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AB265230

Human GIT1 knockout HeLa cell line

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GIT1 KO cell line available to order. KO validated by Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp insertion in exon 3.

View Alternative Names

ARF GAP GIT1, ARF GTPase-activating protein GIT1, Cool-associated and tyrosine-phosphorylated protein 1, G protein coupled receptor kinase interacting ArfGAP 1, G protein-coupled receptor kinase-interactor 1, GIT1_HUMAN, GRK-interacting protein 1

3 Images
Western blot - Human GIT1 knockout HeLa cell line (AB265230)
  • WB

Lab

Western blot - Human GIT1 knockout HeLa cell line (AB265230)

Lanes 1-4 : Merged signal (red and green). Green - ab156001 observed at 98 kDa. Red - loading control ab8245 observed at 36 kDa.

ab156001 Anti-GIT1 antibody [EPR10367] was shown to specifically react with GIT1 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265230 (knockout cell lysate ab257965) was used. Wild-type and GIT1 knockout samples were subjected to SDS-PAGE. ab156001 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4° at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-GIT1 antibody [EPR10367] (<a href='/en-us/products/primary-antibodies/git1-antibody-epr10367-ab156001'>ab156001</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

G1T1 knockout HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

Western blot - Human GIT1 knockout HeLa cell line (ab265230)

Lane 3:

Human brain tissue nuclear fraction lysate at 20 µg

Lane 4:

SH-SY5Y (Human neuroblastoma cell line from bone marrow) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 84 kDa

Observed band size: 98 kDa

false

Western blot - Human GIT1 knockout HeLa cell line (AB265230)
  • WB

Lab

Western blot - Human GIT1 knockout HeLa cell line (AB265230)

Lanes 1-4 : Merged signal (red and green). Green - ab171956 observed at 98 kDa. Red - loading control ab8245 observed at 36 kDa.

ab171956 Anti-GIT1 antibody [EPR10368] was shown to specifically react with GIT1 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265230 (knockout cell lysate ab257965) was used. Wild-type and GIT1 knockout samples were subjected to SDS-PAGE. ab171956 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4° at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-GIT1 antibody [EPR10368] (<a href='/en-us/products/primary-antibodies/git1-antibody-epr10368-ab171956'>ab171956</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

G1T1 knockout HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

Western blot - Human GIT1 knockout HeLa cell line (ab265230)

Lane 3:

Human brain tissue nuclear fraction lysate at 20 µg

Lane 4:

SH-SY5Y (Human neuroblastoma cell line from bone marrow) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 84 kDa

Observed band size: 98 kDa

false

Sanger Sequencing - Human GIT1 knockout HeLa cell line (AB265230)
  • Sanger seq

Unknown

Sanger Sequencing - Human GIT1 knockout HeLa cell line (AB265230)

Homozygous : 1 bp insertion in exon 3.

Key facts

Cell type

HeLa

Species or organism

Human

Tissue

Cervix

Form

Liquid

form

Knockout validation

Sanger Sequencing,Western blot

Mutation description

Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp insertion in exon 3

Disease

Adenocarcinoma

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Reactivity data

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Product details

Recommended control: Human wild-type HeLa cell line (ab255928). Please note a wild-type cell line is not automatically included with a knockout cell line order, if required please add recommended wild-type cell line at no additional cost using the code WILDTYPE-TMTK1.

We will provide viable cells that proliferate on revival.

This product is subject to limited use licenses from The Broad Institute, ERS Genomics Limited and Sigma-Aldrich Co. LLC, and is developed with patented technology. For full details of the licenses and patents please refer to our limited use license and patent pages.

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What's included?

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Properties and storage information

Gene name
GIT1
Gene editing type
Knockout
Gene editing method
CRISPR technology
Knockout validation
Sanger Sequencing, Western blot
Zygosity
Homozygous
Shipped at conditions
Dry Ice
Appropriate short-term storage conditions
-196°C
Appropriate long-term storage conditions
-196°C
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Handling procedures

Initial handling guidelines

Upon arrival, the vial should be stored in liquid nitrogen vapor phase and not at -80°C. Storage at -80°C may result in loss of viability.

1. Thaw the vial in 37°C water bath for approximately 1-2 minutes.
2. Transfer the cell suspension (0.8 mL) to a 15 mL/50 mL conical sterile polypropylene centrifuge tube containing 8.4 mL pre-warmed culture medium, wash vial with an additional 0.8 mL culture medium (total volume 10 mL) to collect remaining cells, and centrifuge at 201 x g (rcf) for 5 minutes at room temperature. 10 mL represents minimum recommended dilution. 20 mL represents maximum recommended dilution.
3. Resuspend the cell pellet in 5 mL pre-warmed culture medium and count using a haemocytometer or alternative cell counting method seed all remaining cells into a T25.
4. Incubate the culture at 37°C incubator with 5% CO2. Check the culture one day after revival and continue to check until 80% confluent. Media change can be given if needed.
5. Once confluent passage into an appropriate flask at a density of 2x104 cells/cm2. Seeding density is given as a guide only and should be scaled to align with individual lab schedules. Cultures should be monitored daily.

Subculture guidelines
  • All seeding densities should be based on cell counts gained by established methods.
  • A guide seeding density of 2x104 cells/cm2 is recommended.
  • Cells should be passaged when they have achieved 80-90% confluence.
Culture medium

DMEM (High Glucose) + 10% FBS

Cryopreservation medium

Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.

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Supplementary information

This supplementary information is collated from multiple sources and compiled automatically.

GIT1 also known as G protein-coupled receptor kinase interacting protein 1 is a multi-domain protein with a mass of approximately 90 kDa. It plays a central role in cellular signaling processes. GIT1 contains a GTPase-activating protein (GAP) domain and a number of other domains that interact with protein partners. This protein is expressed ubiquitously in various tissues with higher expression levels in the brain and heart. Its presence in different cell types indicates its involvement in numerous cellular functions.
Biological function summary

GIT1 interacts with several proteins to form multi-protein complexes essential for cellular dynamics. It functions in regulating the actin cytoskeleton impacting cell adhesion migration and membrane trafficking. GIT1 participates in the reorganization of the cytoskeleton by activating small GTPases which are important for these processes. Additionally it interacts with paxillin and ARF GTPases further playing a role in focal adhesion signaling.

Pathways

GIT1 serves an important role in the ERK and JNK signaling pathways. It acts as a scaffolding protein facilitating the assembly of signaling complexes that promote the transfer of signals from the membrane to the nucleus. GIT1 modulates the activity of proteins such as MEK1/2 and paxillin which ultimately regulate cellular responses like proliferation and differentiation. This function positions GIT1 at an important junction of pathways controlling cellular architecture and stress responses.

GIT1 has been linked to cardiovascular diseases and neurological disorders. Dysregulation of GIT1 and its associated pathways can lead to pathological changes in the heart contributing to conditions such as cardiac hypertrophy. In the nervous system altered GIT1 activity can affect synaptic functions associated with disorders like schizophrenia. In these contexts interactions between GIT1 and other proteins such as beta-arrestins become significant impacting disease progression and therapeutic targeting.
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Quality control

STR analysis

CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX

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Cell culture

Biosafety level

EU: 2 US: 2

Adherent/suspension

Adherent

Gender

Female

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Product protocols

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