antibodies against git1  (Boster Bio)

Journal: Science Advances

Article Title: Genetics of mirror movements identifies a multifunctional complex required for Netrin-1 guidance and lateralization of motor control

doi: 10.1126/sciadv.add5501

Figure Lengend Snippet: ( A ) The mean mRNA expression, fragments per kilobase of transcript per million mapped reads (fpkm), (± SEM) of Arhgef7 and Dcc in dissociated commissural neurons ( n = 3). ( B ) Dissociated commissural neurons were fixed and immunostained for Arhgef7 and Dcc. Scale bar, 6 μm. ( C ) Mouse E10.5 and E11.5 neural tube cross sections were immunostained for Arhgef7 and Robo3, a marker of commissural neurons. Scale bar, 100 μm; zoom, 25 μm. ( D and E ) Commissural neurons were transfected with scrambled shRNA or shRNA against Arhgef7 , together with empty vector, ARHGEF7 WT or ARHGEF7 mut expression vectors as indicated and then exposed to a gradient of Netrin-1 (0.1 μg/ml in the outer well) in the Dunn chamber. (D) The mean angle turned (±SEM) was quantified. (E) Scatter plots of the turned angle versus the original angle between the axons and the direction of the Netrin-1 gradient for neurons under the indicated conditions. Arhgef7 knockdown inhibits the turning of axons up a Netrin-1 gradient, while expression of ARHGEF7 WT but not ARHGEF7 mut completely rescued Netrin-1–mediated growth cone turning. n = 132, 152, 63, and 115 respectively from three independent experiments. One-way analysis of variance (ANOVA), Tukey’s multiple comparison post-test, * P < 0.05. See also fig. S1.

Article Snippet: The samples were blocked with 10% BSA [immunoglobulin G (IgG) free] and 0.1% Triton X-100 in PBS (pH 7.4) for 1 hour at room temperature and then incubated with antibodies against DCC (1:50) (Santa Cruz Biotechnology sc-6535) and ARHGEF7 (1:50) (MilliporeSigma, 07-1450-I) or GIT1 (1:50) (Novus Bio NBP2-22423), diluted in PBS with 1% BSA (IgG free) and 0.1% Triton X-100, overnight at 4°C.

Techniques: Expressing, Marker, Transfection, shRNA, Plasmid Preparation, Knockdown, Comparison

Journal: Science Advances

Article Title: Genetics of mirror movements identifies a multifunctional complex required for Netrin-1 guidance and lateralization of motor control

doi: 10.1126/sciadv.add5501

Figure Lengend Snippet: ( A ) Schematic of the ARHGEF7 constructs used. ( B and D ) Cos7 cells were transfected with tagged Dcc and ARHGEF7 expression vectors as indicated. The cell lysates were immunoprecipitated (IP) with an anti-Flag antibody and the immunoprecipitates analyzed by immunoblotting (IB) with the indicated antibodies. ( C ) The relative amount (mean ± SEM) of Dcc interacting with ARHGEF7 WT and ARHGEF7 mut . Dcc has significantly less interaction with ARHGEF7 mut compared to ARHGEF7 WT . n = 5, unpaired t test, **** P < 0.0001. ( E ) The relative amount (mean ± SEM) of Dcc binding to the ARHGEF7 constructs. n = 4, one-way ANOVA, Tukey’s multiple comparison post-test, * P < 0.05, ** P < 0.01, *** P < 0.001. The interaction between ARHGEF7 and Dcc requires the KER and LZ domains. ( F ) Schematic of the DCC and Frazzled cytoplasmic domain constructs. ( G ) Purified GST fusion proteins coupled to glutathione-agarose beads were incubated with in vitro translated ARHGEF7. Pulled-down proteins were analyzed by Western blotting with an anti-ARHGEF7 antibody. ARHGEF7 directly binds to the P1 domain of DCC. ( H ) The relative binding (mean ± SEM) of ARHGEF7 to DCC/Frazzled. n ≥ 4, one-way ANOVA with Dunnett’s multiple comparison test. * P < 0.05, ** P < 0.01. ( I and L ) Dissociated commissural neurons were treated with 0.1 μg/ml BSA or Netrin-1 for 2 and 5 min respectively, then fixed. The PLA assay was performed for Dcc and Arhgef7. Arrowheads indicate some examples of PLA puncta. Scale bar, 7 μm. ( J and M ) The relative number of PLA puncta per growth cone and ( K and N ) the mean (±SEM) intensity of the PLA signal in the growth cone after 2 min (J and K) and 5 min (M and N) of Netrin-1 stimulation. n = 4 experiments, 15 growth cones per condition, per experiment. Paired t test, * P < 0.05, ** P < 0.01. See also fig. S2.

Article Snippet: The samples were blocked with 10% BSA [immunoglobulin G (IgG) free] and 0.1% Triton X-100 in PBS (pH 7.4) for 1 hour at room temperature and then incubated with antibodies against DCC (1:50) (Santa Cruz Biotechnology sc-6535) and ARHGEF7 (1:50) (MilliporeSigma, 07-1450-I) or GIT1 (1:50) (Novus Bio NBP2-22423), diluted in PBS with 1% BSA (IgG free) and 0.1% Triton X-100, overnight at 4°C.

Techniques: Construct, Transfection, Expressing, Immunoprecipitation, Western Blot, Binding Assay, Comparison, Purification, Incubation, In Vitro

Journal: Science Advances

Article Title: Genetics of mirror movements identifies a multifunctional complex required for Netrin-1 guidance and lateralization of motor control

doi: 10.1126/sciadv.add5501

Figure Lengend Snippet: ( A ) Cos7 cells were transfected with ARHGEF7 and Git1 expression vectors. Cell lysates were immunoprecipitated with an anti-ARHGEF7 antibody and analyzed by Western blotting. ( B ) Relative amount (mean ± SEM) of Git1 interacting with ARHGEF7 WT or ARHGEF7 mut . Git1 has significantly less interaction with ARHGEF7 mut compared to ARHGEF7 WT . n = 3, paired t test, ** P < 0.01. ( C ) The mean mRNA expression (± SEM) of Git1 , Git2 and Dcc in dissociated commissural neurons ( n = 3). ( D ) Commissural neurons immunostained for Git1, Arhgef7 and Dcc. Scale bar, 6 μm. ( E ) Mouse E10.5 and E11.5 neural tube cross sections were immunostained for Git1 and Robo3, a marker of commissural neurons. Scale bars, 100 μm; zoom, 25 μm. ( F ) Purified GST or GST-Dcc coupled to glutathione-agarose beads were incubated with in vitro translated Git1, ARHGEF7 WT and ARHGEF7 mut as indicated. Pulled-down proteins were analyzed by Western blotting. ARHGEF7 WT and Git1, but not ARHGEF7 mut , directly bind to Dcc. ( G and H ) The relative binding (mean ± SEM) of ARHGEF7 and Git1 to Dcc. n = 4, two-way ANOVA with Sidak’s multiple comparison test. ** P < 0.01, *** P < 0.001; one-way ANOVA with Tukey’s multiple comparison post-test. ns, not significant. ( I ) Commissural neuron lysates were immunoprecipitated with an anti-DCC antibody or IgG control and analyzed by Western blotting. ( J and M ) Commissural neurons were treated with BSA or Netrin-1 for 2 and 5 min and then fixed. The PLA assay was performed for Dcc and Git1. Scale bar, 7 μm. ( K and N ) The relative number of PLA puncta per growth cone and ( L and O ) the mean (±SEM) intensity of the PLA signal in the growth cone after 2 min (K and L) and 5 min (N and O) of Netrin-1 stimulation. n = 6 experiments, 15 growth cones per condition, per experiment. Paired t test, * P < 0.05, ** P < 0.01. See also fig. S3.

Article Snippet: The samples were blocked with 10% BSA [immunoglobulin G (IgG) free] and 0.1% Triton X-100 in PBS (pH 7.4) for 1 hour at room temperature and then incubated with antibodies against DCC (1:50) (Santa Cruz Biotechnology sc-6535) and ARHGEF7 (1:50) (MilliporeSigma, 07-1450-I) or GIT1 (1:50) (Novus Bio NBP2-22423), diluted in PBS with 1% BSA (IgG free) and 0.1% Triton X-100, overnight at 4°C.

Techniques: Transfection, Expressing, Immunoprecipitation, Western Blot, Marker, Purification, Incubation, In Vitro, Binding Assay, Comparison, Control

Journal: Science Advances

Article Title: Genetics of mirror movements identifies a multifunctional complex required for Netrin-1 guidance and lateralization of motor control

doi: 10.1126/sciadv.add5501

Figure Lengend Snippet: ( A ) Commissural neurons were stimulated for 1, 2, 5 and 15 min with 0.1 μg/ml Netrin-1 or BSA and fixed. Extracellular Dcc (Dcc ex ) was detected by immunostaining under nonpermeabilizing conditions. ( B ) Mean intensity (±SEM) of Dcc ex fluorescence in growth cones. Netrin-1 increases Dcc ex . n = 3 experiments, 15 growth cones per condition, per experiment. Two-way ANOVA, Sidak’s multiple comparisons test. ( C and E ) Commissural neurons were transfected as indicated. Cells were stimulated with 0.1 μg/ml Netrin-1 or BSA for 5 min and fixed. Dcc ex was detected as in (A). ( D ) The mean intensity (±SEM) of Dcc ex fluorescence in growth cones. Arhgef7 knockdown blocks the Netrin-1–induced increase in Dcc ex . This is rescued by ARHGEF7 WT but not ARHGEF7 mut . n = 4 experiments, 15 growth cones per condition, per experiment. Two-way ANOVA, Tukey’s multiple comparisons test. ( F ) Mean intensity (±SEM) of Dcc ex fluorescence in growth cones. Git1 R39A blocks Netrin-1–induced increase in Dcc ex . n = 3 experiments, 15 growth cones per condition, per experiment. Two-way ANOVA, Sidak’s multiple comparisons test. ( G ) Commissural neurons were treated with vehicle or Rac inhibitor EHT1864 (20 μM, 2 hours). Cells were stimulated with Netrin-1 and Dcc ex was detected as in (C). ( H ) Mean intensity (±SEM) of Dcc ex fluorescence in growth cones. Rac inhibition does not block Netrin-1–induced increase in Dcc ex . n = 3 experiments, 15 growth cones per condition, per experiment. Two-way ANOVA, Tukey’s multiple comparisons test. ( I ) Commissural neurons were transfected as indicated. Cells were stimulated with Netrin-1 and Dcc ex was detected as in (C). ( J ) Mean intensity (±SEM) of Dcc ex fluorescence in growth cones. Arf1 knockdown blocks the Netrin-1–induced increase in Dcc ex , and this is rescued by ARF1 WT but not ARF1 T31N . n = 4 experiments, 15 growth cones per condition, per experiment. One-way ANOVA, Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001. Scale bars, 20 μm (A) and 7 μm (C, E, G, and I). See also figs. S5 to S8.

Article Snippet: The samples were blocked with 10% BSA [immunoglobulin G (IgG) free] and 0.1% Triton X-100 in PBS (pH 7.4) for 1 hour at room temperature and then incubated with antibodies against DCC (1:50) (Santa Cruz Biotechnology sc-6535) and ARHGEF7 (1:50) (MilliporeSigma, 07-1450-I) or GIT1 (1:50) (Novus Bio NBP2-22423), diluted in PBS with 1% BSA (IgG free) and 0.1% Triton X-100, overnight at 4°C.

Techniques: Immunostaining, Fluorescence, Transfection, Knockdown, Inhibition, Blocking Assay

Journal: Science Advances

Article Title: Genetics of mirror movements identifies a multifunctional complex required for Netrin-1 guidance and lateralization of motor control

doi: 10.1126/sciadv.add5501

Figure Lengend Snippet: ( A ) Weight of control ( n = 10) and Arhgef7 het ( n = 13) male mice. ( B ) Weight of control ( n = 13) and Arhgef7 het ( n = 11) female mice. Mann-Whitney test. ( C and D ) The total number of errors made in the horizontal ladder rung test, for males and females. ( E ) Images of mice walking on the ladder rungs. Red arrows indicate the paw positions. Top: Image of a control mouse with typical asymmetrical (alternating) paw placements. Bottom: Image of an Arhgef7 het mouse with symmetrical paw placements. ( F and G ) The number of symmetrical paw placements during the ladder rung test for males and females. (C, D, F, and G) Number of males: control ( n = 6) and Arhgef7 het ( n = 11), and females: control ( n = 10) and Arhgef7 het ( n = 7). Mann-Whitney, *** P < 0.001. ( H ) Arhgef7 forms a multifunctional effector complex required for Netrin-1–mediated axon guidance and lateralization of motor control. Arhgef7/Git1 directly bind to Dcc. Netrin-1 activates Rac1 and inactivates Arf1 in an Arhgef7/Git1-dependent manner. Arf1 inactivation increases cell surface Dcc. Arhgef7 mut does not bind to Dcc and Git1. When Arhgef7 mut is expressed, Netrin-1 fails to activate Rac1 and inactivate Arf1. Consequently, axon guidance is impaired resulting in lateralization defects.

Article Snippet: The samples were blocked with 10% BSA [immunoglobulin G (IgG) free] and 0.1% Triton X-100 in PBS (pH 7.4) for 1 hour at room temperature and then incubated with antibodies against DCC (1:50) (Santa Cruz Biotechnology sc-6535) and ARHGEF7 (1:50) (MilliporeSigma, 07-1450-I) or GIT1 (1:50) (Novus Bio NBP2-22423), diluted in PBS with 1% BSA (IgG free) and 0.1% Triton X-100, overnight at 4°C.

Techniques: Control, MANN-WHITNEY

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: Figure 1. 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: Figure 3. 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, ++

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: Figure 5. 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 3-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. aap < 0.01, aaap < 0.001 versus NC mimics; bbbp < 0.001 versus NC inhibitor; ddp < 0.01 versus NC mimics + GIT1 3UTR (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: Figure 6. 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. XXXp < 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: Figure 7. 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: 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: Animal Cells and Systems

Article Title: The ATP-dependent RNA helicase, DDX42 interacts with paxillin and regulates apoptosis and polarization of Ba/F3 cells

doi: 10.1080/19768354.2019.1567580

Figure Lengend Snippet: (A) Overexpression of His6-tagged wild-type and mutant paxillins. The expression of paxillin in non-infected Ba/F3 (Control) and in Ba/F3-derived cell lines overexpressing His6-tagged wild-type (Paxillin WT) and mutant paxillin (S273D and S273A) were compared by western blot analysis of cell extracts (20 μg protein) using anti-paxillin and anti-actin antibodies. (B) Comparison of protein bands of the pulled-down samples after electrophoresis and silver staining. Cell lysates from mock-transfected (Mock) and His6-tagged wild-type paxillin-overexpressing (His6-paxillin) cells, incubated with Ni + -NTA agarose beads, were loaded on an 8% polyacrylamide gel, and silver-stained. Differences in band patterns were observed in two regions (1 and 2), highlighted by rectangles, and are shown as magnified images on the right.

Article Snippet: Antibodies against paxillin (610052), GIT1 (sc-13961), actin (A2066), and DDX42 (A303-354A) were from BD Korea (Seoul Korea), Santa Cruz Biotechnology (Santa Cruz, CA, USA), Sigma Aldrich Co. (St. Louis, MO, USA), and Bethyl Laboratories (Montgomery, TX, USA), respectively.

Techniques: Over Expression, Mutagenesis, Expressing, Infection, Control, Derivative Assay, Western Blot, Comparison, Electrophoresis, Silver Staining, Transfection, Incubation, Staining

Journal: Animal Cells and Systems

Article Title: The ATP-dependent RNA helicase, DDX42 interacts with paxillin and regulates apoptosis and polarization of Ba/F3 cells

doi: 10.1080/19768354.2019.1567580

Figure Lengend Snippet: Results of mass spectrometry analysis. Gel slices were cut out of two regions of each lanes in Figure 1 (B) and sent for LC–MS analysis for peptide identification to the facility service in Yonsei Proteomic Research Center (Seoul, Korea).

Article Snippet: Antibodies against paxillin (610052), GIT1 (sc-13961), actin (A2066), and DDX42 (A303-354A) were from BD Korea (Seoul Korea), Santa Cruz Biotechnology (Santa Cruz, CA, USA), Sigma Aldrich Co. (St. Louis, MO, USA), and Bethyl Laboratories (Montgomery, TX, USA), respectively.

Techniques: Mass Spectrometry, Control

Journal: Animal Cells and Systems

Article Title: The ATP-dependent RNA helicase, DDX42 interacts with paxillin and regulates apoptosis and polarization of Ba/F3 cells

doi: 10.1080/19768354.2019.1567580

Figure Lengend Snippet: Interaction of DDX42 with paxillin. (A) His6-tagged wild-type (WT) and mutant (S273D and S273A) paxillin were overexpressed in Ba/F3 cells. The overexpressed proteins were pulled-down from each cell lysate with Ni + -NTA beads, and loaded on an 8% SDS-PAGE gel (Pulled-down). Pulled-down and co-precipitated proteins were detected by immunoblot with anti-DDX42, anti-GIT1, and anti-paxillin antibodies. DDX42 co-precipitated only with wild-type and S273A mutant paxillin, not with S273D. Whole cell lysates (20 μg protein) were also loaded on another gel and the indicated proteins were detected by immunoblot using the respective antibodies (Lysate). (B) His6-tagged wild-type (WT) and mutant (D407A) DDX42 were overexpressed and analyzed as described above (Pulled-down). Paxillin co-precipitated only with wild-type (WT), but not the DDX42 mutant, D407A. The amount of proteins was normalized by immunoblot of the lysates (Lysate).

Article Snippet: Antibodies against paxillin (610052), GIT1 (sc-13961), actin (A2066), and DDX42 (A303-354A) were from BD Korea (Seoul Korea), Santa Cruz Biotechnology (Santa Cruz, CA, USA), Sigma Aldrich Co. (St. Louis, MO, USA), and Bethyl Laboratories (Montgomery, TX, USA), respectively.

Techniques: Mutagenesis, SDS Page, Western Blot

Journal: Animal Cells and Systems

Article Title: The ATP-dependent RNA helicase, DDX42 interacts with paxillin and regulates apoptosis and polarization of Ba/F3 cells

doi: 10.1080/19768354.2019.1567580

Figure Lengend Snippet: DDX42 and paxillin interact with each other through the DEAD box and S273, respectively. Both proteins showed anti-apoptotic effects when overexpressed in Ba/F3 cells. (A) Illustration of DDX42 and paxillin structure showing their main motifs and domains. (B) hypothetical model of the impact of the DDX42/paxillin interaction on cell apoptosis and polarization. Phosphorylation of S273 might abolish not only the anti-apoptotic action of paxillin but also its interaction with DDX42. Mutational inactivation of the DEAD box in DDX42 also prevented the interaction with paxillin.

Article Snippet: Antibodies against paxillin (610052), GIT1 (sc-13961), actin (A2066), and DDX42 (A303-354A) were from BD Korea (Seoul Korea), Santa Cruz Biotechnology (Santa Cruz, CA, USA), Sigma Aldrich Co. (St. Louis, MO, USA), and Bethyl Laboratories (Montgomery, TX, USA), respectively.

Techniques: Phospho-proteomics

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