6 K1, S6 K1, p4E-BP1, and 4E-BP1 were analyzed by immunoblotting. (b) The correlation plot of gankyrin and PGK1 mRNA level in 414 gastric cancer samples was presented by analyzing the TCGA cancer genome database.

    Article Snippet: Moreover, the mRNA level of gankyrin and PGK1 was found to be correlated in 414 gastric cancer samples according to the TCGA cancer genome database by Pearson's correlation analysis ( ).

    Techniques:

    Rrp12 is required for the export of pre-40S particles out of the nucleus. ( A to G ) Top panels, epifluorescence microscopy analysis of the subcellular distribution of GFP-tagged Enp1 (A), Dim1 (B), Tsr1 (C), Pno1 (D), Ltv1 (E), Nob1 (F) and Rio2 (G) in the indicated yeast strains and culture conditions (top). Bottom panels, differential interference contrast (DIC) images of the above preparations. ( H ) Western blot analysis showing the distribution of Rio2-GFP (top panel) and Pgk1 (bottom panel) in whole cell lysates (W), cytosolic (C) and nuclear (F) fractions obtained from either control rio2-GFP cells (lanes 1 to 3) or GAL::HA-rrp12/rio2-GFP cells growing in galactose-containing medium (lanes 4 to 6) or upon a shift to glucose-containing medium for 9 hours (lanes 7 to 9).

    Journal: PLoS Genetics

    Article Title: Rrp12 and the Exportin Crm1 Participate in Late Assembly Events in the Nucleolus during 40S Ribosomal Subunit Biogenesis

    doi: 10.1371/journal.pgen.1004836

    Figure Lengend Snippet: Rrp12 is required for the export of pre-40S particles out of the nucleus. ( A to G ) Top panels, epifluorescence microscopy analysis of the subcellular distribution of GFP-tagged Enp1 (A), Dim1 (B), Tsr1 (C), Pno1 (D), Ltv1 (E), Nob1 (F) and Rio2 (G) in the indicated yeast strains and culture conditions (top). Bottom panels, differential interference contrast (DIC) images of the above preparations. ( H ) Western blot analysis showing the distribution of Rio2-GFP (top panel) and Pgk1 (bottom panel) in whole cell lysates (W), cytosolic (C) and nuclear (F) fractions obtained from either control rio2-GFP cells (lanes 1 to 3) or GAL::HA-rrp12/rio2-GFP cells growing in galactose-containing medium (lanes 4 to 6) or upon a shift to glucose-containing medium for 9 hours (lanes 7 to 9).

    Article Snippet: Other antibodies used in Western blot analysis were: anti-MYC (Roche), anti-GFP (Clontech), anti-HA (Covance), anti-Nop1 (Pierce), anti-Mex67 (kind gift of C. Dargemont, Institut Jacques Monod), anti-Rps3 (kind gift of M. Seedorf, University of Heidelberg), anti-Rps8 (kind gift of G. Dieci, University of Parma), anti-Rpl1 (kind gift of F. Lacroute, Centre de Génétique Moléculaire, Gif-sur-Yvette), anti-Pgk1 (Abcam), and anti-Cdc11 (Santa Cruz).

    Techniques: Epifluorescence Microscopy, Western Blot

    Effect of gene deletion on translation and transcription of β-galactosidase mRNA. (A) The relative β-galactosidase activity is determined by normalizing the activity of the mutant strains to that of the WT strain. Blue bars represent β-galactosidase activity under the translational control of URE2 -IRES. Red bars represent β-galactosidase activity via cap-dependent translation. (B) The relative β-galactosidase mRNA level quantified by normalizing the mRNA content of the mutant strains to those in the wild type. The house keeping gene PGK1 was used as an internal control. Deletion of ITT1 or RPS1A had no effect on the normalized β-galactosidase mRNA content. Each experiment was repeated at least three times. Error bars represent standard deviations. * Indicates statistically significant differences (t-test) between WT cells and mutant cells (p

    Journal: PLoS ONE

    Article Title: Heavy metal sensitivities of gene deletion strains for ITT1 and RPS1A connect their activities to the expression of URE2, a key gene involved in metal detoxification in yeast

    doi: 10.1371/journal.pone.0198704

    Figure Lengend Snippet: Effect of gene deletion on translation and transcription of β-galactosidase mRNA. (A) The relative β-galactosidase activity is determined by normalizing the activity of the mutant strains to that of the WT strain. Blue bars represent β-galactosidase activity under the translational control of URE2 -IRES. Red bars represent β-galactosidase activity via cap-dependent translation. (B) The relative β-galactosidase mRNA level quantified by normalizing the mRNA content of the mutant strains to those in the wild type. The house keeping gene PGK1 was used as an internal control. Deletion of ITT1 or RPS1A had no effect on the normalized β-galactosidase mRNA content. Each experiment was repeated at least three times. Error bars represent standard deviations. * Indicates statistically significant differences (t-test) between WT cells and mutant cells (p

    Article Snippet: Mouse anti-Pgk1 (Abcam® ) was used to detect Pgk1p levels [ ].

    Techniques: Activity Assay, Mutagenesis, T-Test

    The relative URE2 mRNA level quantified by normalizing the mRNA content of the mutant strains to those in the wild type. The house keeping gene PGK1 was used as an internal control. Deletion of ITT1 or RPS1A had no effect on the normalized URE2 mRNA content. Each experiment was repeated at least three times. Error bars represent standard deviations.

    Journal: PLoS ONE

    Article Title: Heavy metal sensitivities of gene deletion strains for ITT1 and RPS1A connect their activities to the expression of URE2, a key gene involved in metal detoxification in yeast

    doi: 10.1371/journal.pone.0198704

    Figure Lengend Snippet: The relative URE2 mRNA level quantified by normalizing the mRNA content of the mutant strains to those in the wild type. The house keeping gene PGK1 was used as an internal control. Deletion of ITT1 or RPS1A had no effect on the normalized URE2 mRNA content. Each experiment was repeated at least three times. Error bars represent standard deviations.

    Article Snippet: Mouse anti-Pgk1 (Abcam® ) was used to detect Pgk1p levels [ ].

    Techniques: Mutagenesis

    Polysome-bound mRNA analysis of itt1Δ , rps1aΔ and WT strains. The amount of URE2 mRNA in each fraction was determined by RT-qPCR and the percentage of total URE2 mRNA on the gradient is plotted for each fraction. The profiles of PGK1 mRNA, used as an internal control, were similar for deletion and WT strains and were used to normalize other values. Each experiment was repeated at least three times. Error bars represent standard deviations.

    Journal: PLoS ONE

    Article Title: Heavy metal sensitivities of gene deletion strains for ITT1 and RPS1A connect their activities to the expression of URE2, a key gene involved in metal detoxification in yeast

    doi: 10.1371/journal.pone.0198704

    Figure Lengend Snippet: Polysome-bound mRNA analysis of itt1Δ , rps1aΔ and WT strains. The amount of URE2 mRNA in each fraction was determined by RT-qPCR and the percentage of total URE2 mRNA on the gradient is plotted for each fraction. The profiles of PGK1 mRNA, used as an internal control, were similar for deletion and WT strains and were used to normalize other values. Each experiment was repeated at least three times. Error bars represent standard deviations.

    Article Snippet: Mouse anti-Pgk1 (Abcam® ) was used to detect Pgk1p levels [ ].

    Techniques: Quantitative RT-PCR

    Overexpression of RAD53 in HSP90 -overexpressing cell restores DNA damage hypersensitivity. (A) Percentage survivability of four different strains upon 0.03% MMS treatment plotted as indicated along the x -axis. Average viability ± SD for each strain from five independent experiments. (B) Percentage survivability to UV radiation for the same sets of strains as in A against different UV doses as indicated along the x -axis. (C) Representative Western blots, showing the restoration of Rad51p up-regulation upon treatment with MMS in the WT + p HSP90 + p RAD53 strain compared with the HSP90 -overexpressing strain. The increased level of Rad53p in the RAD53 -overexpressing strain is shown by probing with anti-Flag antibody. –, MMS-untreated fraction; +, fractions treated with 0.05% MMS for 2 h. Pgk1 acts as loading control. Rad53p level was monitored using anti-Flag antibody. (D) Quantification of band intensities from two independent experiments showing that the extent of up-regulation of Rad51p in the wild-type strain (WT + pEMPTY(2)) is similar to that for the WT + p HSP90 + p RAD53 strain. Each lane was normalized against actin. Average band intensities ± SD. (E, F) Real time RT-PCR showing relative abundance of CLN1 and CLN2 transcripts, respectively, for the strains noted along the x -axis. There was no significant change between the levels of G1-cyclins from the 30-min, MMS-treated samples in G1-arrested cells and the 120-min post–MMS treatment samples. (G) Representative cell images for WT, the strain overexpressing RAD53 against the HSP90 -overexpression background (WT + p HSP90 + p RAD53 ), and the RAD53 -overexpressing (WT + p RAD53 ) strain. (H) Average cell size ( y -axis) for each strain type ( x -axis). Sample size > 100. Cell sizes all lie in the same range. Error bar indicates ±SD. *, p

    Journal: Molecular Biology of the Cell

    Article Title: Hsp90 induces increased genomic instability toward DNA-damaging agents by tuning down RAD53 transcription

    doi: 10.1091/mbc.E15-12-0867

    Figure Lengend Snippet: Overexpression of RAD53 in HSP90 -overexpressing cell restores DNA damage hypersensitivity. (A) Percentage survivability of four different strains upon 0.03% MMS treatment plotted as indicated along the x -axis. Average viability ± SD for each strain from five independent experiments. (B) Percentage survivability to UV radiation for the same sets of strains as in A against different UV doses as indicated along the x -axis. (C) Representative Western blots, showing the restoration of Rad51p up-regulation upon treatment with MMS in the WT + p HSP90 + p RAD53 strain compared with the HSP90 -overexpressing strain. The increased level of Rad53p in the RAD53 -overexpressing strain is shown by probing with anti-Flag antibody. –, MMS-untreated fraction; +, fractions treated with 0.05% MMS for 2 h. Pgk1 acts as loading control. Rad53p level was monitored using anti-Flag antibody. (D) Quantification of band intensities from two independent experiments showing that the extent of up-regulation of Rad51p in the wild-type strain (WT + pEMPTY(2)) is similar to that for the WT + p HSP90 + p RAD53 strain. Each lane was normalized against actin. Average band intensities ± SD. (E, F) Real time RT-PCR showing relative abundance of CLN1 and CLN2 transcripts, respectively, for the strains noted along the x -axis. There was no significant change between the levels of G1-cyclins from the 30-min, MMS-treated samples in G1-arrested cells and the 120-min post–MMS treatment samples. (G) Representative cell images for WT, the strain overexpressing RAD53 against the HSP90 -overexpression background (WT + p HSP90 + p RAD53 ), and the RAD53 -overexpressing (WT + p RAD53 ) strain. (H) Average cell size ( y -axis) for each strain type ( x -axis). Sample size > 100. Cell sizes all lie in the same range. Error bar indicates ±SD. *, p

    Article Snippet: We also used anti-DDDDKK tag antibody, which recognizes Flag fusions (Abcam), and anti-Pgk1 antibody (Novus Biologicals) at 1:3000 dilution.

    Techniques: Over Expression, Western Blot, Quantitative RT-PCR

    Heat shock or HSP90 overexpression causes hypersensitivity to MMS and UV radiation. (A) Percentage survivability of cells upon treatment with 0.03% MMS for 2 h for wild-type strain (harboring pRS313 empty vector) at normal temperature 30°C and upon heat shock at 40°C. One set of cells (both untreated and treated fractions) was then shifted to 40°C for 1 h and shifted to 30°C for another 1 h of MMS treatment. Equal numbers of cells were then spread on agar plates. Average of three independent experiments ± SD. (B) Percentage survivability to UV radiation for wild-type strain (pRS313 empty vector) at normal temperature 30°C and upon heat shock at 40°C for different UV doses as indicated along the x -axis. The cells were grown to mid log phase at 30°C. One set was then shifted to 40°C for 1-h heat shock. Equal numbers of cells were spread on agar plates and immediately exposed to various UV doses. Average of three independent experiments ± SD. (C) Percentage survivability of cells upon treatment with MMS was repeated for HSP90 -overexpressing strain pHSP90 , a strain overexpressing G170Dhsp82 mutant, Δrad51 strain, and the isogenic wild-type strain (pEMPTY). Average of five independent experiments ± SD. (D) Percentage survivability to UV radiation for overexpressing HSP90 strain pHSP90 ( CEN ) plotted along with its isogenic wild-type strain (harboring pRS313 empty vector) against different UV doses as indicated along the x -axis. Average of four independent experiments ± SD. (E) Western blot comparing the increased levels of Hsp90 upon heat shock and through CEN plasmid against the wild-type strain. Pgk1 acts as loading control. (F) Quantification of band intensities from three independent batches of cells showing comparable up-regulation of Hsp90 upon heat shock and through overexpression from CEN plasmid. **, p

    Journal: Molecular Biology of the Cell

    Article Title: Hsp90 induces increased genomic instability toward DNA-damaging agents by tuning down RAD53 transcription

    doi: 10.1091/mbc.E15-12-0867

    Figure Lengend Snippet: Heat shock or HSP90 overexpression causes hypersensitivity to MMS and UV radiation. (A) Percentage survivability of cells upon treatment with 0.03% MMS for 2 h for wild-type strain (harboring pRS313 empty vector) at normal temperature 30°C and upon heat shock at 40°C. One set of cells (both untreated and treated fractions) was then shifted to 40°C for 1 h and shifted to 30°C for another 1 h of MMS treatment. Equal numbers of cells were then spread on agar plates. Average of three independent experiments ± SD. (B) Percentage survivability to UV radiation for wild-type strain (pRS313 empty vector) at normal temperature 30°C and upon heat shock at 40°C for different UV doses as indicated along the x -axis. The cells were grown to mid log phase at 30°C. One set was then shifted to 40°C for 1-h heat shock. Equal numbers of cells were spread on agar plates and immediately exposed to various UV doses. Average of three independent experiments ± SD. (C) Percentage survivability of cells upon treatment with MMS was repeated for HSP90 -overexpressing strain pHSP90 , a strain overexpressing G170Dhsp82 mutant, Δrad51 strain, and the isogenic wild-type strain (pEMPTY). Average of five independent experiments ± SD. (D) Percentage survivability to UV radiation for overexpressing HSP90 strain pHSP90 ( CEN ) plotted along with its isogenic wild-type strain (harboring pRS313 empty vector) against different UV doses as indicated along the x -axis. Average of four independent experiments ± SD. (E) Western blot comparing the increased levels of Hsp90 upon heat shock and through CEN plasmid against the wild-type strain. Pgk1 acts as loading control. (F) Quantification of band intensities from three independent batches of cells showing comparable up-regulation of Hsp90 upon heat shock and through overexpression from CEN plasmid. **, p

    Article Snippet: We also used anti-DDDDKK tag antibody, which recognizes Flag fusions (Abcam), and anti-Pgk1 antibody (Novus Biologicals) at 1:3000 dilution.

    Techniques: Over Expression, Plasmid Preparation, Mutagenesis, Western Blot

    Higher abundance of HSP90 mitigates DNA damage–dependent cell cycle arrest. (A) Western blots showing the status of Rad53p phosphorylation in WT and HSP90 -overexpressing (WT + pHSP90 ) strains upon treatment with DNA-damaging agent. The blot represents the protein isolated from both strains before and after treatment with 0.05% MMS for 2 h. The Rad53-phosphorylated form is marked as Rad53*. Actin acts a loading control. (B) Western blot analysis showed up-regulation of Rad51p in WT, but no such up-regulation was observed in WT + pHSP90 strain upon 0.05% MMS treatment (M); also shown are levels of Rad52p in untreated (U) and 0.15% MMS-treated (M) samples in these strains. PGK1 and actin acts as loading controls for monitoring Rad51p and Rad52p levels, respectively. (C) Quantification of band intensities from three independent experiments displayed 2.5-fold up-regulation for Rad51p in the wild-type condition upon MMS treatment, but no such up-regulation is evident in the HSP90 -overexpressing strain. The band intensities in each lane are normalized against PGK1, and mean densities ± SD are plotted. (D) Western blot analysis showing up-regulation of Rad51p and Rad52p in WT and WT + pHSP90 strains from untreated (–) and treated (+) fractions with 3-kJ/m 2 dose of UV radiation followed by growth for 3 h. Actin acts as loading control. (E) Quantification of band intensity from three independent experiments normalized against actin. Mean densities ± SD reveal minimal up-regulation of Rad51p in the HSP90 -overexpressing strain. (F, G) Real-time RT-PCR shows relative abundance of CLN1 and CLN2 transcripts, respectively, for WT (WT + pEMPTY ) and HSP90 -overexpressing strains. The reaccumulation status of both the transcripts was studied by comparing the mRNA levels from one fraction exposed to 0.2% MMS for 30 min (30 min + MMS) with a fraction in which the cells were allowed to grow for 120 min after MMS treatment in fresh medium. (H) Microscopic images showing the relative cell sizes for WT (WT + pEMPTY ) and HSP90 -overexpressing cells. The experiment was performed twice with > 100 cells. Representative images. (I) Different cell sizes observed for the strains, showing that the average cell size for the HSP90 -overexpressing strain was significantly smaller than that of the wild-type strain. The p value was calculated as

    Journal: Molecular Biology of the Cell

    Article Title: Hsp90 induces increased genomic instability toward DNA-damaging agents by tuning down RAD53 transcription

    doi: 10.1091/mbc.E15-12-0867

    Figure Lengend Snippet: Higher abundance of HSP90 mitigates DNA damage–dependent cell cycle arrest. (A) Western blots showing the status of Rad53p phosphorylation in WT and HSP90 -overexpressing (WT + pHSP90 ) strains upon treatment with DNA-damaging agent. The blot represents the protein isolated from both strains before and after treatment with 0.05% MMS for 2 h. The Rad53-phosphorylated form is marked as Rad53*. Actin acts a loading control. (B) Western blot analysis showed up-regulation of Rad51p in WT, but no such up-regulation was observed in WT + pHSP90 strain upon 0.05% MMS treatment (M); also shown are levels of Rad52p in untreated (U) and 0.15% MMS-treated (M) samples in these strains. PGK1 and actin acts as loading controls for monitoring Rad51p and Rad52p levels, respectively. (C) Quantification of band intensities from three independent experiments displayed 2.5-fold up-regulation for Rad51p in the wild-type condition upon MMS treatment, but no such up-regulation is evident in the HSP90 -overexpressing strain. The band intensities in each lane are normalized against PGK1, and mean densities ± SD are plotted. (D) Western blot analysis showing up-regulation of Rad51p and Rad52p in WT and WT + pHSP90 strains from untreated (–) and treated (+) fractions with 3-kJ/m 2 dose of UV radiation followed by growth for 3 h. Actin acts as loading control. (E) Quantification of band intensity from three independent experiments normalized against actin. Mean densities ± SD reveal minimal up-regulation of Rad51p in the HSP90 -overexpressing strain. (F, G) Real-time RT-PCR shows relative abundance of CLN1 and CLN2 transcripts, respectively, for WT (WT + pEMPTY ) and HSP90 -overexpressing strains. The reaccumulation status of both the transcripts was studied by comparing the mRNA levels from one fraction exposed to 0.2% MMS for 30 min (30 min + MMS) with a fraction in which the cells were allowed to grow for 120 min after MMS treatment in fresh medium. (H) Microscopic images showing the relative cell sizes for WT (WT + pEMPTY ) and HSP90 -overexpressing cells. The experiment was performed twice with > 100 cells. Representative images. (I) Different cell sizes observed for the strains, showing that the average cell size for the HSP90 -overexpressing strain was significantly smaller than that of the wild-type strain. The p value was calculated as

    Article Snippet: We also used anti-DDDDKK tag antibody, which recognizes Flag fusions (Abcam), and anti-Pgk1 antibody (Novus Biologicals) at 1:3000 dilution.

    Techniques: Western Blot, Isolation, Quantitative RT-PCR

    Rpd3 deacetylates Rad53 upon HO-induced DSB. (A) RPD3 deletion increases acetylation levels of Rad53 and Rfa1. Wild-type or rpd3 Δ cells expressing Rad53-FLAG, Rfa1-FLAG, or Cdk1-FLAG were lysed and subjected to antiacetyllysine immunoprecipitation and then anti-FLAG immunoblotting to detect protein acetylation (left) or to direct immunoblotting to verify protein expression (right). (B) Same as panel A except that cell lysates were subjected to anti-FLAG immunoprecipitation (IP) and then antiacetyllysine immunoblotting to detect protein acetylation. WB, Western blotting. (C) Rad53 is deacetylated by Rpd3 in vitro . Rad53-FLAG immunoprecipitated from rpd3 Δ cells expressing Rad53-FLAG was mixed with purified Rpd3-GST or deacetylase-deficient Rpd3((H150A; H151A))-GST to carry out in vitro deacetylation. Rad53 acetylation was assessed by antiacetyllysine immunoblotting (top). Anti-FLAG (middle) or anti-GST (bottom) immunoblotting was used to confirm Rad53 or Rpd3 protein levels. 2HA stands for the H150A and H151A double mutations. Ac-K, acetylated lysine. (D) Rpd3 interacts with Rad53. The extracts of untreated or MMS-treated cells expressing Rpd3-HA and/or Rad53-FLAG were subjected to anti-HA immunoprecipitation and then anti-FLAG immunoblotting (top) or to direct immunoblotting to verify protein expression (middle and bottom). (E) Rad53 is deacetylated after HO induction in a Rpd3-dependent manner. Wild-type or rpd3 Δ cells expressing Rad53-FLAG were grown on YEP-lactate medium to an OD 660 of 0.6 to 0.8, and HO endonuclease was induced by 2% galactose. Samples were taken at the indicated times and subjected to antiacetyllysine immunoprecipitation as described above for panel A. Pgk1 is shown as a loading control. (F) Identification of Rad53 Lys22 acetylation by HPLC-MS/MS analysis. The MS/MS spectrum of a dual-charged ion at m/z 1,126.57 for MH22 + corresponding to the mass of the acetylated peptide FLIEK(Ac)FSQEQIGENIVC(carboxyamido)R is shown. All major fragment ions match the predicted b and y ions of the acetylated peptide. (G) Mutation of Lys22 and Lys213 reduces acetylation of Rad53. sml1 Δ rad53 Δ rpd3 Δ cells transfected with the indicated plasmids under the control of the Rad53 promoter were lysed and subjected to antiacetyllysine immunoprecipitation as described above for panel A. (H) K235 of human Chk2 is conserved with K213 of yeast Rad53. The conserved amino acids are highlighted in gray.

    Journal: Molecular and Cellular Biology

    Article Title: Deacetylase Rpd3 Facilitates Checkpoint Adaptation by Preventing Rad53 Overactivation

    doi: 10.1128/MCB.00618-13

    Figure Lengend Snippet: Rpd3 deacetylates Rad53 upon HO-induced DSB. (A) RPD3 deletion increases acetylation levels of Rad53 and Rfa1. Wild-type or rpd3 Δ cells expressing Rad53-FLAG, Rfa1-FLAG, or Cdk1-FLAG were lysed and subjected to antiacetyllysine immunoprecipitation and then anti-FLAG immunoblotting to detect protein acetylation (left) or to direct immunoblotting to verify protein expression (right). (B) Same as panel A except that cell lysates were subjected to anti-FLAG immunoprecipitation (IP) and then antiacetyllysine immunoblotting to detect protein acetylation. WB, Western blotting. (C) Rad53 is deacetylated by Rpd3 in vitro . Rad53-FLAG immunoprecipitated from rpd3 Δ cells expressing Rad53-FLAG was mixed with purified Rpd3-GST or deacetylase-deficient Rpd3((H150A; H151A))-GST to carry out in vitro deacetylation. Rad53 acetylation was assessed by antiacetyllysine immunoblotting (top). Anti-FLAG (middle) or anti-GST (bottom) immunoblotting was used to confirm Rad53 or Rpd3 protein levels. 2HA stands for the H150A and H151A double mutations. Ac-K, acetylated lysine. (D) Rpd3 interacts with Rad53. The extracts of untreated or MMS-treated cells expressing Rpd3-HA and/or Rad53-FLAG were subjected to anti-HA immunoprecipitation and then anti-FLAG immunoblotting (top) or to direct immunoblotting to verify protein expression (middle and bottom). (E) Rad53 is deacetylated after HO induction in a Rpd3-dependent manner. Wild-type or rpd3 Δ cells expressing Rad53-FLAG were grown on YEP-lactate medium to an OD 660 of 0.6 to 0.8, and HO endonuclease was induced by 2% galactose. Samples were taken at the indicated times and subjected to antiacetyllysine immunoprecipitation as described above for panel A. Pgk1 is shown as a loading control. (F) Identification of Rad53 Lys22 acetylation by HPLC-MS/MS analysis. The MS/MS spectrum of a dual-charged ion at m/z 1,126.57 for MH22 + corresponding to the mass of the acetylated peptide FLIEK(Ac)FSQEQIGENIVC(carboxyamido)R is shown. All major fragment ions match the predicted b and y ions of the acetylated peptide. (G) Mutation of Lys22 and Lys213 reduces acetylation of Rad53. sml1 Δ rad53 Δ rpd3 Δ cells transfected with the indicated plasmids under the control of the Rad53 promoter were lysed and subjected to antiacetyllysine immunoprecipitation as described above for panel A. (H) K235 of human Chk2 is conserved with K213 of yeast Rad53. The conserved amino acids are highlighted in gray.

    Article Snippet: Proteins were resolved by 8% SDS-PAGE, and immunoblotting was performed with anti-FLAG M2 monoclonal (Sigma) or anti-Pgk1 (Nordic Immunological Laboratories) antibodies.

    Techniques: Expressing, Immunoprecipitation, Western Blot, In Vitro, Purification, Histone Deacetylase Assay, High Performance Liquid Chromatography, Mass Spectrometry, Mutagenesis, Transfection

    Deacetylation of Rad53 by Rpd3 prevents its overactivation and facilitates adaptation. (A and B) Deletion of RPD3 increases Rad53 kinase activity. Wild-type (A) or rpd3 Δ (B) cells expressing Rad53-FLAG were collected at the indicated times after HO induction and analyzed by anti-FLAG or anti-Pgk1 (loading control) immunoblotting or subjected to an in situ autophosphorylation assay under identical experimental conditions and with identical exposure times. (C and D) Deletion of RPD3 enhances MMS-induced Rad53 phosphorylation. Wild-type or rpd3 Δ cells expressing Rad53-FLAG were treated with 0.02% (C) or 0.05% (D) MMS for the indicated times and analyzed by anti-FLAG or anti-Pgk1 (loading control) immunoblotting. (E and F) The 2KR mutation suppresses Rad53 overactivation in rpd3 Δ cells during the checkpoint response. sml1 Δ rad53 Δ rpd3 Δ cells expressing wild-type Rad53-FLAG (E) or Rad53(2KR)-FLAG (F) under the control of its own promoter were collected at the indicated time points after HO induction. Immunoblotting and in situ autophosphorylation assays were carried out under identical experimental conditions and with identical exposure times. (G) Overexpression of Rad53 delays adaptation. Wild-type JKM179 cells containing either the empty vector or plasmids expressing Rad53 under the control of the GAL1 promoter were grown on SD-Trp medium containing raffinose. HO endonuclease and Rad53 expression were induced by the addition of 2% galactose. At 6 h post-HO induction, a cell aliquot was sonicated and plated onto SD-Trp plates containing 2% raffinose and 2% galactose. Adaptation was monitored at 24 h after HO induction by counting the buds and cells of microcolonies. (H) Elimination of Rad53 acetylation partially rescues the adaptation defect induced by RPD3 deletion. An adaptation assay was performed as described above for panel G. Adaptation was monitored at multiple time points after HO induction. The percentage of cells adapted at each time point was calculated. Error bars reflect the standard deviations of values from 3 independent plates. The asterisks indicate a statistically significant difference between sml1 Δ rad53 Δ rpd3 Δ cells expressing wild-type Rad53 and sml1 Δ rad53 Δ rpd3 Δ cells expressing Rad53(2KR), which was analyzed by a Student t test (∗∗, P

    Journal: Molecular and Cellular Biology

    Article Title: Deacetylase Rpd3 Facilitates Checkpoint Adaptation by Preventing Rad53 Overactivation

    doi: 10.1128/MCB.00618-13

    Figure Lengend Snippet: Deacetylation of Rad53 by Rpd3 prevents its overactivation and facilitates adaptation. (A and B) Deletion of RPD3 increases Rad53 kinase activity. Wild-type (A) or rpd3 Δ (B) cells expressing Rad53-FLAG were collected at the indicated times after HO induction and analyzed by anti-FLAG or anti-Pgk1 (loading control) immunoblotting or subjected to an in situ autophosphorylation assay under identical experimental conditions and with identical exposure times. (C and D) Deletion of RPD3 enhances MMS-induced Rad53 phosphorylation. Wild-type or rpd3 Δ cells expressing Rad53-FLAG were treated with 0.02% (C) or 0.05% (D) MMS for the indicated times and analyzed by anti-FLAG or anti-Pgk1 (loading control) immunoblotting. (E and F) The 2KR mutation suppresses Rad53 overactivation in rpd3 Δ cells during the checkpoint response. sml1 Δ rad53 Δ rpd3 Δ cells expressing wild-type Rad53-FLAG (E) or Rad53(2KR)-FLAG (F) under the control of its own promoter were collected at the indicated time points after HO induction. Immunoblotting and in situ autophosphorylation assays were carried out under identical experimental conditions and with identical exposure times. (G) Overexpression of Rad53 delays adaptation. Wild-type JKM179 cells containing either the empty vector or plasmids expressing Rad53 under the control of the GAL1 promoter were grown on SD-Trp medium containing raffinose. HO endonuclease and Rad53 expression were induced by the addition of 2% galactose. At 6 h post-HO induction, a cell aliquot was sonicated and plated onto SD-Trp plates containing 2% raffinose and 2% galactose. Adaptation was monitored at 24 h after HO induction by counting the buds and cells of microcolonies. (H) Elimination of Rad53 acetylation partially rescues the adaptation defect induced by RPD3 deletion. An adaptation assay was performed as described above for panel G. Adaptation was monitored at multiple time points after HO induction. The percentage of cells adapted at each time point was calculated. Error bars reflect the standard deviations of values from 3 independent plates. The asterisks indicate a statistically significant difference between sml1 Δ rad53 Δ rpd3 Δ cells expressing wild-type Rad53 and sml1 Δ rad53 Δ rpd3 Δ cells expressing Rad53(2KR), which was analyzed by a Student t test (∗∗, P

    Article Snippet: Proteins were resolved by 8% SDS-PAGE, and immunoblotting was performed with anti-FLAG M2 monoclonal (Sigma) or anti-Pgk1 (Nordic Immunological Laboratories) antibodies.

    Techniques: Activity Assay, Expressing, In Situ, Mutagenesis, Over Expression, Plasmid Preparation, Sonication