Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Microtubule nucleation in mouse bone marrow-derived mast cells is regulated by the concerted action of GIT1/βPIX proteins and calcium.

doi: 10.4049/jimmunol.1402459

Figure Lengend Snippet: FIGURE 2. Subcellular localization of GFP-tagged bPIX and GIT1 in BMMCLs. Mitotic BMMCL-gTb stained for a-tubulin (A) to visua- lize localization of TagRFP-tagged g-tubulin (B). (C) Superposition of images (a-tubulin, green; g-tubulin, red), methanol-fixed cells. Locali- zation of bPIX-GFP (D) and g-tubulin–TagRFP (E) in live BMMCL-gTb cells. (F) Superposition of images (bPIX, green; g-tubulin, red). Local- ization of GIT1-GFP (G) and g-tubulin–TagRFP (H) in live BMMCL- gTb. (I) Superposition of images (GIT1, green; g-tubulin, red). Locali- zation of control mitochondrial Mito-GFP (J) and g-tubulin-TagRFP (K) in live BMMCL-gTb. (L) Superposition of images (Mito-GFP, green; g-tubulin, red). In (D)–(L), the best centrosomal plane is shown. Scale bars, 5 mm.

Article Snippet: A cassette encoding mouse bPIX (mbPIX; Arhgef7, tv3; RefSeq ID: NM_017402.4) fused to GFP was amplified from the TurboGFP-tagged Arhgef7 cDNA ORF clone (MG223397; OriGene Technologies) using forward 59-TATGCTAGCGTCGACTGGATCCGG-39 and reverse 59-GCCGGGAATTCGTTTAAACTCTTTC-39 primers.

Techniques: Staining, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Microtubule nucleation in mouse bone marrow-derived mast cells is regulated by the concerted action of GIT1/βPIX proteins and calcium.

doi: 10.4049/jimmunol.1402459

Figure Lengend Snippet: FIGURE 5. Effect of Ca2+ on bPIX and GIT1 binding to the C-terminal domain of g-tubulin. (A) bPIX and GIT1 interact with whole-length g-tubulin but not with its truncated form lacking a C-terminal region. Extracts from HEK cells expressing either GFP-tagged whole-length (aa 1–451; lanes 1 and 3) or truncated (aa 1–382; lanes 2 and 4) g-tubulin were precipitated with Protein A–immobilized Abs specific to bPIX or GIT1. Blots were probed with Ab to GFP. Loads (lanes 1 and 2), immu- noprecipitated proteins (lanes 3 and 4). (B) Binding of bPIX to the C-terminal region of g-tubulin is affected by Ca2+. Extracts from BMMCLs (Load) were incubated in the presence or absence of 0.8 mM Ca2+ with the GST-tagged C-terminal region of g-tubulin (aa 362–451) or GST alone immobilized on glutathione-Sepharose beads. Blots of bound proteins were probed with Abs to bPIX, GIT1, actin, or GST. Lines on the left indicate the positions of molecular mass markers (in kDa).

Article Snippet: A cassette encoding mouse bPIX (mbPIX; Arhgef7, tv3; RefSeq ID: NM_017402.4) fused to GFP was amplified from the TurboGFP-tagged Arhgef7 cDNA ORF clone (MG223397; OriGene Technologies) using forward 59-TATGCTAGCGTCGACTGGATCCGG-39 and reverse 59-GCCGGGAATTCGTTTAAACTCTTTC-39 primers.

Techniques: Binding Assay, Expressing, Incubation

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