Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: β-PIX plays an important role in regulation of intestinal epithelial restitution by interacting with GIT1 and Rac1 after wounding

doi: 10.1152/ajpgi.00296.2017

Figure Lengend Snippet: β-PIX (p21-activated kinase-interacting exchange factor) and GIT1 (G-protein-coupled receptor kinase-interacting protein 1) protein expression and their interactions in different lines of intestinal epithelial cells (IECs). A: representative immunoblots of β-PIX, GIT1, and Rac1 in IEC-6 (undifferentiated IECs), IEC-Cdx2L1 [differentiated IECs (line of IEC-Cdx2L1)], and Caco-2 cells. Levels of total β-PIX and GIT1 were examined by Western blot analysis, and GAPDH immunoblotting was performed as an internal control for equal loading. B: levels of GIT1 and β-PIX in materials immunoprecipitated (IP) by the anti-β-PIX antibody (Ab) in cells described in A. After whole cell lysates (400 µg) were IP by the specific antibody against β-PIX, precipitates were separated by performing SDS-PAGE gels. Levels of GIT1 and β-PIX were measured using Western blot analysis with specific antibodies. Three separate experiments were performed that showed similar results.

Article Snippet: The β-PIX or GIT1 expression vector that contains the full-length wild-type β-PIX was purchased from Origene Technologies (Rockville, MD; cat. no. SC318985), or GIT1 cDNA was from Addgene (Cambridge, MA; cat. no. 15225), in which β-PIX or GIT1 expression was directed by the pCMV promoter.

Techniques: Expressing, Western Blot, Immunoprecipitation, SDS Page

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: β-PIX plays an important role in regulation of intestinal epithelial restitution by interacting with GIT1 and Rac1 after wounding

doi: 10.1152/ajpgi.00296.2017

Figure Lengend Snippet: Changes in the levels of β-PIX, GIT1, Rac1, and β-PIX/GIT1/Rac1 association after wounding. After IEC-Cdx2L1 cells were grown to confluence, epithelial restitution was induced by removing part of the monolayer, as described in materials and methods. A: levels of β-PIX, GIT1, and Rac1 proteins in total cell lysates isolated at various times after wounding and detected by Western blot analysis. GAPDH immunoblotting was performed as an internal control for equal loading. B: levels of β-PIX, GIT1, and Rac1 proteins in IP materials by the anti-GIT1 Ab from the samples described in A. C: newly synthesized β-PIX and GIT1 proteins as measured by Click-IT protein analysis using l-azidohomoalanine (AHA) in samples described in A. Three experiments were performed that showed similar results.

Article Snippet: The β-PIX or GIT1 expression vector that contains the full-length wild-type β-PIX was purchased from Origene Technologies (Rockville, MD; cat. no. SC318985), or GIT1 cDNA was from Addgene (Cambridge, MA; cat. no. 15225), in which β-PIX or GIT1 expression was directed by the pCMV promoter.

Techniques: Isolation, Western Blot, Synthesized

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: β-PIX plays an important role in regulation of intestinal epithelial restitution by interacting with GIT1 and Rac1 after wounding

doi: 10.1152/ajpgi.00296.2017

Figure Lengend Snippet: β-PIX silencing on β-PIX/GIT1/Rac1 interactions and cell migration. A: representative immunoblots of β-PIX, GIT1 and Rac1. IEC-Cdx2L1 cells were transfected with control siRNA (C-siRNA) or siβ-PIX by LipofectAMINE 2000, and whole cell lysates were harvested 48 h thereafter. Levels of β-PIX, GIT1, and Rac1 proteins were measured by Western immunoblot analysis, and GAPDH immunoblotting was performed as an internal control for equal loading. Three separate experiments were performed that showed similar results. B: quantitative analysis of β-PIX immunoblots by densitometry that were corrected for GAPDH loading from cells described above. Values are means ± SE. * P < 0.05 vs. C-siRNA-transfected cells. C: levels of GIT1, Rac1, and β-PIX proteins in IP materials by the anti-β-PIX Ab from the samples described in A. D: summarized data of cell migration 6 h after wounding in cells transfected with siβ-PIX for 48 h. Data are means ± SE from 6 dishes. *P < 0.05 vs. cells transfected with C-siRNA.

Article Snippet: The β-PIX or GIT1 expression vector that contains the full-length wild-type β-PIX was purchased from Origene Technologies (Rockville, MD; cat. no. SC318985), or GIT1 cDNA was from Addgene (Cambridge, MA; cat. no. 15225), in which β-PIX or GIT1 expression was directed by the pCMV promoter.

Techniques: Migration, Western Blot, Transfection

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: β-PIX plays an important role in regulation of intestinal epithelial restitution by interacting with GIT1 and Rac1 after wounding

doi: 10.1152/ajpgi.00296.2017

Figure Lengend Snippet: Ectopic overexpression of β-PIX on β-PIX/GIT1/Rac1 association and cell migration. A: representative immunoblots of β-PIX and GIT1. Caco-2 cells were transfected with β-PIX expression vector or empty vector (Null) by LipofectAMINE 2000 for 48 and 72 h, and levels of β-PIX and GIT1 proteins were examined by Western immunoblotting analysis. Three separate experiments were performed that showed similar results. B: quantitative analysis of β-PIX immunoblots by densitometry that were corrected for GAPDH loading from cells described above. Values are means ± SE. *P < 0.05 vs. Null cells. C: levels of GIT1, Rac1, and β-PIX proteins in IP materials by the anti-β-PIX Ab from the samples described in A. D: summarized data showing cell migration 6 h after wounding in cells described in A. Values are means ± SE from 6 dishes. *P < 0.05 vs. cells transfected with Null.

Article Snippet: The β-PIX or GIT1 expression vector that contains the full-length wild-type β-PIX was purchased from Origene Technologies (Rockville, MD; cat. no. SC318985), or GIT1 cDNA was from Addgene (Cambridge, MA; cat. no. 15225), in which β-PIX or GIT1 expression was directed by the pCMV promoter.

Techniques: Over Expression, Migration, Western Blot, Transfection, Expressing, Plasmid Preparation

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: β-PIX plays an important role in regulation of intestinal epithelial restitution by interacting with GIT1 and Rac1 after wounding

doi: 10.1152/ajpgi.00296.2017

Figure Lengend Snippet: Changes in levels of β-PIX, GIT1, and Rac1 expression, their association, and cell migration after increasing levels of cellular polyamines. A: changes in β-PIX, GIT1 and Rac1 protein levels in clonal (C) populations of stable line of IECs overexpressing ornithine decarboxylase (ODC) (ODC-IEC) cells and control cells (Vector). IEC-6 cells were infected with either the retroviral vector containing the sequence encoding mouse ODC cDNA or control retroviral vector lacking ODC cDNA. Clones resistant to the selection medium containing 0.6 mg/ml G418 were isolated and screened for ODC expression. Levels of β-PIX, GIT1, and Rac1 proteins were measured by Western blot analysis, and equal loading was monitored by GAPDH immunoblotting. Three separate experiments were performed that showed similar results. B: levels of GIT1, Rac1, and β-PIX proteins in IP materials by the anti-β-PIX Ab from samples described in A. C: summarized data showing cell migration 6 h after wounding in cells described in A. Values are means ± SE of data from 6 dishes. *P < 0.05 vs. Vector cells.

Article Snippet: The β-PIX or GIT1 expression vector that contains the full-length wild-type β-PIX was purchased from Origene Technologies (Rockville, MD; cat. no. SC318985), or GIT1 cDNA was from Addgene (Cambridge, MA; cat. no. 15225), in which β-PIX or GIT1 expression was directed by the pCMV promoter.

Techniques: Expressing, Migration, Plasmid Preparation, Infection, Sequencing, Clone Assay, Selection, Isolation, Western Blot

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: β-PIX plays an important role in regulation of intestinal epithelial restitution by interacting with GIT1 and Rac1 after wounding

doi: 10.1152/ajpgi.00296.2017

Figure Lengend Snippet: Changes in levels of β-PIX, GIT1, and Rac1 proteins, their association, and cell migration after polyamine depletion. A: representative immunoblots of β-PIX, GIT1, and Rac1 proteins. Cells were grown in DMEM containing l-α-difluoromethylornithine (DFMO; 5 mM) alone or DFMO plus putrescine (Put, 10 μM) for 4 days. Levels of β-PIX, GIT1, and Rac1 proteins were measured by Western blot analysis, and equal loading was monitored by GAPDH immunoblotting. Three separate experiments were performed that showed similar results. B: levels of β-PIX, GIT1, and Rac1 proteins in the complex IP by the anti-GIT1 Ab from samples described in A. C: summarized data showing cell migration 6 h after wounding in cells described in A. Values are means ± SE of data from 6 dishes. *P < 0.05 vs. control cells or cells treated with DFMO plus Put.

Article Snippet: The β-PIX or GIT1 expression vector that contains the full-length wild-type β-PIX was purchased from Origene Technologies (Rockville, MD; cat. no. SC318985), or GIT1 cDNA was from Addgene (Cambridge, MA; cat. no. 15225), in which β-PIX or GIT1 expression was directed by the pCMV promoter.

Techniques: Migration, Western Blot

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: β-PIX plays an important role in regulation of intestinal epithelial restitution by interacting with GIT1 and Rac1 after wounding

doi: 10.1152/ajpgi.00296.2017

Figure Lengend Snippet: Effect of β-PIX and GIT1 overexpression on cell migration in polyamine-deficient cells. A: representative immunoblots of β-PIX and GIT1. Cells were exposed to 5 mM DFMO for 2 days and then transfected with either β-PIX or GIT1 expression vector or empty vector (Null) for 48 h in the presence of DFMO. Levels of β-PIX and GIT1 protein levels were measured by Western blot analysis, and equal loading was monitored by GAPDH immunoblotting. Three experiments were performed that showed similar results. B: quantitative analysis of β-PIX immunoblots by densitometry that were corrected for GAPDH loading from cells described above. Values are means ± SE. *P < 0.05 vs. control cells; +P < 0.05 vs. DFMO alone. C: summarized data showing cell migration 6 h after wounding in cells described in A. Values are means ± SE of data from 6 dishes. *P < 0.05 vs. control cells; +P < 0.05 vs. DFMO alone.

Article Snippet: The β-PIX or GIT1 expression vector that contains the full-length wild-type β-PIX was purchased from Origene Technologies (Rockville, MD; cat. no. SC318985), or GIT1 cDNA was from Addgene (Cambridge, MA; cat. no. 15225), in which β-PIX or GIT1 expression was directed by the pCMV promoter.

Techniques: Over Expression, Migration, Western Blot, Transfection, Expressing, Plasmid Preparation

Journal: Bioengineered

Article Title: Inhibition of micro RNA miR-122-5p prevents lipopolysaccharide-induced myocardial injury by inhibiting oxidative stress, inflammation and apoptosis via targeting GIT1

doi: 10.1080/21655979.2021.1926201

Figure Lengend Snippet: Inhibition of micro RNA miR-122-5p on lipopolysaccharide-induced myocardial injury. Wistar rats were intravenously injected with miR-122-5p antagomir, followed by lipopolysaccharide (LPS) stimulation (n = 6 rats per group). (a-b) After LPS treatment for 12 h, heart tissues were harvested for the relative expression levels of miR-122-5p and G-protein-coupled receptor kinase interacting protein-1 (GIT1) using real-time quantitative PCR (RT-qPCR) or western blot assay. (c) The ratio of heart weight/body weight (HW/BW) was calculated. (d) Hematein and eosin (H&E) staining revealed the effect of miR-122-5p on LPS-induced histopathological changes in heart. (e) Levels of cardiac troponin I (cTnI) and lactate dehydrogenase (LDH) were examined by enzyme-linked immunosorbent assay (ELISA). *** p < 0.001 versus control; ## p < 0.01, ### p < 0.001 versus LPS + NC antagomir

Article Snippet: After blocking with 5% nonfat milk for 1 h, the proteins were incubated overnight at 4°C with corresponding primary antibodies against GIT1 (1: 500; Boster, USA), caspase 3 (1: 1000; CST, USA), nuclear factor erythroid 2-related factor 2 (Nrf-2; 1: 500; Affinity, China) and GAPDH (1: 10000; Proteintech, China).

Techniques: Inhibition, Injection, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Staining, Enzyme-linked Immunosorbent Assay, Control

Journal: Bioengineered

Article Title: Inhibition of micro RNA miR-122-5p prevents lipopolysaccharide-induced myocardial injury by inhibiting oxidative stress, inflammation and apoptosis via targeting GIT1

doi: 10.1080/21655979.2021.1926201

Figure Lengend Snippet: In vitro analysis for beneficial role of inhibiting micro RNA miR-122-5p in lipopolysaccharide (LPS)-induced apoptosis. (a-b) Rat H9c2 cells were treated with LPS for 12 h or 24 h, and the expression levels of miR-122-5p and G-protein-coupled receptor kinase interacting protein-1 (GIT1) were assessed by real-time quantitative PCR (RT-qPCR) or western blot analysis. (c-d) H9c2 cells were transfected with NC inhibitor or miR-122-5p inhibitor for 24 h, followed by LPS treatment for another 24 h under proper culture conditions. After that, miR-122-5p and GIT1 expression levels were measured. (e) The contents of cardiac troponin I (cTnI) and lactate dehydrogenase (LDH) were detected by appropriate kits. (f) Flow cytometry showed the apoptosis of LPS-stimulated myocardial cells. (g) Western blot analysis illustrated the changes of caspase-3 expression. ** p < 0.01, *** p < 0.001 versus control; ++ p < 0.01, +++ p < 0.001 versus LPS + NC inhibitor

Article Snippet: After blocking with 5% nonfat milk for 1 h, the proteins were incubated overnight at 4°C with corresponding primary antibodies against GIT1 (1: 500; Boster, USA), caspase 3 (1: 1000; CST, USA), nuclear factor erythroid 2-related factor 2 (Nrf-2; 1: 500; Affinity, China) and GAPDH (1: 10000; Proteintech, China).

Techniques: In Vitro, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Transfection, Flow Cytometry, Control

Journal: Bioengineered

Article Title: Inhibition of micro RNA miR-122-5p prevents lipopolysaccharide-induced myocardial injury by inhibiting oxidative stress, inflammation and apoptosis via targeting GIT1

doi: 10.1080/21655979.2021.1926201

Figure Lengend Snippet: Potential downstream target gene of micro RNA miR-122-5p. H9c2 cells were transfected with NC mimics, miR-122-5p mimics, NC inhibitor and miR-122-5p inhibitor for 48 h. (a-b) The expression levels of miR-122-5p and G-protein-coupled receptor kinase interacting protein-1 (GIT1) were verified by real-time quantitative PCR (RT-qPCR) assay. (c) The predicted binding sites of miR-122-5p in the 3ʹ-UTR of GIT1, and the sequence information of miR-122-5p and GIT1 (wild- or mutant- type) was displayed. (d) Luciferase assay verified the correlation between miR-122-5p and GIT1. aa p < 0.01, aaa p < 0.001 versus NC mimics; bbb p < 0.001 versus NC inhibitor; dd p < 0.01 versus NC mimics + GIT1 3ʹUTR (WT)

Article Snippet: After blocking with 5% nonfat milk for 1 h, the proteins were incubated overnight at 4°C with corresponding primary antibodies against GIT1 (1: 500; Boster, USA), caspase 3 (1: 1000; CST, USA), nuclear factor erythroid 2-related factor 2 (Nrf-2; 1: 500; Affinity, China) and GAPDH (1: 10000; Proteintech, China).

Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Binding Assay, Sequencing, Mutagenesis, Luciferase

Journal: Bioengineered

Article Title: Inhibition of micro RNA miR-122-5p prevents lipopolysaccharide-induced myocardial injury by inhibiting oxidative stress, inflammation and apoptosis via targeting GIT1

doi: 10.1080/21655979.2021.1926201

Figure Lengend Snippet: G-protein-coupled receptor kinase interacting protein-1 (GIT1) deficiency attenuates the effects of micro RNA miR-122-5p loss on myocardial injury. (a) H9c2 cells were transfected with GIT1 siRNA to downregulate GIT1 expression. (b) The cells were transfected with GIT1 siRNA and/or miR-122-5p inhibitor, and then induced by lipopolysaccharide (LPS). GIT1 expression at mRNA and protein levels was then measured using real-time quantitative PCR (RT-qPCR) or western blot. (c) Apoptosis of myocardial cells was analyzed by flow cytometry. (d) Reactive oxygen species (ROS) production was examined using flow cytometry. (e-g) The contents of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and tumor necrosis factor alpha (TNF-α) were assessed by the enzyme-linked immunosorbent assay (ELISA) kits. XXX p < 0.001 versus NC siRNA; ^ p < 0.05, ^^ p < 0.01, ^^^ p < 0.001 versus LPS + miR-122-5p inhibitor + NC siRNA

Article Snippet: After blocking with 5% nonfat milk for 1 h, the proteins were incubated overnight at 4°C with corresponding primary antibodies against GIT1 (1: 500; Boster, USA), caspase 3 (1: 1000; CST, USA), nuclear factor erythroid 2-related factor 2 (Nrf-2; 1: 500; Affinity, China) and GAPDH (1: 10000; Proteintech, China).

Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Bioengineered

Article Title: Inhibition of micro RNA miR-122-5p prevents lipopolysaccharide-induced myocardial injury by inhibiting oxidative stress, inflammation and apoptosis via targeting GIT1

doi: 10.1080/21655979.2021.1926201

Figure Lengend Snippet: G-protein-coupled receptor kinase interacting protein-1 (GIT1) deficiency inhibits nuclear factor erythroid 2-related factor 2 (Nrf-2) activation. (a) Real-time quantitative PCR (RT-qPCR) assay was used to measure the heme oxygenase-1 (HO-1) and NAD(p)H: quinone oxidoreductase 1 (NQO-1) expression. (b) Nuclear Nrf-2 level was revealed using western blot analysis. ^^ p < 0.01 versus LPS + miR-122-5p inhibitor + NC siRNA

Article Snippet: After blocking with 5% nonfat milk for 1 h, the proteins were incubated overnight at 4°C with corresponding primary antibodies against GIT1 (1: 500; Boster, USA), caspase 3 (1: 1000; CST, USA), nuclear factor erythroid 2-related factor 2 (Nrf-2; 1: 500; Affinity, China) and GAPDH (1: 10000; Proteintech, China).

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Western Blot