human git1  (Addgene inc)

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: To observe localization and activity of Git1, YFP was fused to the N terminus of human Git1 (Addgene, 15225) and cloned into FUGW backbone (Addgene, 14883).

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: To observe localization and activity of Git1, YFP was fused to the N terminus of human Git1 (Addgene, 15225) and cloned into FUGW backbone (Addgene, 14883).

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: To observe localization and activity of Git1, YFP was fused to the N terminus of human Git1 (Addgene, 15225) and cloned into FUGW backbone (Addgene, 14883).

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: To observe localization and activity of Git1, YFP was fused to the N terminus of human Git1 (Addgene, 15225) and cloned into FUGW backbone (Addgene, 14883).

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: To observe localization and activity of Git1, YFP was fused to the N terminus of human Git1 (Addgene, 15225) and cloned into FUGW backbone (Addgene, 14883).

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

Journal: Bioengineered

Article Title: GIT1 overexpression promotes epithelial-mesenchymal transition and predicts poor prognosis in hepatocellular carcinoma

doi: 10.1080/21655979.2020.1855914

Figure Lengend Snippet: GIT1 expression in human cancers

Article Snippet: These antibodies included rabbit anti-human GIT1 (Abcam, Cat. No. ab171956); rabbit anti-p-ERK (Cat. No. #4370), anti-ERK (Cat. No. #4695), anti-E-cadherin (Cat. No. #3195), anti-p-AKT (Cat. No. #4685), anti-Vimentin (Cat. No. #5741), anti-β-cadherin (Cat. No. #8480), anti-AKT (Cat. No. #9272), and anti-GAPDH (Cat. No. #5174), all bought from Cell signaling, and diluted to 1:1000.

Techniques: Expressing

Journal: Bioengineered

Article Title: GIT1 overexpression promotes epithelial-mesenchymal transition and predicts poor prognosis in hepatocellular carcinoma

doi: 10.1080/21655979.2020.1855914

Figure Lengend Snippet: Expression pattern of GIT1 in HCC tissues

Article Snippet: These antibodies included rabbit anti-human GIT1 (Abcam, Cat. No. ab171956); rabbit anti-p-ERK (Cat. No. #4370), anti-ERK (Cat. No. #4695), anti-E-cadherin (Cat. No. #3195), anti-p-AKT (Cat. No. #4685), anti-Vimentin (Cat. No. #5741), anti-β-cadherin (Cat. No. #8480), anti-AKT (Cat. No. #9272), and anti-GAPDH (Cat. No. #5174), all bought from Cell signaling, and diluted to 1:1000.

Techniques: Expressing

Journal: Bioengineered

Article Title: GIT1 overexpression promotes epithelial-mesenchymal transition and predicts poor prognosis in hepatocellular carcinoma

doi: 10.1080/21655979.2020.1855914

Figure Lengend Snippet: GIT1 overexpression promoted HCC cell invasion, proliferation, and migration

Article Snippet: These antibodies included rabbit anti-human GIT1 (Abcam, Cat. No. ab171956); rabbit anti-p-ERK (Cat. No. #4370), anti-ERK (Cat. No. #4695), anti-E-cadherin (Cat. No. #3195), anti-p-AKT (Cat. No. #4685), anti-Vimentin (Cat. No. #5741), anti-β-cadherin (Cat. No. #8480), anti-AKT (Cat. No. #9272), and anti-GAPDH (Cat. No. #5174), all bought from Cell signaling, and diluted to 1:1000.

Techniques: Over Expression, Migration

Journal: Bioengineered

Article Title: GIT1 overexpression promotes epithelial-mesenchymal transition and predicts poor prognosis in hepatocellular carcinoma

doi: 10.1080/21655979.2020.1855914

Figure Lengend Snippet: GIT1 promoted HCC tumorigenesis in vivo

Article Snippet: These antibodies included rabbit anti-human GIT1 (Abcam, Cat. No. ab171956); rabbit anti-p-ERK (Cat. No. #4370), anti-ERK (Cat. No. #4695), anti-E-cadherin (Cat. No. #3195), anti-p-AKT (Cat. No. #4685), anti-Vimentin (Cat. No. #5741), anti-β-cadherin (Cat. No. #8480), anti-AKT (Cat. No. #9272), and anti-GAPDH (Cat. No. #5174), all bought from Cell signaling, and diluted to 1:1000.

Techniques: In Vivo

Journal: Bioengineered

Article Title: GIT1 overexpression promotes epithelial-mesenchymal transition and predicts poor prognosis in hepatocellular carcinoma

doi: 10.1080/21655979.2020.1855914

Figure Lengend Snippet: GIT1 promoted EMT in HCC cells through the ERK1/2

Article Snippet: These antibodies included rabbit anti-human GIT1 (Abcam, Cat. No. ab171956); rabbit anti-p-ERK (Cat. No. #4370), anti-ERK (Cat. No. #4695), anti-E-cadherin (Cat. No. #3195), anti-p-AKT (Cat. No. #4685), anti-Vimentin (Cat. No. #5741), anti-β-cadherin (Cat. No. #8480), anti-AKT (Cat. No. #9272), and anti-GAPDH (Cat. No. #5174), all bought from Cell signaling, and diluted to 1:1000.

Techniques:

Journal: Bioengineered

Article Title: GIT1 overexpression promotes epithelial-mesenchymal transition and predicts poor prognosis in hepatocellular carcinoma

doi: 10.1080/21655979.2020.1855914

Figure Lengend Snippet: The expression of GIT1 and its prognosis value in 158 HCC patients

Article Snippet: These antibodies included rabbit anti-human GIT1 (Abcam, Cat. No. ab171956); rabbit anti-p-ERK (Cat. No. #4370), anti-ERK (Cat. No. #4695), anti-E-cadherin (Cat. No. #3195), anti-p-AKT (Cat. No. #4685), anti-Vimentin (Cat. No. #5741), anti-β-cadherin (Cat. No. #8480), anti-AKT (Cat. No. #9272), and anti-GAPDH (Cat. No. #5174), all bought from Cell signaling, and diluted to 1:1000.

Techniques: Expressing

Journal: Bioengineered

Article Title: GIT1 overexpression promotes epithelial-mesenchymal transition and predicts poor prognosis in hepatocellular carcinoma

doi: 10.1080/21655979.2020.1855914

Figure Lengend Snippet: Correlations between GIT1 with clinicopathologic features in 158 HCC patients

Article Snippet: These antibodies included rabbit anti-human GIT1 (Abcam, Cat. No. ab171956); rabbit anti-p-ERK (Cat. No. #4370), anti-ERK (Cat. No. #4695), anti-E-cadherin (Cat. No. #3195), anti-p-AKT (Cat. No. #4685), anti-Vimentin (Cat. No. #5741), anti-β-cadherin (Cat. No. #8480), anti-AKT (Cat. No. #9272), and anti-GAPDH (Cat. No. #5174), all bought from Cell signaling, and diluted to 1:1000.

Techniques:

Journal: Bioengineered

Article Title: GIT1 overexpression promotes epithelial-mesenchymal transition and predicts poor prognosis in hepatocellular carcinoma

doi: 10.1080/21655979.2020.1855914

Figure Lengend Snippet: Univariate and multivariate analyses of factors associated with recurrence and survival

Article Snippet: These antibodies included rabbit anti-human GIT1 (Abcam, Cat. No. ab171956); rabbit anti-p-ERK (Cat. No. #4370), anti-ERK (Cat. No. #4695), anti-E-cadherin (Cat. No. #3195), anti-p-AKT (Cat. No. #4685), anti-Vimentin (Cat. No. #5741), anti-β-cadherin (Cat. No. #8480), anti-AKT (Cat. No. #9272), and anti-GAPDH (Cat. No. #5174), all bought from Cell signaling, and diluted to 1:1000.

Techniques: