Journal: Molecular medicine reports

Article Title: miR-195 inhibits the proliferation and migration of chondrocytes by targeting GIT1.

doi: 10.3892/mmr.2016.5982

Figure Lengend Snippet: Figure 1. Prediction of miR‑195 target site in GIT1 3'‑UTR. (A) miR‑195 target sites in the conservative sequence of the GIT1 3'‑UTR were identified using common bioinformatic algorithms (miRanda, TargetScanHuman, miRBase and miRWalk), as indicated by capital letters. The putative miR‑195 binding site was mutated (underlined nucleotides). (B) Luciferase expression in HEK293A cells transfected with vectors containing the wt GIT1 3'‑UTR or mu GIT1 3'‑UTR together with miR‑195 mimics or controls. (C) The Ago2 monoclonal antibody was immobilized on Protein‑G‑Sepharose beads and incubated with HEK 293 cell lysates obtained following transfection with miR‑195 mimics. After stringent washing, co‑immunoprecipitated Ago‑bound RNAs were subject to RT‑qPCR to detect GIT1 mRNA expression. Data are presented as the mean ± standard deviation (n=3). *P<0.01 vs. the controls. miR‑195, microRNA‑195; GIT1, G‑protein‑coupled receptor kinase interacting protein‑1; UTR, untranslated region; RT‑qPCR, reverse transcription‑quantitative polymerase chain reaction; NC, negative control miRNA transfection group; wt, wild‑type; mu, mutant.

Article Snippet: Membranes were blocked with 5% non-fat milk for 1 h at room temperature and then incubated with antibodies against GIT1 (cat. no. 2919; dilution, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) or GAPDH (cat. no. 2118; 1:1,000; Cell Signaling Technology, Inc.) in 5% non-fat milk overnight at 4 ̊C.

Techniques: Sequencing, Binding Assay, Luciferase, Expressing, Transfection, Incubation, Standard Deviation, Polymerase Chain Reaction, Negative Control, Mutagenesis

Journal: Molecular medicine reports

Article Title: miR-195 inhibits the proliferation and migration of chondrocytes by targeting GIT1.

doi: 10.3892/mmr.2016.5982

Figure Lengend Snippet: Figure 3. Role of miR‑195 and GIT1 in chondrocyte proliferation. Chondrocytes were transfected with GIT1‑expression vectors, miR‑195 mimics, miR‑195 inhibitors or GIT1 siRNA and cultured for 72 h. Cell proliferation was measured using a BrdU assay. The results are presented as the mean ± standard deviation, (n=3). *P<0.01 vs. the control. miR‑195, microRNA‑195; GIT1, G‑protein‑coupled receptor kinase interacting pro tein‑1; NC, negative control; UTR, untranslated region; wt, wild‑type; mut, mutant; siRNA, small‑interfering RNA.

Article Snippet: Membranes were blocked with 5% non-fat milk for 1 h at room temperature and then incubated with antibodies against GIT1 (cat. no. 2919; dilution, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) or GAPDH (cat. no. 2118; 1:1,000; Cell Signaling Technology, Inc.) in 5% non-fat milk overnight at 4 ̊C.

Techniques: Transfection, Cell Culture, BrdU Staining, Standard Deviation, Control, Negative Control, Mutagenesis, Small Interfering RNA

Journal: Molecular medicine reports

Article Title: miR-195 inhibits the proliferation and migration of chondrocytes by targeting GIT1.

doi: 10.3892/mmr.2016.5982

Figure Lengend Snippet: Figure 2. miR‑195 inhibits the expression of GIT1 in chondrocytes. Human chondrocytes were transfected with miR‑195 mimics or inhibitors. The mRNA expression levels of (A) miR‑195 and (B) GIT1 were measured by RT‑qPCR analysis. (C) Western blot analysis and (D) quantification of band intensities of GIT1 protein expression in chondrocytes following transfection with miR‑195 mimics or inhibitors. GAPDH was used as a loading control. Experiments were performed at least in triplicate, and the results are expressed as the mean ± standard deviation. *P<0.01 vs. the control. miR‑195, microRNA‑195; GIT1, G‑protein‑coupled receptor kinase interacting protein‑1; RT‑qPCR, reverse transcription‑quantitative polymerase chain reaction; NC, negative control.

Article Snippet: Membranes were blocked with 5% non-fat milk for 1 h at room temperature and then incubated with antibodies against GIT1 (cat. no. 2919; dilution, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) or GAPDH (cat. no. 2118; 1:1,000; Cell Signaling Technology, Inc.) in 5% non-fat milk overnight at 4 ̊C.

Techniques: Expressing, Transfection, Western Blot, Control, Standard Deviation, Polymerase Chain Reaction, Negative Control

Journal: Molecular medicine reports

Article Title: miR-195 inhibits the proliferation and migration of chondrocytes by targeting GIT1.

doi: 10.3892/mmr.2016.5982

Figure Lengend Snippet: Figure 4. Role of miR‑195 and GIT1 in chondrocyte migration. Cell migration capabilities were analyzed using a transwell assay. (A) Light microscope images of chondrocytes transfected with GIT1‑overexpressing vectors, miR‑195a‑3p mimics, miR‑195a‑3p inhibitors or GIT1 siRNA, that were seeded onto matrigel‑coated filters of cell culture inserts and incubated for 24 h. The images shown are representative of three independent experiments (magnification, x200). Cells that had migrated to the underside of the filters toward FBS‑containing media were counted. (B) Quantitative analysis of the effects of miR‑195 and/or GIT1 expression on chondrocyte migration capabilities. Data are presented as the mean ± standard deviation (n=3). *P<0.01 vs. the control. miR‑195, microRNA‑195; GIT1, G‑protein‑coupled receptor kinase interacting protein‑1; FBS, fetal bovine serum; siRNA, small interfering RNA; NC, negative control.

Article Snippet: Membranes were blocked with 5% non-fat milk for 1 h at room temperature and then incubated with antibodies against GIT1 (cat. no. 2919; dilution, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) or GAPDH (cat. no. 2118; 1:1,000; Cell Signaling Technology, Inc.) in 5% non-fat milk overnight at 4 ̊C.

Techniques: Migration, Transwell Assay, Light Microscopy, Transfection, Cell Culture, Incubation, Expressing, Standard Deviation, Control, Small Interfering RNA, Negative 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: 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

Journal: Neural Regeneration Research

Article Title: GIT1 enhances neurite outgrowth by stimulating microtubule assembly

doi: 10.4103/1673-5374.179054

Figure Lengend Snippet: GIT1 was highly expressed in the nervous system. (A) Expression profiles of Git1 and Git2 in 2-month-old mice, as assessed by RT-PCR. Ctrl: Negative control. (B) The relative mRNA expression levels of Git1 and Git2 in 2-month-old mice, normalized to Gapdh . The data are expressed as optical density ratio of Git / Gapdh ( n = 3; data are given as mean ± SEM). (C) GIT1 protein expression in the whole brain from embryonic day (E) 10.5 to postnatal day (P) 60. (D) GIT1 expression in the mouse hippocampus at P0, 7, 14, 30 and 60. Quantification of GIT1 expression in the whole brain (E) and hippocampus (F) were normalized to β-actin. Data represent the mean ± SEM of three independent observations.

Article Snippet: After blocking in milk, the blots were incubated in primary antibody against GIT1 (goat, 1:800; Santa Cruz Biotechnology), Clasp2 (rabbit, 1:600; Biorbyt), CRMP2 (rabbit, 1:1,000; Proteintech, Chicago IL, USA), MAP2 (rabbit, 1:1,000; Cell Signaling Technology), Tuj1 (rabbit, 1:1,000; Sigma-Aldrich), TUBGCP3 (rabbit, 1:1,000; Proteintech), MAP1B (rabbit, 1:1,000; Proteintech), Tau (rabbit, 1:1,000; Sigma-Aldrich) or beta-actin (mouse, 1:2,000; Millipore, Darmstadt, Germany) at 4°C overnight.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Negative Control

Journal: Neural Regeneration Research

Article Title: GIT1 enhances neurite outgrowth by stimulating microtubule assembly

doi: 10.4103/1673-5374.179054

Figure Lengend Snippet: Neurite growth was impaired in GIT1 knockout neurons. (A) Hippocampal neurons isolated from wild type (WT), GIT1 heterozygote (HET) and GIT1 knockout (KO) mice at postnatal day (P) 0 were cultured for 5 days in control medium (left) or medium containing 50 ng/mL nerve growth factor (NGF) (right). Neurons were immunostained with anti-Tuj1 (green) antibody and DAPI (blue). Scale bar: 20 μm. Quantification of total neurite length per neuron (B) and average length of axon-like processes (D) of WT, HET and KO hippocampal neurons in vitro . The effects of NGF treatment are shown in Figure D. n = 30–50 cells; data are expressed as the mean ± SEM. The experiments were repeated three times. (C) Western blot of lysates from P0 WT and HET hippocampal neurons cultured for 5 days in the presence of 0, 25 or 50 ng/mL NGF. Anti-GIT1 and anti-actin antibodies were used to detect target protein expression. * P < 0.05, ** P < 0.01, *** P < 0.001. One-way analysis of variance followed by Tukey's test.

Article Snippet: After blocking in milk, the blots were incubated in primary antibody against GIT1 (goat, 1:800; Santa Cruz Biotechnology), Clasp2 (rabbit, 1:600; Biorbyt), CRMP2 (rabbit, 1:1,000; Proteintech, Chicago IL, USA), MAP2 (rabbit, 1:1,000; Cell Signaling Technology), Tuj1 (rabbit, 1:1,000; Sigma-Aldrich), TUBGCP3 (rabbit, 1:1,000; Proteintech), MAP1B (rabbit, 1:1,000; Proteintech), Tau (rabbit, 1:1,000; Sigma-Aldrich) or beta-actin (mouse, 1:2,000; Millipore, Darmstadt, Germany) at 4°C overnight.

Techniques: Knock-Out, Isolation, Cell Culture, Control, In Vitro, Western Blot, Expressing

Journal: Neural Regeneration Research

Article Title: GIT1 enhances neurite outgrowth by stimulating microtubule assembly

doi: 10.4103/1673-5374.179054

Figure Lengend Snippet: Axonal growth in GIT1 knockout (KO) hippocampal neurons and the effects of overexpression of GIT1 or GIT1 deletion constructs. (A) Schematic diagram of the full-length GIT1 and different GIT1 deletion constructs: cGIT1, CDΔAS/SLD, nGIT1 and GIT1ΔSLD. The Flag-tag was attached to the N terminal of the constructs. (B) KO hippocampal neurons were cultured for 5 days after transfection with the constructs, and then stained with anti-Flag (green) and anti-Tuj1 (red) antibodies and DAPI (blue). Scale bars: 20 μm. (C) Average length of axon-like processes in KO hippocampal neurons cultured for 5 days after transfection with the GIT1 deletion constructs. Cells were analyzed using ImageJ software. n = 3 cells; data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. One-way analysis of variance followed by Tukey's test. The experiments were repeated three times. Con: Control.

Article Snippet: After blocking in milk, the blots were incubated in primary antibody against GIT1 (goat, 1:800; Santa Cruz Biotechnology), Clasp2 (rabbit, 1:600; Biorbyt), CRMP2 (rabbit, 1:1,000; Proteintech, Chicago IL, USA), MAP2 (rabbit, 1:1,000; Cell Signaling Technology), Tuj1 (rabbit, 1:1,000; Sigma-Aldrich), TUBGCP3 (rabbit, 1:1,000; Proteintech), MAP1B (rabbit, 1:1,000; Proteintech), Tau (rabbit, 1:1,000; Sigma-Aldrich) or beta-actin (mouse, 1:2,000; Millipore, Darmstadt, Germany) at 4°C overnight.

Techniques: Knock-Out, Over Expression, Construct, FLAG-tag, Cell Culture, Transfection, Staining, Software, Control

Journal: Neural Regeneration Research

Article Title: GIT1 enhances neurite outgrowth by stimulating microtubule assembly

doi: 10.4103/1673-5374.179054

Figure Lengend Snippet: GIT1 interacted with tubulins and microtubule-associated proteins, and stimulated microtubule assembly. (A) Brain extract from 8-week-old mice was immunoprecipitated with the GIT1 antibody, and western blots were probed by antibodies against CRMP2, Clasp2, α-tubulin, Tuj1, TUBGCP3, MAP2, Tau, MAP1B and GIT1. The images show the colocalization of Tuj1 (red) and GIT1 (green) (B), Clasp2 (red) and GIT1 (green) (C), and MAP2 (red) and GIT1 (green) (D) in primary cultured hippocampal neurons on postnatal day (P) 0. Scale bars: 20 μm. (E) Expression levels of Tuj1, MAP2 and GIT1 in the cortex and hippocampus of wild-type (WT) and GIT1 heterozygote (HET) mice on postnatal day (P) 0, P15 and P30. (F) GIT1 activates tubulin polymerization in vitro . Result represents means of three independent observations. min: Minutes.

Article Snippet: After blocking in milk, the blots were incubated in primary antibody against GIT1 (goat, 1:800; Santa Cruz Biotechnology), Clasp2 (rabbit, 1:600; Biorbyt), CRMP2 (rabbit, 1:1,000; Proteintech, Chicago IL, USA), MAP2 (rabbit, 1:1,000; Cell Signaling Technology), Tuj1 (rabbit, 1:1,000; Sigma-Aldrich), TUBGCP3 (rabbit, 1:1,000; Proteintech), MAP1B (rabbit, 1:1,000; Proteintech), Tau (rabbit, 1:1,000; Sigma-Aldrich) or beta-actin (mouse, 1:2,000; Millipore, Darmstadt, Germany) at 4°C overnight.

Techniques: Immunoprecipitation, Western Blot, Cell Culture, Expressing, In Vitro

Journal: Journal of Cellular and Molecular Medicine

Article Title: Decreased long non‐coding RNA lincFOXF1 indicates poor progression and promotes cell migration and metastasis in osteosarcoma

doi: 10.1111/jcmm.15828

Figure Lengend Snippet: GIT1 is a potential target of lincFOXF1 and inversely correlated with lincFOXF1 expression in osteosarcoma tissues. (A) and (B) qRT‐PCR was used to detect expression of cell adhesion and extracellular matrix molecules, including N‐cadherin, Intergrin, CD44, ICAM‐1, Vimentin, Fibronectin, GIT1, and host gene FOXF1 after lincFOXF1 was knocked down or overexpressed in MNNG‐HOS cells. C, Western blot analyses were performed to confirm the gene expression in lincFOXF1‐overexpressing 143B cells or knocked down MNNG‐HOS cells compared with control cells. D, The inverse correlation between lincFOXF1 expression (ΔCt value) and GIT1 expression (ΔCt value) was analysed by qRT‐PCR in osteosarcoma tissues. All experiments were performed in triplicate. Bars: SD; * P < 0.05 and ** P < 0.01

Article Snippet: The membranes were then blocked in 5% non‐fat dry milk and incubated with the antibodies against GIT1 (1:1000, Abcam, ab153958) and GAPDH (1:5000, Bioworld, #AP0066).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Gene Expression, Control

Journal: bioRxiv

Article Title: Proximity based proteomics reveals Git1 as a regulator of Smoothened signaling

doi: 10.1101/2025.01.06.631593

Figure Lengend Snippet: (A) Relative Git1 intensity in the Smo-TurboID proteomic results. (B) Git1 is biotinylated by Smo-TurboID independent of Shh treatment. Smo-V5-TurboID stable cells were infected with lentiviruses expressing YFP-Git1. Cells were treated with Shh for 1h, and cell lysates were used for purification with Streptavidin beads. a-Tubulin is used as loading control. (C) Git1 localizes to the basal body. NIH3T3 cells were transfected with YFP-Git1, and stained for primary cilium (Arl13b, red), basal body (Pericentrin, magenta), and nucleus (DAPI, blue). In addition to its putative distribution in the cytosol, Git1 also localizes to the basal body. Scale bar, 5 μm, 2 μm (inset). (D, F) Immunofluorescence imaging of Smo and phosphorylated Smo (pSmo) in WT and Git1-null cells. The cells are treated with 1 μg/ml recombinant Shh or vehicle. Primary cilium is highlighted by acetylated Tubulin (red). Scale bar, 5 μm, 2 μm (inset). (E, G) Quantification of ciliary Smo and pSmo immunofluorescence intensity (AU). n = 100-150 cells/condition from three biological replicates. (H) Representative images of immunofluorescence staining in Smo-TurboID cells transfected with control shRNA or shRNA against Git1. Cells were fixed 72hr after lentiviral infection and stained with PKA-C (green), Arl13b (red) and nucleus (DAPI, blue). Scale bar, 5 μm, 2 μm (inset). (I) Left: Quantification of ciliary PKA-C immunofluorescence intensity (AU), n = 90 cells/condition from three biological replicates; Right: Quantification of % ciliary PKA-C relative to total nucleus in the field. n= 15 fields per condition. Statistics in E, G, I (Left): two-way ANOVA followed by Tukey’s multiple comparison test. Statistics in I (Right) is assessed by one-way ANOVA followed by Sidak’s multiple comparison test. *p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant.

Article Snippet: The following antibodies were used in this study: Rabbit anti-pSmo (7TM antibodies, 7TM0239A-IC), Rabbit anti-Smo (gift from M. Scotts, Stanford University), Mouse anti-acetylated tubulin (Sigma,T6793), Rabbit anti-Arl13b (Proteintech, 17711–1-AP), Rat anti-Arl13b (BiCell Scientific, 90413), rabbit anti-IFT88 (Proteintech, 13967-1-AP), Goat anti-Gli2 (R&D Systems, AF3635,), Goat anti-Gli1 (R&D Systems, AF3455), Rabbit anti-Git1 (Novus Biologicals, NBP1-86144), Chicken anti-GFP (Aves labs, GFP-1020), Rabbit anti-GFP (Thermo Fisher Scientific, A-11122), Mouse-anti-Flag (Sigma, F3165), Rabbit anti-HA (Cell Signaling Technology, 3724), Mouse anti-GAPDH (Thermo Fisher Scientific, MA5-15738), Mouse anti-V5 (Thermo Fisher Scientific, R960-25), Mouse anti-PKACα (BD Biosciences, 610980), Rabbit anti-PKACα ( Cell Signaling, D38C6), Mouse-anti-pericentrin (BD Biosciences, 611814), Mouse anti-gamma Tubulin (Proteintech, 66320-1-Ig), DAPI ((Thermo Fisher Scientific, D21490), Donkey anti-rabbit Rhodamine (Jackson ImmunoResearch Labs, 711-025-152), Donkey anti-rabbit Alexa 488 (Jackson ImmunoResearch Labs, 711-545-152), Donkey anti-mouse Alexa 488 (Jackson ImmunoResearch Labs, 715-025-151), Donkey anti-mouse Rhodamine (Jackson ImmunoResearch Labs, 715-545-151), Donkey anti-Chicken AlexaFluor 488 (Jackson ImmunoResearch Labs, 703-545-155), Donkey anti-Goat AlexaFluor 488 (Jackson ImmunoResearch Labs, 705-545-003), Donkey anti-Goat AlexaFluro 647 (Jackson ImmunoResearch Labs, 705-605-147), Alexa Fluor 647 Streptavidin (Jackson ImmunoResearch Labs, 016-600-084), Donkey-anti, HRP-Conjugated Streptavidin (Thermo Fisher Scientific, N100).

Techniques: Infection, Expressing, Purification, Control, Transfection, Staining, Immunofluorescence, Imaging, Recombinant, shRNA, Comparison

Journal: bioRxiv

Article Title: Proximity based proteomics reveals Git1 as a regulator of Smoothened signaling

doi: 10.1101/2025.01.06.631593

Figure Lengend Snippet: (A) guide RNA was designed to target exon 2 of mouse Git1 . (B) The gRNA targeting region in mouse genomic was amplified by genomic PCR, ligated into TOPO vector, and transfected into chemically competent cells. 20 bacterial colonies of each cell clones were randomly picked and sequenced. The Sanger sequencing results were aligned with the genome sequence of the M. musculus. Single base pair deletion and insertion are identified, resulting in biallelic frameshift or early termination. (C) Immunoblot of Git1-null cell lines showing that Git1 protein is not detected in knockout cells. GAPDH is used as the loading control. (D) Cilium staining in WT and Git1-null cell colonies. Primary cilium is marked with Arl13b (red). Scale bar, 10 μm. (E) Quantification of cilium length, n = 60 cells/condition from 3 biological replicates. (F) Git1 transcript levels in cells transfected with shRNA against Git1. Statistics in (E-F): one-way ANOVA followed by Sidak’s multiple comparisons test. *p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant.

Article Snippet: The following antibodies were used in this study: Rabbit anti-pSmo (7TM antibodies, 7TM0239A-IC), Rabbit anti-Smo (gift from M. Scotts, Stanford University), Mouse anti-acetylated tubulin (Sigma,T6793), Rabbit anti-Arl13b (Proteintech, 17711–1-AP), Rat anti-Arl13b (BiCell Scientific, 90413), rabbit anti-IFT88 (Proteintech, 13967-1-AP), Goat anti-Gli2 (R&D Systems, AF3635,), Goat anti-Gli1 (R&D Systems, AF3455), Rabbit anti-Git1 (Novus Biologicals, NBP1-86144), Chicken anti-GFP (Aves labs, GFP-1020), Rabbit anti-GFP (Thermo Fisher Scientific, A-11122), Mouse-anti-Flag (Sigma, F3165), Rabbit anti-HA (Cell Signaling Technology, 3724), Mouse anti-GAPDH (Thermo Fisher Scientific, MA5-15738), Mouse anti-V5 (Thermo Fisher Scientific, R960-25), Mouse anti-PKACα (BD Biosciences, 610980), Rabbit anti-PKACα ( Cell Signaling, D38C6), Mouse-anti-pericentrin (BD Biosciences, 611814), Mouse anti-gamma Tubulin (Proteintech, 66320-1-Ig), DAPI ((Thermo Fisher Scientific, D21490), Donkey anti-rabbit Rhodamine (Jackson ImmunoResearch Labs, 711-025-152), Donkey anti-rabbit Alexa 488 (Jackson ImmunoResearch Labs, 711-545-152), Donkey anti-mouse Alexa 488 (Jackson ImmunoResearch Labs, 715-025-151), Donkey anti-mouse Rhodamine (Jackson ImmunoResearch Labs, 715-545-151), Donkey anti-Chicken AlexaFluor 488 (Jackson ImmunoResearch Labs, 703-545-155), Donkey anti-Goat AlexaFluor 488 (Jackson ImmunoResearch Labs, 705-545-003), Donkey anti-Goat AlexaFluro 647 (Jackson ImmunoResearch Labs, 705-605-147), Alexa Fluor 647 Streptavidin (Jackson ImmunoResearch Labs, 016-600-084), Donkey-anti, HRP-Conjugated Streptavidin (Thermo Fisher Scientific, N100).

Techniques: Amplification, Plasmid Preparation, Transfection, Clone Assay, Sequencing, Western Blot, Knock-Out, Control, Staining, shRNA

Journal: bioRxiv

Article Title: Proximity based proteomics reveals Git1 as a regulator of Smoothened signaling

doi: 10.1101/2025.01.06.631593

Figure Lengend Snippet: (A) Immunofluorescence image of Grk2-V5 (red) and YFP-Git1 (green) in NIH3T3. γTub labels centrosome (magenta). Scale bars, 5 μm and 2 μm (inset). (B) Co-immunoprecipitation blot showing interaction between Git1-Flag and Grk2-V5 when co-expressed in 293T cells. (C) Representative images of Grk2 levels at the basal body and in the cilium after Shh stimulation in WT and Git1-null cells. (D, E) Quantification of basal body and ciliary Grk2 intensity in WT and Git1-null cells. n=90 cells/condition from three biological replicates. Data are shown as mean ± SD. Statistics in D and E: two-way ANOVA followed by Tukey’s multiple comparison test. ** or ## p < 0.001, ****p < 0.0001, versus time 0.

Article Snippet: The following antibodies were used in this study: Rabbit anti-pSmo (7TM antibodies, 7TM0239A-IC), Rabbit anti-Smo (gift from M. Scotts, Stanford University), Mouse anti-acetylated tubulin (Sigma,T6793), Rabbit anti-Arl13b (Proteintech, 17711–1-AP), Rat anti-Arl13b (BiCell Scientific, 90413), rabbit anti-IFT88 (Proteintech, 13967-1-AP), Goat anti-Gli2 (R&D Systems, AF3635,), Goat anti-Gli1 (R&D Systems, AF3455), Rabbit anti-Git1 (Novus Biologicals, NBP1-86144), Chicken anti-GFP (Aves labs, GFP-1020), Rabbit anti-GFP (Thermo Fisher Scientific, A-11122), Mouse-anti-Flag (Sigma, F3165), Rabbit anti-HA (Cell Signaling Technology, 3724), Mouse anti-GAPDH (Thermo Fisher Scientific, MA5-15738), Mouse anti-V5 (Thermo Fisher Scientific, R960-25), Mouse anti-PKACα (BD Biosciences, 610980), Rabbit anti-PKACα ( Cell Signaling, D38C6), Mouse-anti-pericentrin (BD Biosciences, 611814), Mouse anti-gamma Tubulin (Proteintech, 66320-1-Ig), DAPI ((Thermo Fisher Scientific, D21490), Donkey anti-rabbit Rhodamine (Jackson ImmunoResearch Labs, 711-025-152), Donkey anti-rabbit Alexa 488 (Jackson ImmunoResearch Labs, 711-545-152), Donkey anti-mouse Alexa 488 (Jackson ImmunoResearch Labs, 715-025-151), Donkey anti-mouse Rhodamine (Jackson ImmunoResearch Labs, 715-545-151), Donkey anti-Chicken AlexaFluor 488 (Jackson ImmunoResearch Labs, 703-545-155), Donkey anti-Goat AlexaFluor 488 (Jackson ImmunoResearch Labs, 705-545-003), Donkey anti-Goat AlexaFluro 647 (Jackson ImmunoResearch Labs, 705-605-147), Alexa Fluor 647 Streptavidin (Jackson ImmunoResearch Labs, 016-600-084), Donkey-anti, HRP-Conjugated Streptavidin (Thermo Fisher Scientific, N100).

Techniques: Immunofluorescence, Immunoprecipitation, Comparison

Journal: bioRxiv

Article Title: Proximity based proteomics reveals Git1 as a regulator of Smoothened signaling

doi: 10.1101/2025.01.06.631593

Figure Lengend Snippet: (A) qPCR measurement of Gli1 transcript levels in wild-type NIH3T3 (WT) and Git1-null cell colonies. Cells were stimulated with Shh or vehicle for 24h. (B) Immunoblot of Gli3, Gli1 in WT and Git1-null cell colonies. a-Tubulin (aTub) is used as the loading control. (C and D) Quantification of immunoblot intensity of Gli3 and Gli1. n =4 independent experiments. (E) Immunofluorescent imaging of WT and Git1-null cells with or without Shh treatment. Cells were stained for Gli2 (green), and primary cilium (acTub, red). Scale bars, 5 μm and 2 μm (inset). (F) Quantification of Gli2 signal at the ciliary tip in WT and Git1-null cells. n=150 cells from 3 biological replicates. (G) Representative images of Git1-null cells infected with lentiviruses expressing Grk2-V5-Δ1Arl13b. (H) qPCR measurement of Gli1 transcript levels in WT and Git1-null cell colonies that express the indicated construct. The control plasmid refers to V5-Δ1Arl13b backbone. Statistics in A, C, D, F, H: two-way ANOVA followed by Tukey’s multiple comparison test. *p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant.

Article Snippet: The following antibodies were used in this study: Rabbit anti-pSmo (7TM antibodies, 7TM0239A-IC), Rabbit anti-Smo (gift from M. Scotts, Stanford University), Mouse anti-acetylated tubulin (Sigma,T6793), Rabbit anti-Arl13b (Proteintech, 17711–1-AP), Rat anti-Arl13b (BiCell Scientific, 90413), rabbit anti-IFT88 (Proteintech, 13967-1-AP), Goat anti-Gli2 (R&D Systems, AF3635,), Goat anti-Gli1 (R&D Systems, AF3455), Rabbit anti-Git1 (Novus Biologicals, NBP1-86144), Chicken anti-GFP (Aves labs, GFP-1020), Rabbit anti-GFP (Thermo Fisher Scientific, A-11122), Mouse-anti-Flag (Sigma, F3165), Rabbit anti-HA (Cell Signaling Technology, 3724), Mouse anti-GAPDH (Thermo Fisher Scientific, MA5-15738), Mouse anti-V5 (Thermo Fisher Scientific, R960-25), Mouse anti-PKACα (BD Biosciences, 610980), Rabbit anti-PKACα ( Cell Signaling, D38C6), Mouse-anti-pericentrin (BD Biosciences, 611814), Mouse anti-gamma Tubulin (Proteintech, 66320-1-Ig), DAPI ((Thermo Fisher Scientific, D21490), Donkey anti-rabbit Rhodamine (Jackson ImmunoResearch Labs, 711-025-152), Donkey anti-rabbit Alexa 488 (Jackson ImmunoResearch Labs, 711-545-152), Donkey anti-mouse Alexa 488 (Jackson ImmunoResearch Labs, 715-025-151), Donkey anti-mouse Rhodamine (Jackson ImmunoResearch Labs, 715-545-151), Donkey anti-Chicken AlexaFluor 488 (Jackson ImmunoResearch Labs, 703-545-155), Donkey anti-Goat AlexaFluor 488 (Jackson ImmunoResearch Labs, 705-545-003), Donkey anti-Goat AlexaFluro 647 (Jackson ImmunoResearch Labs, 705-605-147), Alexa Fluor 647 Streptavidin (Jackson ImmunoResearch Labs, 016-600-084), Donkey-anti, HRP-Conjugated Streptavidin (Thermo Fisher Scientific, N100).

Techniques: Western Blot, Control, Imaging, Staining, Infection, Expressing, Construct, Plasmid Preparation, Comparison

Journal: bioRxiv

Article Title: Proximity based proteomics reveals Git1 as a regulator of Smoothened signaling

doi: 10.1101/2025.01.06.631593

Figure Lengend Snippet: (A) Experimental workflow of GNP primary culture and treatment. (B) qPCR measurement of Git1 transcript levels in GNPs at the end of the experiment. (C) Hh signaling intensity is assessed by Gli1 transcript levels in GNPs at the end of the experiment. (D) Immunostaining of Edu (magenta) incorporation in primary cultured GNPs. Cells are infected with lentivirus expressing control or Git1 shRNA. (E) quantification of Edu incorporation into the GNP nucleus. n = 10 fields for each condition. (F) Schematic view of Git1’s function in Hh signaling. Git1 at the ciliary base facilitates Grk2’s interaction with Smo; it also promotes Grk2 translocation into the cilium to effectively phosphorylate Smo. Without Git1, Grk2 fails to phosphorylate Smo, leading to reduced Hh signaling. Data in B, C, E are shown as Mean ± SD. Statistics: two-way ANOVA followed by Tukey’s multiple comparison test. *p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant.

Article Snippet: The following antibodies were used in this study: Rabbit anti-pSmo (7TM antibodies, 7TM0239A-IC), Rabbit anti-Smo (gift from M. Scotts, Stanford University), Mouse anti-acetylated tubulin (Sigma,T6793), Rabbit anti-Arl13b (Proteintech, 17711–1-AP), Rat anti-Arl13b (BiCell Scientific, 90413), rabbit anti-IFT88 (Proteintech, 13967-1-AP), Goat anti-Gli2 (R&D Systems, AF3635,), Goat anti-Gli1 (R&D Systems, AF3455), Rabbit anti-Git1 (Novus Biologicals, NBP1-86144), Chicken anti-GFP (Aves labs, GFP-1020), Rabbit anti-GFP (Thermo Fisher Scientific, A-11122), Mouse-anti-Flag (Sigma, F3165), Rabbit anti-HA (Cell Signaling Technology, 3724), Mouse anti-GAPDH (Thermo Fisher Scientific, MA5-15738), Mouse anti-V5 (Thermo Fisher Scientific, R960-25), Mouse anti-PKACα (BD Biosciences, 610980), Rabbit anti-PKACα ( Cell Signaling, D38C6), Mouse-anti-pericentrin (BD Biosciences, 611814), Mouse anti-gamma Tubulin (Proteintech, 66320-1-Ig), DAPI ((Thermo Fisher Scientific, D21490), Donkey anti-rabbit Rhodamine (Jackson ImmunoResearch Labs, 711-025-152), Donkey anti-rabbit Alexa 488 (Jackson ImmunoResearch Labs, 711-545-152), Donkey anti-mouse Alexa 488 (Jackson ImmunoResearch Labs, 715-025-151), Donkey anti-mouse Rhodamine (Jackson ImmunoResearch Labs, 715-545-151), Donkey anti-Chicken AlexaFluor 488 (Jackson ImmunoResearch Labs, 703-545-155), Donkey anti-Goat AlexaFluor 488 (Jackson ImmunoResearch Labs, 705-545-003), Donkey anti-Goat AlexaFluro 647 (Jackson ImmunoResearch Labs, 705-605-147), Alexa Fluor 647 Streptavidin (Jackson ImmunoResearch Labs, 016-600-084), Donkey-anti, HRP-Conjugated Streptavidin (Thermo Fisher Scientific, N100).

Techniques: Immunostaining, Cell Culture, Infection, Expressing, Control, shRNA, Translocation Assay, Comparison