Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Protective Effects of Scutellarin on Human Cardiac Microvascular Endothelial Cells against Hypoxia-Reoxygenation Injury and Its Possible Target-Related Proteins

doi: 10.1155/2015/278014

Figure Lengend Snippet: Results of Western blotting assay of protein levels of EIF6, HSPD1, and CCT6A in cells of control group, SCU group, HR group, and SCU + HR group shown were representative results of three independent experiments.

Article Snippet: The primary antibody for CCT6A was rabbit polyclonal anti-CCT6 antibody (number ab155541, 1 : 800) bought from Abcam company (Cambridge, USA).

Techniques: Western Blot, Control

Journal: The Journal of Clinical Investigation

Article Title: Upregulated YB-1 protein promotes glioblastoma growth through a YB-1/CCT4/mLST8/mTOR pathway

doi: 10.1172/JCI146536

Figure Lengend Snippet: ( A and B ) Western blot analysis of the molecular markers for mTORC1 and mTORC2 signaling in control or YB-1–knockdown U251 ( A ) and U87 ( B ) cells. ( C and D ) Western blot analysis of mLST8 in control or YB-1–knockdown U251 ( C ) and U87 ( D ) cells. ( E and F ) Western blot analysis of YB-1, mLST8, and mTOR markers in control, YB-1–knockdown cells, or YB-1 knockdown complemented with mLST8 in U251 ( E ) and U87 ( F ) cells. ( G ) RT-qPCR analysis of mLST8 mRNA expression in control or YB-1–knockdown U251 cells. Data are presented as mean ± SEM ( n = 3). ( H ) Western blot analysis of mLST8 in control or YB-1–knockdown U251 cells treated with CHX for the times indicated. Data represent 3 independent experiments. ( I ) Quantification of the relative mLST8 protein levels in H .

Article Snippet: The primary antibodies used for this study are anti-GAPDH (AC033, clone AMC0062), anti-CCT1 (A13364), anti-CCT3 (A6547), anti-CCT4 (A6548), anti-CCT5 (A6549), anti-CCT6 (A3589), anti-CCT7 (A12146), anti-CCT8 (A4449), anti-mLST8 (A1059), anti–pan S6K1 (A16658), anti-p62 (A0682), anti-PCBP1 (A1044), anti-FASN (A0461), anti-G6PD (A1537), anti-TPI1 (A2579), anti-PGAM1 (A4015), anti-DEPTOR (A9447), anti–pan 4EBP1 (A1248), anti–p-4EBP1 (T37/46; AP0030) from ABclonal; anti–hnRNP A1 (sc-32301, clone 4B10), anti–hnRNP LL (sc-132712), anti-CCT2 (sc-374152, clone D-8) from Santa Cruz Biotechnology; anti–YB-1 (Y0396), anti-FLAG (F3165, clone M2), anti-LC3B (L7543), anti–hnRNP L (R4903, clone 4D11) from Sigma-Aldrich; anti–p-S6K1 (T389; 9234S), anti–p-AKT (S473; 9271S), anti–pan AKT (4691P), anti-PKM2 (4053T), anti-RICTOR (2114T), anti-RAPTOR (2280T) from Cell Signaling Technology; anti–pan mTOR (66888-1-Ig, clone 1G11A3) from Proteintech; anti–p-mTOR [S2481; ab137133, clone EPR427(N)] from Abcam; and anti-GFP (11814460001, clones 7.1 and 13.1) from Roche.

Techniques: Western Blot, Control, Knockdown, Quantitative RT-PCR, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Upregulated YB-1 protein promotes glioblastoma growth through a YB-1/CCT4/mLST8/mTOR pathway

doi: 10.1172/JCI146536

Figure Lengend Snippet: ( A ) Immunopurification of mLST8-interacting proteins from U251 cells stably expressing FLAG-tagged mLST8 followed by SDS-PAGE and visualization with silver staining. The major specific interacting proteins are indicated by red dots. ( B ) The components of mTOR and CCT complexes and tubulin proteins were identified by mass spectrometry as mLST8-interacting proteins with high confidence. The percentage of peptide coverage and the number of peptide spectra matched for the protein are shown in the parentheses. ( C ) Immunoprecipitation of CCT components from HEK293T cells transiently transfected with a vector or FLAG-tagged mLST8 expression construct using an anti-FLAG antibody followed by immunoblotting analysis. ( D ) Western blot analysis of CCT components in control or YB-1–knockdown U251 cells. ( E ) Western blot analysis of CCT4 and mLST8 in U251 cells transfected with control or CCT4-specific siRNA. ( F ) RT-qPCR analysis of mLST8 mRNA in U251 cells expressing control or CCT4-specific shRNA. Data are presented as mean ± SEM ( n = 3). ( G ) Western blot analysis of mLST8 in control or CCT4-knockdown U251 cells treated with Baf A1 followed by incubation with thermolysin for the indicated time. Data represent 3 independent experiments. ( H ) Quantitation of G . ( I and J ) Western blot analysis of YB-1, CCT4, mLST8, and the markers for mTORC1 and mTORC2 signaling in control, YB-1–knockdown, or YB-1 knockdown complemented with CCT4 in U251 ( I ) and U87 ( J ) cells.

Article Snippet: The primary antibodies used for this study are anti-GAPDH (AC033, clone AMC0062), anti-CCT1 (A13364), anti-CCT3 (A6547), anti-CCT4 (A6548), anti-CCT5 (A6549), anti-CCT6 (A3589), anti-CCT7 (A12146), anti-CCT8 (A4449), anti-mLST8 (A1059), anti–pan S6K1 (A16658), anti-p62 (A0682), anti-PCBP1 (A1044), anti-FASN (A0461), anti-G6PD (A1537), anti-TPI1 (A2579), anti-PGAM1 (A4015), anti-DEPTOR (A9447), anti–pan 4EBP1 (A1248), anti–p-4EBP1 (T37/46; AP0030) from ABclonal; anti–hnRNP A1 (sc-32301, clone 4B10), anti–hnRNP LL (sc-132712), anti-CCT2 (sc-374152, clone D-8) from Santa Cruz Biotechnology; anti–YB-1 (Y0396), anti-FLAG (F3165, clone M2), anti-LC3B (L7543), anti–hnRNP L (R4903, clone 4D11) from Sigma-Aldrich; anti–p-S6K1 (T389; 9234S), anti–p-AKT (S473; 9271S), anti–pan AKT (4691P), anti-PKM2 (4053T), anti-RICTOR (2114T), anti-RAPTOR (2280T) from Cell Signaling Technology; anti–pan mTOR (66888-1-Ig, clone 1G11A3) from Proteintech; anti–p-mTOR [S2481; ab137133, clone EPR427(N)] from Abcam; and anti-GFP (11814460001, clones 7.1 and 13.1) from Roche.

Techniques: Immu-Puri, Stable Transfection, Expressing, SDS Page, Silver Staining, Mass Spectrometry, Immunoprecipitation, Transfection, Plasmid Preparation, Construct, Western Blot, Control, Knockdown, Quantitative RT-PCR, shRNA, Incubation, Quantitation Assay

Journal: The Journal of Clinical Investigation

Article Title: Upregulated YB-1 protein promotes glioblastoma growth through a YB-1/CCT4/mLST8/mTOR pathway

doi: 10.1172/JCI146536

Figure Lengend Snippet: ( A and B ) Western blot analysis of YB-1, CCT4, mLST8, and mTOR markers in GSCWL1 ( A ) and GSC456 ( B ) cells expressing control shRNA, YB-1–specific shRNA, or YB-1 shRNA supplemented with CCT4 or mLST8. ( C and D ) Cell viability analysis of GSCWL1 ( C ) and GSC456 ( D ) cells described in A and B . Data are presented as mean ± SEM ( n = 3). *** P < 0.001 by 1-way ANOVA followed by Dunnett’s test. ( E and F ) Relative numbers of tumor spheres formed in GSCWL1 ( E ) and GSC456 ( F ) cells described in A and B ( n = 5). *** P < 0.001 by 1-way ANOVA with Dunnett’s test. ( G and H ) In vitro extreme limiting dilution assays were performed in GSCWL1 ( G ) and GSC456 ( H ) cells described in A and B .

Article Snippet: The primary antibodies used for this study are anti-GAPDH (AC033, clone AMC0062), anti-CCT1 (A13364), anti-CCT3 (A6547), anti-CCT4 (A6548), anti-CCT5 (A6549), anti-CCT6 (A3589), anti-CCT7 (A12146), anti-CCT8 (A4449), anti-mLST8 (A1059), anti–pan S6K1 (A16658), anti-p62 (A0682), anti-PCBP1 (A1044), anti-FASN (A0461), anti-G6PD (A1537), anti-TPI1 (A2579), anti-PGAM1 (A4015), anti-DEPTOR (A9447), anti–pan 4EBP1 (A1248), anti–p-4EBP1 (T37/46; AP0030) from ABclonal; anti–hnRNP A1 (sc-32301, clone 4B10), anti–hnRNP LL (sc-132712), anti-CCT2 (sc-374152, clone D-8) from Santa Cruz Biotechnology; anti–YB-1 (Y0396), anti-FLAG (F3165, clone M2), anti-LC3B (L7543), anti–hnRNP L (R4903, clone 4D11) from Sigma-Aldrich; anti–p-S6K1 (T389; 9234S), anti–p-AKT (S473; 9271S), anti–pan AKT (4691P), anti-PKM2 (4053T), anti-RICTOR (2114T), anti-RAPTOR (2280T) from Cell Signaling Technology; anti–pan mTOR (66888-1-Ig, clone 1G11A3) from Proteintech; anti–p-mTOR [S2481; ab137133, clone EPR427(N)] from Abcam; and anti-GFP (11814460001, clones 7.1 and 13.1) from Roche.

Techniques: Western Blot, Expressing, Control, shRNA, In Vitro

Journal: The Journal of Clinical Investigation

Article Title: Upregulated YB-1 protein promotes glioblastoma growth through a YB-1/CCT4/mLST8/mTOR pathway

doi: 10.1172/JCI146536

Figure Lengend Snippet: ( A ) H&E-stained sections of tumor-bearing mouse brains intracranially injected with GSCWL1 cells expressing control shRNA, YB-1–specific shRNA, or YB-1 shRNA supplemented with CCT4 or mLST8. Scale bar: 2 mm. ( B ) Bioluminescence images of tumor-bearing mouse brains described in A . Colored scale bar represents photons/s/cm 2 /steradian. ( C ) Total flux (photons/s) was detected by bioluminescence imaging (BLI) at times indicated in mouse brains described in A . Data are presented as mean ± SEM. *** P < 0.001 by 2-way ANOVA. ( D ) Western blot analysis of YB-1, CCT4, mLST8, and mTOR markers in tumors derived from nude mice intracranially implanted GSCWL1 cells described in A . ( E ) Kaplan-Meier survival curves of nude mice intracranially implanted GSCWL1 cells described in A . *** P < 0.001 by Mantel-Cox log-rank test.

Article Snippet: The primary antibodies used for this study are anti-GAPDH (AC033, clone AMC0062), anti-CCT1 (A13364), anti-CCT3 (A6547), anti-CCT4 (A6548), anti-CCT5 (A6549), anti-CCT6 (A3589), anti-CCT7 (A12146), anti-CCT8 (A4449), anti-mLST8 (A1059), anti–pan S6K1 (A16658), anti-p62 (A0682), anti-PCBP1 (A1044), anti-FASN (A0461), anti-G6PD (A1537), anti-TPI1 (A2579), anti-PGAM1 (A4015), anti-DEPTOR (A9447), anti–pan 4EBP1 (A1248), anti–p-4EBP1 (T37/46; AP0030) from ABclonal; anti–hnRNP A1 (sc-32301, clone 4B10), anti–hnRNP LL (sc-132712), anti-CCT2 (sc-374152, clone D-8) from Santa Cruz Biotechnology; anti–YB-1 (Y0396), anti-FLAG (F3165, clone M2), anti-LC3B (L7543), anti–hnRNP L (R4903, clone 4D11) from Sigma-Aldrich; anti–p-S6K1 (T389; 9234S), anti–p-AKT (S473; 9271S), anti–pan AKT (4691P), anti-PKM2 (4053T), anti-RICTOR (2114T), anti-RAPTOR (2280T) from Cell Signaling Technology; anti–pan mTOR (66888-1-Ig, clone 1G11A3) from Proteintech; anti–p-mTOR [S2481; ab137133, clone EPR427(N)] from Abcam; and anti-GFP (11814460001, clones 7.1 and 13.1) from Roche.

Techniques: Staining, Injection, Expressing, Control, shRNA, Imaging, Western Blot, Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: Upregulated YB-1 protein promotes glioblastoma growth through a YB-1/CCT4/mLST8/mTOR pathway

doi: 10.1172/JCI146536

Figure Lengend Snippet: ( A and B ) MTT analysis of cell growth in U251 ( A ) and U87 ( B ) cells expressing control or YB-1–specific shRNA with or without control or YB-1–specific (YBX1-1) decoy oligonucleotides. Data are presented as mean ± SEM ( n = 3). *** P < 0.001 by 1-way ANOVA followed by Dunnett’s test. ( C ) SDS-PAGE of material pulled down without added oligonucleotides (mock) or with biotinylated scrambled or YB-1–specific (YBX1-2) decoy oligonucleotides followed by silver staining. ( D ) Western blotting for YB-1, hnRNP L, hnRNP LL, hnRNP A1, and PCBP1 in the material pulled down by biotinylated scrambled or YB-1 RNA decoy oligonucleotides from U87 cell extracts. ( E – G ) Western blot analysis of YB-1, CCT4, mLST8, and mTOR markers in U251 ( E ), U87 ( F ), and GSCWL1 ( G ) cells transfected with scrambled or YB-1 decoy oligonucleotides. ( H ) Effects of scrambled or YB-1 decoy oligonucleotides on cell proliferation were tested in GSCWL1 cells. Data are presented as mean ± SEM ( n = 3). *** P < 0.001 by unpaired, 2-tailed Student’s t test. ( I ) In vitro extreme limiting dilution assays were performed in GSCWL1 cells transfected with scrambled or YB-1 decoy oligonucleotides. ( J ) H&E-stained sections of tumor-bearing mouse brains. Tumors were formed by intracranial injection of GSCWL cells transfected with scrambled or YB-1 decoy oligonucleotides (scrambled, n = 8; YBX1-2, n = 7). Scale bar: 2 mm. ( K ) Bioluminescence images of tumor-bearing mouse brains described in J . Colored scale bar represents photons/s/cm 2 /steradian. ( L ) Total flux (photons/s) was determined by bioluminescence imaging (BLI) for the times indicated after intracranial injection of GSCWL1 cells described in J . Data are presented as mean ± SEM. *** P < 0.001 by 2-way ANOVA. ( M ) Kaplan-Meier survival curves of nude mice described in J . P value was determined by Mantel-Cox log-rank test. ( N ) Schematic illustration of the working model.

Article Snippet: The primary antibodies used for this study are anti-GAPDH (AC033, clone AMC0062), anti-CCT1 (A13364), anti-CCT3 (A6547), anti-CCT4 (A6548), anti-CCT5 (A6549), anti-CCT6 (A3589), anti-CCT7 (A12146), anti-CCT8 (A4449), anti-mLST8 (A1059), anti–pan S6K1 (A16658), anti-p62 (A0682), anti-PCBP1 (A1044), anti-FASN (A0461), anti-G6PD (A1537), anti-TPI1 (A2579), anti-PGAM1 (A4015), anti-DEPTOR (A9447), anti–pan 4EBP1 (A1248), anti–p-4EBP1 (T37/46; AP0030) from ABclonal; anti–hnRNP A1 (sc-32301, clone 4B10), anti–hnRNP LL (sc-132712), anti-CCT2 (sc-374152, clone D-8) from Santa Cruz Biotechnology; anti–YB-1 (Y0396), anti-FLAG (F3165, clone M2), anti-LC3B (L7543), anti–hnRNP L (R4903, clone 4D11) from Sigma-Aldrich; anti–p-S6K1 (T389; 9234S), anti–p-AKT (S473; 9271S), anti–pan AKT (4691P), anti-PKM2 (4053T), anti-RICTOR (2114T), anti-RAPTOR (2280T) from Cell Signaling Technology; anti–pan mTOR (66888-1-Ig, clone 1G11A3) from Proteintech; anti–p-mTOR [S2481; ab137133, clone EPR427(N)] from Abcam; and anti-GFP (11814460001, clones 7.1 and 13.1) from Roche.

Techniques: Expressing, Control, shRNA, SDS Page, Silver Staining, Western Blot, Transfection, In Vitro, Staining, Injection, Imaging