β actin  (Abcam)

Journal: The Journal of Veterinary Medical Science

Article Title: Intravenous human endothelial progenitor cell administration into aged mice enhances embryo development and oocyte quality by reducing inflammation, endoplasmic reticulum stress and apoptosis

doi: 10.1292/jvms.18-0242

Figure Lengend Snippet: Protein expression levels of PERK, IRE1α and ATF6 in ovaries of aged mice. Representative western blot images (a) and quantification of PERK, IRE1α and ATF6 protein expression levels (b). β-ACTIN served as an internal control. Data are expressed as the mean ± SEM, expressed by error bars.

Article Snippet: The loading and blotting of the amount of protein was verified by reprobing the membrane with anti-β actin antibody (1:5,000; Abcam) followed by Coomassie Blue staining.

Techniques: Expressing, Mouse Assay, Western Blot

Journal: International Journal of Molecular Medicine

Article Title: Gastrodin protects MC3T3-E1 osteoblasts from dexamethasone-induced cellular dysfunction and promotes bone formation via induction of the NRF2 signaling pathway

doi: 10.3892/ijmm.2018.3414

Figure Lengend Snippet: Gastrodin (GSTD) increased osteogenic transcription Runx2, decreased adipogenic factor peroxisome proliferator-activated receptor (PPAR)γ isoform 2 and activated nuclear factor-like 2 (NRF2) pathway protein expression levels. Representative western blot analyses for total protein was analyzed by western blotting in dexamethasone (DEX)-treated MC3T3-E1 cells with or without GSTD pretreatment at different doses. The relative protein expression levels were measured using the fold-change in each protein relative to β-actin from the same sample. The data are expressed as the mean ± standard deviation. ## P

Article Snippet: Rabbit anti-HO-1 (cat. no. ab68477; 1:1,000), rabbit anti-NQO-1 (cat. no. ab76956; 1:1,000), and mouse anti-β-actin (cat. no. ab8245; 1:1,000), Anti-OCN (cat. no. ab93876; 1:1,000) monoclonal antibodies were purchased from Abcam (Cambridge, MA, USA).

Techniques: Expressing, Western Blot, Standard Deviation

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Hirudin Protects Ang II-Induced Myocardial Fibroblasts Fibrosis by Inhibiting the Extracellular Signal-Regulated Kinase1/2 (ERK1/2) Pathway

doi: 10.12659/MSM.909044

Figure Lengend Snippet: Hirudin regulated fibrosis-related factors in Ang II-induced myocardial fibroblasts. ( A ) The relative mRNA levels of MMP-2, MMP-9, TIMP-2 was evaluated by qRT-PCR. ( B ) The relative protein levels of MMP-2, MMP-9, TIMP-2 was examined by western blot. ( C ) qRT-PCR was performed to determine the mRNA levels of FN, TGF-β1, COL-I and COL-III. ( D ) Western blot was carried out to detect the protein levels of FN, TGF-β1, COL-I and COL-III, and normalized to β-actin expression. Gray value was detected and counted by quality one. * P

Article Snippet: The membranes were incubated with anti-matrix metalloproteinase-2 (MMP-2) (AmyJet Scientific, 12015-01, 1: 2000), anti-MMP-9 (R & D, AF909-SP, 1: 1000), anti-tissue inhibitor of metalloproteinases-2 (TIMP-2) (R & D, AF971, 1: 1200), anti-fibronectin (FN) (Abcam, ab2413, 1: 800), anti-transforming growth factor beta 1 (TGF-β1) (R & D, FAB766G, 1: 700), anti-collagen-I (COL-I) (Abcam, ab90395, 1: 1000), anti-collagen III (COL-III) (Abcam, ab7778, 1: 800), anti-p-ERK1/2 (CST, 8544, 1: 1000), anti-ERK1/2 (CST, 9102, 1: 700), and anti-β-actin (Abcam, ab8226, 1: 2000) at 4°C for 24 hours.

Techniques: Quantitative RT-PCR, Western Blot, Expressing

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Hirudin Protects Ang II-Induced Myocardial Fibroblasts Fibrosis by Inhibiting the Extracellular Signal-Regulated Kinase1/2 (ERK1/2) Pathway

doi: 10.12659/MSM.909044

Figure Lengend Snippet: Hirudin downregulated ERK1/2 pathway in Ang II-induced myocardial fibroblasts. ( A ) The proteins expression levels of ERK1/2 and p-ERK1/2 were examined by western blot. ( B ) Quantification of protein expression was carried out with GraphPad prism 7.0. β-actin was used as internal control. The gray value was evaluated and calculated by quality one. * P

Article Snippet: The membranes were incubated with anti-matrix metalloproteinase-2 (MMP-2) (AmyJet Scientific, 12015-01, 1: 2000), anti-MMP-9 (R & D, AF909-SP, 1: 1000), anti-tissue inhibitor of metalloproteinases-2 (TIMP-2) (R & D, AF971, 1: 1200), anti-fibronectin (FN) (Abcam, ab2413, 1: 800), anti-transforming growth factor beta 1 (TGF-β1) (R & D, FAB766G, 1: 700), anti-collagen-I (COL-I) (Abcam, ab90395, 1: 1000), anti-collagen III (COL-III) (Abcam, ab7778, 1: 800), anti-p-ERK1/2 (CST, 8544, 1: 1000), anti-ERK1/2 (CST, 9102, 1: 700), and anti-β-actin (Abcam, ab8226, 1: 2000) at 4°C for 24 hours.

Techniques: Expressing, Western Blot

Journal: Oncology Reports

Article Title: Inhibition of autophagy enhances adenosine-induced apoptosis in human hepatoblastoma HepG2 cells

doi: 10.3892/or.2018.6899

Figure Lengend Snippet: Ado inhibits cell viability and triggers ER stress in HepG2 cells. (A) Time- and dose-dependent cytotoxic effects of Ado on HepG2 cells. Cells were treated with different concentrations of Ado (1.0–4.0 mM) for 12, 24, or 48 h. Cell viability was determined by MTT assay. Results are expressed as percentages of cell growth relative to initial number of viable cells. (B) Cells were treated with 2.0 mM for 6, 12, or 24 h. The cell cycle distribution was evaluated using flow cytometric assay by PI staining. (C) HepG2 cells were treated with 2.0 mM Ado for 24 h. Cells were collected and subjected to total RNA extraction. The mRNA levels of ER stress-related genes GRP78, PERK, ATF4, CHOP, Cyt C and caspase-3 were assessed by RT-PCR. (D) HepG2 cells underwent the aforementioned treatment and were collected and subjected to western blot analyses with specific antibodies directed against GRP78, PERK, ATF4, CHOP, Cyt C and cleaved caspase-3, or β-actin. The density of the corresponding bands was assessed quantitatively using image analysis software and corrected by reference to the value of β-actin. Bar graphs represent the mean fluorescence intensity. *P

Article Snippet: ATF4 (cat. no. ab23760), GRP78 (cat. no. ab21685), PERK (cat. no. ab65142), p-AMPK (cat. no. ab133448), AMPK (cat. no. ab80039), p-ULK1 (cat. no. ab229540), ULK1 (cat. no. ab167139), p-mTOR (cat. no. ab84400), mTOR (cat. no. ab32028), CHOP (cat. no. ab11419), cytochrome c (cat. no. ab110325) and β-actin (cat. no. ab8226) were purchased from Abcam (Cambridge, MA, USA).

Techniques: MTT Assay, Flow Cytometry, Staining, RNA Extraction, Reverse Transcription Polymerase Chain Reaction, Western Blot, Software, Fluorescence

Journal: Oncology Reports

Article Title: Inhibition of autophagy enhances adenosine-induced apoptosis in human hepatoblastoma HepG2 cells

doi: 10.3892/or.2018.6899

Figure Lengend Snippet: Inhibition of autophagy enhances ER stress-mediated apoptotic cell death. (A) HepG2 cells were treated with 2.0 mM Ado or 10 µM LY alone. In the combination treatment group, the cells were pre-treated with 10 µM LY for 2 h, followed by 2.0 mM Ado treatment. Cell viability was determined by MTT assay at the indicated time-points. (B) Cell apoptosis was detected by Hoechst staining. Images were captured under a fluorescence microscope (original magnification, ×200). Arrows indicate apoptotic cells. (C) The mRNA levels of Cyt C, AMPK and p62 were assessed by RT-PCR. (D) HepG2 cells underwent the aforementioned treatment and were collected and subjected to western blot analyses with specific antibodies directed against Cyt C, p-AMPK, AMPK, p62, LC3-I and LC3-II, or β-actin. Bar graphs represent the mean protein band intensity. Relative quantity of the aforementioned proteins was computed. (E) Mitochondrial membrane potential (ΔΨm) indicated by Rhodamine-123 was detected by flow cytometry. Bar graphs represent the mean fluorescence intensity. *P

Article Snippet: ATF4 (cat. no. ab23760), GRP78 (cat. no. ab21685), PERK (cat. no. ab65142), p-AMPK (cat. no. ab133448), AMPK (cat. no. ab80039), p-ULK1 (cat. no. ab229540), ULK1 (cat. no. ab167139), p-mTOR (cat. no. ab84400), mTOR (cat. no. ab32028), CHOP (cat. no. ab11419), cytochrome c (cat. no. ab110325) and β-actin (cat. no. ab8226) were purchased from Abcam (Cambridge, MA, USA).

Techniques: Inhibition, MTT Assay, Staining, Fluorescence, Microscopy, Reverse Transcription Polymerase Chain Reaction, Western Blot, Flow Cytometry, Cytometry

Journal: Oncology Reports

Article Title: Inhibition of autophagy enhances adenosine-induced apoptosis in human hepatoblastoma HepG2 cells

doi: 10.3892/or.2018.6899

Figure Lengend Snippet: Ado induces autophagy in HepG2 cells. (A) HepG2 cells were treated with 2.0 mM for 12, or 24 h and cells were stained with MDC. The autophagosomes were detected under fluorescence microscopy (magnification, ×200). (B) The mRNA levels of autophagy-related genes Beclin-1 and p62 were assessed by RT-PCR. (C) HepG2 cells underwent the aforementioned treatment and were collected and subjected to western blot analyses with specific antibodies directed against LC3-I, LC3-II, Beclin-1, p62 or β-actin. Bar graphs represent the mean protein band intensity. Relative quantity of the aforementioned proteins was computed after normalization with β-actin. *P

Article Snippet: ATF4 (cat. no. ab23760), GRP78 (cat. no. ab21685), PERK (cat. no. ab65142), p-AMPK (cat. no. ab133448), AMPK (cat. no. ab80039), p-ULK1 (cat. no. ab229540), ULK1 (cat. no. ab167139), p-mTOR (cat. no. ab84400), mTOR (cat. no. ab32028), CHOP (cat. no. ab11419), cytochrome c (cat. no. ab110325) and β-actin (cat. no. ab8226) were purchased from Abcam (Cambridge, MA, USA).

Techniques: Staining, Fluorescence, Microscopy, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Oncology Reports

Article Title: Inhibition of autophagy enhances adenosine-induced apoptosis in human hepatoblastoma HepG2 cells

doi: 10.3892/or.2018.6899

Figure Lengend Snippet: Inhibition of autophagy by AMPK siRNA enhances Ado-induced apoptosis and inhibits the AMPK/mTOR/ULK1 pathway. (A) HepG2 cells were transfected with AMPK siRNA (20 µM) or control siRNA (transfected with empty vector, 20 µM), then exposed to 2.0 mM Ado or not for 24 h and then the cell apoptotic ratio was determined by flow cytometry (FACS) with Annexin V-FITC and PI double staining. (B) HepG2 cells underwent the aforementioned treatment and were collected and the protein levels of the AMPK/mTOR/ULK1 pathway were assessed by western blotting. The phosphorylation ratios of p-AMPK/AMPK, p-ULK1/ULK1 and p-mTOR/mTOR were computed (C) The mRNA and protein levels of ER stress-related genes were assessed by RT-PCR and western blotting. HepG2 cells underwent the aforementioned treatment and were collected and subjected to western blot analyses with specific antibodies directed against CHOP, cleaved caspase-3, p62 and Cyt C or β-actin. Bar graphs represent the mean protein band intensity. (D) Mitochondrial membrane potential (ΔΨm) indicated by Rhodamine-123 was detected by flow cytometry. Bar graphs represent the mean fluorescence intensity. *P

Article Snippet: ATF4 (cat. no. ab23760), GRP78 (cat. no. ab21685), PERK (cat. no. ab65142), p-AMPK (cat. no. ab133448), AMPK (cat. no. ab80039), p-ULK1 (cat. no. ab229540), ULK1 (cat. no. ab167139), p-mTOR (cat. no. ab84400), mTOR (cat. no. ab32028), CHOP (cat. no. ab11419), cytochrome c (cat. no. ab110325) and β-actin (cat. no. ab8226) were purchased from Abcam (Cambridge, MA, USA).

Techniques: Inhibition, Transfection, Plasmid Preparation, Flow Cytometry, Cytometry, FACS, Double Staining, Western Blot, Reverse Transcription Polymerase Chain Reaction, Fluorescence