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AB265814

Human ATG4B knockout HeLa cell line

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(1 Publication)

ATG4B KO cell line available to order. KO validated by Western blot. Free of charge wild type control available. Knockout achieved by using CRISPR/Cas9, 1 bp deletion in exon 4 and 1 bp insertion in exon 4. To order both knockout and wild-type control cells: select '2 x 1000000 Cells/vial'. To order only knockout cells: select '1000000 Cells/vial'.
4 Images
Western blot - Human ATG4B knockout HeLa cell line (AB265814)
  • WB

Lab

Western blot - Human ATG4B knockout HeLa cell line (AB265814)

Lanes 1 - 2 : Merged signal (red and green). Green - ab199537 observed at 47 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
ab199537 was shown to react with ATG4B in wild-type HeLa cells in Western blot with loss of signal observed in ATG4B knockout cell line ab260973 (ATG4B knockout cell lysate ab257364). Wild-type and ATG4B knockout HeLa cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with ab199537 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-ATG4B antibody [EPR16572] (<a href='/en-us/products/primary-antibodies/atg4b-antibody-epr16572-ab199537'>ab199537</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

ATG4B knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human ATG4B knockout HeLa cell line (ab265814)

Predicted band size: 44 kDa

Observed band size: 47 kDa

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Western blot - Human ATG4B knockout HeLa cell line (AB265814)
  • WB

Lab

Western blot - Human ATG4B knockout HeLa cell line (AB265814)

Lanes 1 - 2 : Merged signal (red and green). Green - ab154843 observed at 47 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
ab154843 was shown to react with ATG4B in wild-type HeLa cells in Western blot with loss of signal observed in ATG4B knockout cell line ab260973 (ATG4B knockout cell lysate ab257364). Wild-type and ATG4B knockout HEK293T cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with ab154843 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-ATG4B antibody [EPR6436(2)] (<a href='/en-us/products/primary-antibodies/atg4b-antibody-epr64362-ab154843'>ab154843</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

ATG4B knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human ATG4B knockout HeLa cell line (ab265814)

Predicted band size: 44 kDa

Observed band size: 47 kDa

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Sanger Sequencing - Human ATG4B knockout HeLa cell line (AB265814)
  • Sanger seq

Unknown

Sanger Sequencing - Human ATG4B knockout HeLa cell line (AB265814)

Allele-1 : 1 bp deletion in exon 4.

Sanger Sequencing - Human ATG4B knockout HeLa cell line (AB265814)
  • Sanger seq

Unknown

Sanger Sequencing - Human ATG4B knockout HeLa cell line (AB265814)

Allele-2 : 1 bp insertion in exon 4.

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, 1 bp deletion in exon 4 and 1 bp insertion in exon 4

Disease

Adenocarcinoma

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Cell Lines & Lysates

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Advanced Validation

Western blot - Human ATG4A knockout HeLa cell line (AB265738)

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Primary Antibodies

AB154843

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Proteins & Peptides

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Reactivity data

{ "title": "Reactivity Data", "filters": { "stats": ["", "Reactivity", "Dilution Info", "Notes"] }, "values": { "WB": { "reactivity":"TESTED_AND_REACTS", "dilution-info":"", "notes":"<p></p>" } } }
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Product details

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
ATG4B
Gene editing type
Knockout
Gene editing method
CRISPR technology
Knockout validation
Sanger Sequencing, Western blot
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.

ATG4B is a cysteine protease also known as autophagin-1 with a molecular mass of approximately 45 kDa. It is involved in the autophagy process by cleaving ATG8 family proteins. This protease facilitates the lipidation of LC3 an essential component of the autophagosome membrane. You find ATG4B expressed in various tissues especially in the brain liver and muscle where it plays important roles in cellular maintenance.
Biological function summary

ATG4B functions in the cellular autophagic machinery cleaving the C-terminal of ATG8 proteins. This processing is necessary for the conjugation to phosphatidylethanolamine and integration into autophagosomes. ATG4B is part of a larger autophagic complex that also includes other critical proteins like ATG7 and ATG3 helping prepare ATG8 proteins for autophagosome creation.

Pathways

ATG4B participates in the autophagy pathway by refining the cargo processing mechanism. It interacts with the mTOR signaling pathway which influences cell survival and growth based on nutrient availability. The ATG4B enzyme is closely related to ATG7 in the pathway as both contribute to the maturation of autophagosomes.

ATG4B has significant links to cancer and neurodegenerative disorders. In cancer ATG4B activity affects tumor survival since cancer cells exploit autophagy for growth under stress conditions. In neurodegenerative diseases like Alzheimer's alterations in ATG4B activity disrupt cellular homeostasis. This disruption connects to proteins like LC3 which are pivotal in neuron survival and function.
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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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Target data

See full target information ATG4B
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Publications (1)

Recent publications for all applications. Explore the full list and refine your search

BMB reports 57:497-502 PubMed39384175

2024

Differential roles of N- and C-terminal LIR motifs in the catalytic activity and membrane targeting of RavZ and ATG4B proteins.

Applications

Unspecified application

Species

Unspecified reactive species

Sang-Won Park,Ju-Hui Park,Haneul Choi,Pureum Jeon,Seung-Hwan Lee,Won-Dong Shin,Hun-Joo Kim,Jin-A Lee,Deok-Jin Jang
View all publications
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Product promise

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For full details, please see our Terms & Conditions

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