Journal: Oncology Letters

Article Title: α-enolase is highly expressed in liver cancer and promotes cancer cell invasion and metastasis

doi: 10.3892/ol.2020.12003

Figure Lengend Snippet: ENO1 expression in liver cancer tissue and peripheral blood of patients with liver cancer. (A) ENO1 expression in liver cancer tissues (×100). (B) ENO1 was not expressed in benign liver lesions (×100). (C) Serum anti-ENO1 antibody levels in patients with liver cancer were higher than those in patients with benign liver lesions and healthy controls. (D) ENO1 expression in liver cancer tissues (×400). (E) ENO1 was not expressed in benign liver lesion (×400). (F) Receiver operating characteristic curve analysis for liver cancer diagnosis using anti-ENO1 antibody levels. The AUC was 0.741, the sensitivity was 64.3% and the specificity was 85.5%. The red dot represents the cut-off value. **P<0.01; ****P<0.0001 ENO1, α-enolase; AUC, area under the curve.

Article Snippet: The anti-ENO1 antibody ELISA detection reagent kit (cat. no. CSB-EQ027775HU) was purchased from CUSABIO Technology LLC.

Techniques: Expressing

Journal: Oncology Letters

Article Title: α-enolase is highly expressed in liver cancer and promotes cancer cell invasion and metastasis

doi: 10.3892/ol.2020.12003

Figure Lengend Snippet: ENO1 expression in pathological tissues.

Article Snippet: The anti-ENO1 antibody ELISA detection reagent kit (cat. no. CSB-EQ027775HU) was purchased from CUSABIO Technology LLC.

Techniques: Expressing

Journal: Oncology Letters

Article Title: α-enolase is highly expressed in liver cancer and promotes cancer cell invasion and metastasis

doi: 10.3892/ol.2020.12003

Figure Lengend Snippet: Comparison of the serum anti-ENO1 antibody levels among the three groups of participants [P50 (P25-P75)].

Article Snippet: The anti-ENO1 antibody ELISA detection reagent kit (cat. no. CSB-EQ027775HU) was purchased from CUSABIO Technology LLC.

Techniques:

Journal: Oncology Letters

Article Title: α-enolase is highly expressed in liver cancer and promotes cancer cell invasion and metastasis

doi: 10.3892/ol.2020.12003

Figure Lengend Snippet: Validation of the siRNA interference effect on ENO1 expression in liver cancer cells. (A) RT-qPCR validation of the effect of siRNA interference on ENO1 expression in HepG2 cells. The results showed that the relative expression levels of the ENO1 gene in the si-1 and si-2 groups were lower than those in the control group. (B) RT-qPCR validation of the effect of siRNA interference on ENO1 expression in Huh7 cells. The results showed that the relative expression levels of the ENO1 gene in the si-1 and si-2 groups were lower than those in the control group. (C) Western blot validation of the effect of siRNA interference on ENO1 expression in HepG2 cells. The results showed that the relative expression levels of the ENO1 protein in HepG2 cells in the si-1 and si-2 groups were significantly lower than those in the control group. (D) Western blot validation of the effect of siRNA interference on ENO1 expression in Huh7 cells. The results showed that the relative expression levels of the ENO1 protein in Huh7 cells in the si-1 and si-2 groups were significantly lower than those in the control group. **P<0.01; ****P<0.0001. siRNA, small interfering RNA; ENO1, α-enolase; si-1, siRNA interference group 1; si-2, siRNA interference group 2; RT-qPCR, reverse transcription-quantitative PCR.

Article Snippet: The anti-ENO1 antibody ELISA detection reagent kit (cat. no. CSB-EQ027775HU) was purchased from CUSABIO Technology LLC.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Small Interfering RNA, Real-time Polymerase Chain Reaction

Journal: Oncology Letters

Article Title: α-enolase is highly expressed in liver cancer and promotes cancer cell invasion and metastasis

doi: 10.3892/ol.2020.12003

Figure Lengend Snippet: Effect of ENO1 siRNA on liver cancer cell proliferation. (A) HepG2 and (B) Huh7 cell proliferation was suppressed after ENO1 siRNA treatment. Results show that 72 h after transfection in the si-1 and si-2 groups, ENO1 siRNA treatment resulted in proliferation inhibition in HepG2 and Huh7 cells. In HepG2 cells, the differences in cell proliferation between the NC and the siRNA-treated groups were statistically significant (P<0.05 vs. si-2 group and P<0.01 vs. si-1 group). Cell proliferation in Huh7 cells was also significantly different. *P<0.05; **P<0.01. ENO1, α-enolase; siRNA, small interfering RNA; si-1, siRNA interference group 1; si-2, siRNA interference group 2; OD, optical density; NC, negative control.

Article Snippet: The anti-ENO1 antibody ELISA detection reagent kit (cat. no. CSB-EQ027775HU) was purchased from CUSABIO Technology LLC.

Techniques: Transfection, Inhibition, Small Interfering RNA, Negative Control

Journal: Oncology Letters

Article Title: α-enolase is highly expressed in liver cancer and promotes cancer cell invasion and metastasis

doi: 10.3892/ol.2020.12003

Figure Lengend Snippet: Effect of ENO1 siRNA on the migration ability of liver cancer cells. (A) Compared with that of the NC group, HepG2 cell migration after ENO1 siRNA treatment became slower in the si-1 and si-2 groups, and the difference was statistically significant at 48 h after treatment. (B) Gap size was measured using ImageJ software and the ratio of gap size after ENO1 siRNA treatment in HepG2 cells was calculated based on the size of the wound at 0 h. (C) Compared with that of the NC group, Huh7 cell migration after ENO1 siRNA treatment became slower in the si-1 and si-2 groups, and the difference was statistically significant at 48 h after treatment. (D) Gap size was measured with the ImageJ software and the ratio of gap size after ENO1 siRNA treatment in Huh7 cell was calculated based on the size of the wound at 0 h. *P<0.05; **P<0.01. ENO1, α-enolase; siRNA, small interfering RNA; NC, negative control; si-1, siRNA interference group 1; si-2, siRNA interference group 2.

Article Snippet: The anti-ENO1 antibody ELISA detection reagent kit (cat. no. CSB-EQ027775HU) was purchased from CUSABIO Technology LLC.

Techniques: Migration, Software, Small Interfering RNA, Negative Control

Journal: Oncology Letters

Article Title: α-enolase is highly expressed in liver cancer and promotes cancer cell invasion and metastasis

doi: 10.3892/ol.2020.12003

Figure Lengend Snippet: Effect of ENO1 siRNA on the invasion and migration abilities of liver cancer cells. Compared with the NC group, the in vitro invasion and migration abilities of (A) HepG2 and (B) Huh7 cells after ENO1 siRNA treatment decreased in the si-1 and si-2 groups, and the differences were statistically significant. Numbers of (C) HepG2 and (D) Huh7 cells were measured with the ImageJ software after ENO1 siRNA treatment. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. ENO1, α-enolase; siRNA, small interfering RNA; NC, negative control; si-1, siRNA interference group 1; si-2, siRNA interference group 2.

Article Snippet: The anti-ENO1 antibody ELISA detection reagent kit (cat. no. CSB-EQ027775HU) was purchased from CUSABIO Technology LLC.

Techniques: Migration, In Vitro, Software, Small Interfering RNA, Negative Control

Journal: Cell Death & Disease

Article Title: Exosome-derived ENO1 regulates integrin α6β4 expression and promotes hepatocellular carcinoma growth and metastasis

doi: 10.1038/s41419-020-03179-1

Figure Lengend Snippet: A The expression level of ENO1 in LO2exo, HepG2exo, MHCC97Lexo, and HCCLM3exo determined by proteomics analysis. B Western blot analysis of ENO1 levels in LO2, HepG2, MHCC97L, and HCCLM3 cells. A representative of three independent experiments is shown. C Differential expression of ENO1 in 94 pairs of HCC and adjacent non-tumor tissues. D IHC staining of ENO1 expression in normal liver tissues, cirrhosis tissues, paired of HCC and non-tumor tissues and metastasis tissues. Representative photographs of ENO1 staining in different tissues are shown. Scale bar represent 800 μm and 100 μm. E Kaplan–Meier analysis of overall survival of 94 HCC patients with different ENO1 expression levels. F Kaplan–Meier analysis of overall survival of 365 HCC patients in the TCGA cohort. G Forest plot showing the association between ENO1 expression and HCC survival as revealed by univariate and multivariate analyses (HR, hazard ratio; CI, confidence interval). All data are represented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 according to two-tailed Student’s t -test.

Article Snippet: Then, primary antibodies against E-cadherin (1:5000, Proteintech, 20874-1-AP), N-cadherin (1:2000, Proteintech, 22018-1-AP), Vimentin (1:2000, Proteintech, 10366-1-AP), MMP2 (1:1000, Abcam, ab92536), MMP9 (1:1000, Abcam, ab76003), GM130 (1:1000, Abcam, ab52649), GRP94 (1:1000, Abcam, ab238126), ALIX (1:10,000, Abcam, ab186429), TSG101 (1:1000, Abcam, ab125011), CD63 (1:1000, Abcam, ab134045), CD81 (1:1000, Abcam, ab109201), ENO1 (1:1000, Abcam, ab155102), HA (1:4000, Abcam, ab9110), integrin α1 (1:500, Proteintech, 22146-1-AP), integrin α2 (1:10,000, Abcam, ab133557), integrin α3 (1:500, Proteintech, 66070-1-Ig), integrin α5 (1:1000, Abcam, ab150361), integrin α6 (1:2000, Abcam, ab181551), integrin αV (1:5,000, Abcam, ab179475), integrin β1 (1:1000, Abcam, ab52971), integrin β3 (1:1000, Abcam, ab119992), integrin β4 (1:1000, Abcam, ab182120), integrin β6 (1:10,000, Abcam, ab187155), FAK (1:2000, Abcam, ab40794), FAK (Y397) (1:1000, Abcam, ab81298), FAK (Y576 + Y577) (1:50,000, Abcam, ab76244), FAK (Y861) (1:10,000, Abcam, ab81293), FAK (Y925) (1:1000, Abcam, ab230813), Src (1:10,000, Abcam, ab109381), Src (Y418) (1:1000, Abcam, ab40660), Src (Y419) (1:5000, Abcam, ab185617), Src (Y529) (1:5000, Abcam, ab32078), p38MAPK (1:1000, Abcam, ab170099), p38MAPK (T180 + Y182) (1:1000, Abcam, ab195049), GAPDH (1:10,000, Abbkine, A01020, USA), and β-tubulin (1:10,000, Abbkine, A01030) were diluted in primary antibody diluent (KeyGEN BioTECH) and incubated with membranes overnight at 4 °C.

Techniques: Expressing, Western Blot, Immunohistochemistry, Staining, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Exosome-derived ENO1 regulates integrin α6β4 expression and promotes hepatocellular carcinoma growth and metastasis

doi: 10.1038/s41419-020-03179-1

Figure Lengend Snippet: Correlations between ENO1 expression and clinicopathological parameters in patients with HCC.

Article Snippet: Then, primary antibodies against E-cadherin (1:5000, Proteintech, 20874-1-AP), N-cadherin (1:2000, Proteintech, 22018-1-AP), Vimentin (1:2000, Proteintech, 10366-1-AP), MMP2 (1:1000, Abcam, ab92536), MMP9 (1:1000, Abcam, ab76003), GM130 (1:1000, Abcam, ab52649), GRP94 (1:1000, Abcam, ab238126), ALIX (1:10,000, Abcam, ab186429), TSG101 (1:1000, Abcam, ab125011), CD63 (1:1000, Abcam, ab134045), CD81 (1:1000, Abcam, ab109201), ENO1 (1:1000, Abcam, ab155102), HA (1:4000, Abcam, ab9110), integrin α1 (1:500, Proteintech, 22146-1-AP), integrin α2 (1:10,000, Abcam, ab133557), integrin α3 (1:500, Proteintech, 66070-1-Ig), integrin α5 (1:1000, Abcam, ab150361), integrin α6 (1:2000, Abcam, ab181551), integrin αV (1:5,000, Abcam, ab179475), integrin β1 (1:1000, Abcam, ab52971), integrin β3 (1:1000, Abcam, ab119992), integrin β4 (1:1000, Abcam, ab182120), integrin β6 (1:10,000, Abcam, ab187155), FAK (1:2000, Abcam, ab40794), FAK (Y397) (1:1000, Abcam, ab81298), FAK (Y576 + Y577) (1:50,000, Abcam, ab76244), FAK (Y861) (1:10,000, Abcam, ab81293), FAK (Y925) (1:1000, Abcam, ab230813), Src (1:10,000, Abcam, ab109381), Src (Y418) (1:1000, Abcam, ab40660), Src (Y419) (1:5000, Abcam, ab185617), Src (Y529) (1:5000, Abcam, ab32078), p38MAPK (1:1000, Abcam, ab170099), p38MAPK (T180 + Y182) (1:1000, Abcam, ab195049), GAPDH (1:10,000, Abbkine, A01020, USA), and β-tubulin (1:10,000, Abbkine, A01030) were diluted in primary antibody diluent (KeyGEN BioTECH) and incubated with membranes overnight at 4 °C.

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: Exosome-derived ENO1 regulates integrin α6β4 expression and promotes hepatocellular carcinoma growth and metastasis

doi: 10.1038/s41419-020-03179-1

Figure Lengend Snippet: A CCK-8 assays, B colony formation assays, C wound healing assays, D transwell migration and invasion assays of HCCLM3 cells after ENO1 knockdown. E Western blot analysis of E-cadherin, N-cadherin and Vimentin levels in HCCLM3 cells after ENO1 knockdown. f CCK-8 assays, G colony formation assays, H transwell migration and invasion assays of MHCC97L and HepG2 cells after ENO1 overexpression. I Western blot analysis of E-cadherin, N-cadherin and Vimentin levels in MHCC97L and HepG2 cells after ENO1 overexpression. A representative of three independent experiments is shown. All data are represented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 according to two-tailed Student’s t -test.

Article Snippet: Then, primary antibodies against E-cadherin (1:5000, Proteintech, 20874-1-AP), N-cadherin (1:2000, Proteintech, 22018-1-AP), Vimentin (1:2000, Proteintech, 10366-1-AP), MMP2 (1:1000, Abcam, ab92536), MMP9 (1:1000, Abcam, ab76003), GM130 (1:1000, Abcam, ab52649), GRP94 (1:1000, Abcam, ab238126), ALIX (1:10,000, Abcam, ab186429), TSG101 (1:1000, Abcam, ab125011), CD63 (1:1000, Abcam, ab134045), CD81 (1:1000, Abcam, ab109201), ENO1 (1:1000, Abcam, ab155102), HA (1:4000, Abcam, ab9110), integrin α1 (1:500, Proteintech, 22146-1-AP), integrin α2 (1:10,000, Abcam, ab133557), integrin α3 (1:500, Proteintech, 66070-1-Ig), integrin α5 (1:1000, Abcam, ab150361), integrin α6 (1:2000, Abcam, ab181551), integrin αV (1:5,000, Abcam, ab179475), integrin β1 (1:1000, Abcam, ab52971), integrin β3 (1:1000, Abcam, ab119992), integrin β4 (1:1000, Abcam, ab182120), integrin β6 (1:10,000, Abcam, ab187155), FAK (1:2000, Abcam, ab40794), FAK (Y397) (1:1000, Abcam, ab81298), FAK (Y576 + Y577) (1:50,000, Abcam, ab76244), FAK (Y861) (1:10,000, Abcam, ab81293), FAK (Y925) (1:1000, Abcam, ab230813), Src (1:10,000, Abcam, ab109381), Src (Y418) (1:1000, Abcam, ab40660), Src (Y419) (1:5000, Abcam, ab185617), Src (Y529) (1:5000, Abcam, ab32078), p38MAPK (1:1000, Abcam, ab170099), p38MAPK (T180 + Y182) (1:1000, Abcam, ab195049), GAPDH (1:10,000, Abbkine, A01020, USA), and β-tubulin (1:10,000, Abbkine, A01030) were diluted in primary antibody diluent (KeyGEN BioTECH) and incubated with membranes overnight at 4 °C.

Techniques: CCK-8 Assay, Migration, Western Blot, Over Expression, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Exosome-derived ENO1 regulates integrin α6β4 expression and promotes hepatocellular carcinoma growth and metastasis

doi: 10.1038/s41419-020-03179-1

Figure Lengend Snippet: A Western blot analysis of ENO1 level in exosomes derived from HCCLM3-NC, HCCLM3-shENO1, MHCC97L-NC, MHCC97L-ENO1, HepG2-NC, and HepG2-ENO1 cells, and HA protein expression in MHCC97L-ENO1exos and HepG2-ENO1exos. B NTA analysis of the impact of altered ENO1 expression in HCC cells on the number of exosomes released. C Fluorescence microscopy analysis of PKH67-labeled exosomes with high ENO1 expression incorporation (green) by HCCLM3-shENO1, MHCC97L-NC, and HepG2-NC cells with relatively low ENO1 expression. Exosomes were isolated from HCCLM3-NC, MHCC97L-ENO1 and HepG2-ENO1 cells. Scale bar represent 10 μm. D Cell CCK-8 assays, E transwell migration and invasion assays of HCCLM3-shENO1, MHCC97L-NC, and HepG2-NC cells educated with HCCLM3-NCexos, MHCC97L-ENO1exos, HepG2-ENO1exos or self-secreted exosomes (50 μg/ml). F Western blot analysis of ENO1, HA, E-cadherin, N-cadherin and Vimentin levels in HCCLM3-shENO1, MHCC97L-NC, and HepG2-NC cells educated with HCCLM3-NCexos, MHCC97L-ENO1exos, HepG2-ENO1exos or self-secreted exosomes (50 μg/ml). G Evaluation of HCC metastasis by tumor size and number of nude mice with lung metastasis in mice injected in the tail vein with HCCLM3-shENO1 and MHCC97L-NC cells following education with HCCLM3-NCexos, MHCC97L-ENO1exos or self-secreted exosomes. For the in vivo experiments, four nude mice per group were used. A representative of three independent experiments is shown. All data are represented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 according to two-tailed Student’s t -test.

Article Snippet: Then, primary antibodies against E-cadherin (1:5000, Proteintech, 20874-1-AP), N-cadherin (1:2000, Proteintech, 22018-1-AP), Vimentin (1:2000, Proteintech, 10366-1-AP), MMP2 (1:1000, Abcam, ab92536), MMP9 (1:1000, Abcam, ab76003), GM130 (1:1000, Abcam, ab52649), GRP94 (1:1000, Abcam, ab238126), ALIX (1:10,000, Abcam, ab186429), TSG101 (1:1000, Abcam, ab125011), CD63 (1:1000, Abcam, ab134045), CD81 (1:1000, Abcam, ab109201), ENO1 (1:1000, Abcam, ab155102), HA (1:4000, Abcam, ab9110), integrin α1 (1:500, Proteintech, 22146-1-AP), integrin α2 (1:10,000, Abcam, ab133557), integrin α3 (1:500, Proteintech, 66070-1-Ig), integrin α5 (1:1000, Abcam, ab150361), integrin α6 (1:2000, Abcam, ab181551), integrin αV (1:5,000, Abcam, ab179475), integrin β1 (1:1000, Abcam, ab52971), integrin β3 (1:1000, Abcam, ab119992), integrin β4 (1:1000, Abcam, ab182120), integrin β6 (1:10,000, Abcam, ab187155), FAK (1:2000, Abcam, ab40794), FAK (Y397) (1:1000, Abcam, ab81298), FAK (Y576 + Y577) (1:50,000, Abcam, ab76244), FAK (Y861) (1:10,000, Abcam, ab81293), FAK (Y925) (1:1000, Abcam, ab230813), Src (1:10,000, Abcam, ab109381), Src (Y418) (1:1000, Abcam, ab40660), Src (Y419) (1:5000, Abcam, ab185617), Src (Y529) (1:5000, Abcam, ab32078), p38MAPK (1:1000, Abcam, ab170099), p38MAPK (T180 + Y182) (1:1000, Abcam, ab195049), GAPDH (1:10,000, Abbkine, A01020, USA), and β-tubulin (1:10,000, Abbkine, A01030) were diluted in primary antibody diluent (KeyGEN BioTECH) and incubated with membranes overnight at 4 °C.

Techniques: Western Blot, Derivative Assay, Expressing, Fluorescence, Microscopy, Labeling, Isolation, CCK-8 Assay, Migration, Injection, In Vivo, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Exosome-derived ENO1 regulates integrin α6β4 expression and promotes hepatocellular carcinoma growth and metastasis

doi: 10.1038/s41419-020-03179-1

Figure Lengend Snippet: A Immunofluorescence quantification of integrin α6 and β4 expression in arbitrary units (a.u.) in MHCC97L-NC and HepG2-NC cells educated with MHCC97L-ENO1exos, HepG2-ENO1exos or self-secreted exosomes (50 μg/ml). Scale bar represent 25 μm. B Western blot analysis of the impact of exosome-derived ENO1 on the expression of integrin α6β4 and the activation of integrin-mediated FAK/Src-p38MAPK pathway in MHCC97L-NC and HepG2-NC cells educated with MHCC97L-ENO1exos, HepG2-ENO1exos or self-secreted exosomes (50 μg/ml). A representative of three independent experiments is shown. All data are represented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 according to two-tailed Student’s t -test.

Article Snippet: Then, primary antibodies against E-cadherin (1:5000, Proteintech, 20874-1-AP), N-cadherin (1:2000, Proteintech, 22018-1-AP), Vimentin (1:2000, Proteintech, 10366-1-AP), MMP2 (1:1000, Abcam, ab92536), MMP9 (1:1000, Abcam, ab76003), GM130 (1:1000, Abcam, ab52649), GRP94 (1:1000, Abcam, ab238126), ALIX (1:10,000, Abcam, ab186429), TSG101 (1:1000, Abcam, ab125011), CD63 (1:1000, Abcam, ab134045), CD81 (1:1000, Abcam, ab109201), ENO1 (1:1000, Abcam, ab155102), HA (1:4000, Abcam, ab9110), integrin α1 (1:500, Proteintech, 22146-1-AP), integrin α2 (1:10,000, Abcam, ab133557), integrin α3 (1:500, Proteintech, 66070-1-Ig), integrin α5 (1:1000, Abcam, ab150361), integrin α6 (1:2000, Abcam, ab181551), integrin αV (1:5,000, Abcam, ab179475), integrin β1 (1:1000, Abcam, ab52971), integrin β3 (1:1000, Abcam, ab119992), integrin β4 (1:1000, Abcam, ab182120), integrin β6 (1:10,000, Abcam, ab187155), FAK (1:2000, Abcam, ab40794), FAK (Y397) (1:1000, Abcam, ab81298), FAK (Y576 + Y577) (1:50,000, Abcam, ab76244), FAK (Y861) (1:10,000, Abcam, ab81293), FAK (Y925) (1:1000, Abcam, ab230813), Src (1:10,000, Abcam, ab109381), Src (Y418) (1:1000, Abcam, ab40660), Src (Y419) (1:5000, Abcam, ab185617), Src (Y529) (1:5000, Abcam, ab32078), p38MAPK (1:1000, Abcam, ab170099), p38MAPK (T180 + Y182) (1:1000, Abcam, ab195049), GAPDH (1:10,000, Abbkine, A01020, USA), and β-tubulin (1:10,000, Abbkine, A01030) were diluted in primary antibody diluent (KeyGEN BioTECH) and incubated with membranes overnight at 4 °C.

Techniques: Immunofluorescence, Expressing, Western Blot, Derivative Assay, Activation Assay, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Exosome-derived ENO1 regulates integrin α6β4 expression and promotes hepatocellular carcinoma growth and metastasis

doi: 10.1038/s41419-020-03179-1

Figure Lengend Snippet: Exosome-derived ENO1 facilitates the expression of integrin α6β4 and the activation of the FAK/Src pathway, supporting downstream signaling via p38MAPK in HCC cells.

Article Snippet: Then, primary antibodies against E-cadherin (1:5000, Proteintech, 20874-1-AP), N-cadherin (1:2000, Proteintech, 22018-1-AP), Vimentin (1:2000, Proteintech, 10366-1-AP), MMP2 (1:1000, Abcam, ab92536), MMP9 (1:1000, Abcam, ab76003), GM130 (1:1000, Abcam, ab52649), GRP94 (1:1000, Abcam, ab238126), ALIX (1:10,000, Abcam, ab186429), TSG101 (1:1000, Abcam, ab125011), CD63 (1:1000, Abcam, ab134045), CD81 (1:1000, Abcam, ab109201), ENO1 (1:1000, Abcam, ab155102), HA (1:4000, Abcam, ab9110), integrin α1 (1:500, Proteintech, 22146-1-AP), integrin α2 (1:10,000, Abcam, ab133557), integrin α3 (1:500, Proteintech, 66070-1-Ig), integrin α5 (1:1000, Abcam, ab150361), integrin α6 (1:2000, Abcam, ab181551), integrin αV (1:5,000, Abcam, ab179475), integrin β1 (1:1000, Abcam, ab52971), integrin β3 (1:1000, Abcam, ab119992), integrin β4 (1:1000, Abcam, ab182120), integrin β6 (1:10,000, Abcam, ab187155), FAK (1:2000, Abcam, ab40794), FAK (Y397) (1:1000, Abcam, ab81298), FAK (Y576 + Y577) (1:50,000, Abcam, ab76244), FAK (Y861) (1:10,000, Abcam, ab81293), FAK (Y925) (1:1000, Abcam, ab230813), Src (1:10,000, Abcam, ab109381), Src (Y418) (1:1000, Abcam, ab40660), Src (Y419) (1:5000, Abcam, ab185617), Src (Y529) (1:5000, Abcam, ab32078), p38MAPK (1:1000, Abcam, ab170099), p38MAPK (T180 + Y182) (1:1000, Abcam, ab195049), GAPDH (1:10,000, Abbkine, A01020, USA), and β-tubulin (1:10,000, Abbkine, A01030) were diluted in primary antibody diluent (KeyGEN BioTECH) and incubated with membranes overnight at 4 °C.

Techniques: Derivative Assay, Expressing, Activation Assay

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Age-dependent Effects of 17β-estradiol on the Dynamics of Estrogen Receptor β (ERβ) Protein–Protein Interactions in the Ventral Hippocampus

doi: 10.1074/mcp.M113.031559

Figure Lengend Snippet: Identified proteins altered by age and E 2

Article Snippet: Membranes were blocked with 5% bovine serum albumin (BSA) for 1 h before the addition of 1° antibody in TBST with 5% BSA and 0.01% NaN 3 for 1.5 h. All 1° antibodies were used at a 1:1000 dilution: VCP (Pierce, PA5–17486), ERβ (Santa Cruz, Sc-8974x), ENO1 (Santa Cruz, sc-15343), GAPDH (Santa Cruz, sc-25778), GELS (Cell Signaling, #8090), HnRNPH (Santa Cruz, sc-15387), HSP70 (GenTex, GTX-104126), and β-actin (Cell Signaling, Aurora, IL, 4970S).

Techniques: Molecular Weight, Binding Assay

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Age-dependent Effects of 17β-estradiol on the Dynamics of Estrogen Receptor β (ERβ) Protein–Protein Interactions in the Ventral Hippocampus

doi: 10.1074/mcp.M113.031559

Figure Lengend Snippet: DeCyder topography, gel image analysis, and average log standard abundance of α-enolase (ENO1) in response to E2 in young and aged animals. For each panel from top left to right, 3 month: YV representative topography, YE representative topography, YV representative gel image, and YE representative gel image. 18 month: AV representative topography, AE representative topography, AV representative gel image, and AE representative gel image. Graph depicts log transformed average abundance normalized to internal standard and matched to master gel. Average calculated from three independent experiments with a biological variance of four pooled animals per experiment (n = 3, BV = 12). *Significance from vehicle, p < 0.05.

Article Snippet: Membranes were blocked with 5% bovine serum albumin (BSA) for 1 h before the addition of 1° antibody in TBST with 5% BSA and 0.01% NaN 3 for 1.5 h. All 1° antibodies were used at a 1:1000 dilution: VCP (Pierce, PA5–17486), ERβ (Santa Cruz, Sc-8974x), ENO1 (Santa Cruz, sc-15343), GAPDH (Santa Cruz, sc-25778), GELS (Cell Signaling, #8090), HnRNPH (Santa Cruz, sc-15387), HSP70 (GenTex, GTX-104126), and β-actin (Cell Signaling, Aurora, IL, 4970S).

Techniques: Transformation Assay

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Age-dependent Effects of 17β-estradiol on the Dynamics of Estrogen Receptor β (ERβ) Protein–Protein Interactions in the Ventral Hippocampus

doi: 10.1074/mcp.M113.031559

Figure Lengend Snippet: Confirmation of ERβ–VCP interaction and subcellular expression of ERβ-interaction partners. A, confirmation of ERβ–VCP interaction in ventral hippocampus by immunoblot with corresponding input and nonspecific IgG control. B, representative immunoblots for nuclear and cytosolic ERβ, HSP70, GAPDH, VCP, HNRNP H, and ENO1 expression in ventral hippocampus normalized to β-actin. C, quantification of densitometric analysis of protein expression calculated from at least three independent experiments (n = 3). *Significant difference between groups (two-way ANOVA, p < 0.05).

Article Snippet: Membranes were blocked with 5% bovine serum albumin (BSA) for 1 h before the addition of 1° antibody in TBST with 5% BSA and 0.01% NaN 3 for 1.5 h. All 1° antibodies were used at a 1:1000 dilution: VCP (Pierce, PA5–17486), ERβ (Santa Cruz, Sc-8974x), ENO1 (Santa Cruz, sc-15343), GAPDH (Santa Cruz, sc-25778), GELS (Cell Signaling, #8090), HnRNPH (Santa Cruz, sc-15387), HSP70 (GenTex, GTX-104126), and β-actin (Cell Signaling, Aurora, IL, 4970S).

Techniques: Expressing, Western Blot