Heterotrimeric G protein-mediated signal transduction is a dynamically regulated process with the intensity of signal decreasing over time despite the continued presence of the agonist. G protein-coupled receptor kinases (GRKs) are activated by activated G protein-coupled receptors, and they function to phosphorylate and inactivate cell surface receptors in the heterotrimeric G protein signaling cascade. GIT1 (for GRK-interactor 1) and GIT2 are GTPase-activating proteins (GAP) for members of the ADP ribosylation factor (ARF) family of small GTP-binding proteins, which are involved in vesicular trafficking. GIT1 overexpression results in reduced internalization and resensitization of b2-adrenergic receptor, thus reducing b2-adrenergic receptor signaling.
Background References
1. Manabe R et al. GIT1 functions in a motile, multi-molecular signaling complex that regulates protrusive activity and cell migration. J Cell Sci 115:1497-1510 (2002).
2. Nishiya N et al. Hic-5 interacts with GIT1 with a different binding mode from paxillin. J Biochem 132:279-289 (2002).
Post-translational Modification
Phosphorylated on tyrosine residues by PTK2/FAK1 and SRC in growing fibroblasts. Tyrosine-phosphorylation is increased following cell spreading on fibronectin, decreased in cells arrested in mitosis and increased in the ensuing G1 phase (By similarity).
Western blot analysis of GIT1 on different lysates with Rabbit anti-GIT1 antibody (ET7106-91) at 1/2,000 dilution.
Lane 1: C6 cell lysate (10 µg/Lane) Lane 2: Mouse brain tissue lysate (20 µg/Lane) Lane 3: Rat brain tissue lysate (20 µg/Lane) Lane 4: Rat testis tissue lysate (20 µg/Lane)
Predicted band size: 84 kDa Observed band size: 84 kDa
Exposure time: 20 seconds; ECL: K1801;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET7106-91) at 1/2,000 dilution was used in primary antibody dilution (K1803) at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
Western blot analysis of GIT1 on A549 cell lysates with Rabbit anti-GIT1 antibody (ET7106-91) at 1/500 dilution.
Lysates/proteins at 10 µg/Lane.
Predicted band size: 84 kDa Observed band size: 84 kDa
Exposure time: 2 minutes;
8% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET7106-91) at 1/500 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:200,000 dilution was used for 1 hour at room temperature.
Immunohistochemical analysis of paraffin-embedded human colon carcinoma tissue with Rabbit anti-GIT1 antibody (ET7106-91) at 1/400 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET7106-91) at 1/400 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Immunohistochemical analysis of paraffin-embedded human pancreas tissue with Rabbit anti-GIT1 antibody (ET7106-91) at 1/400 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET7106-91) at 1/400 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Immunohistochemical analysis of paraffin-embedded mouse fallopian tubes tissue using anti-GIT1 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes.The tissues were blocked in 1% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET7106-91, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Immunohistochemical analysis of paraffin-embedded rat brain tissue using anti-GIT1 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes.The tissues were blocked in 1% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET7106-91, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Immunohistochemical analysis of paraffin-embedded mouse small intestine tissue with Rabbit anti-GIT1 antibody (ET7106-91) at 1/400 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET7106-91) at 1/400 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
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