Revision 2

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Orders:

877-616-CELL (2355)

[email protected]

Support:

877-678-TECH (8324)

3 Trask Lane | Danvers | Massachusetts | 01923 | USA

For Research Use Only. Not for Use in Diagnostic Procedures.

Product Includes Product # Quantity Mol. Wt Isotype/Source
LC3A/B (D3U4C) Rabbit Monoclonal Antibody1274120 µl14, 16 kDaRabbit IgG
Atg7 (D12B11) Rabbit Monoclonal Antibody855820 µl78 kDaRabbit IgG
Atg4B (D1G2R) Rabbit Monoclonal Antibody1350720 µl48 kDaRabbit IgG
Atg4A (D62C10) Rabbit Monoclonal Antibody761320 µl48-60 kDaRabbit IgG
GABARAP (E1J4E) Rabbit Monoclonal Antibody1373320 µl14, 16 kDaRabbit IgG
Atg3 Antibody341520 µl40 kDaRabbit
Anti-rabbit IgG, HRP-linked Antibody7074100 µlGoat

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

Description

The Autophagy Vesicle Elongation (LC3 Conjugation) Antibody Sampler Kit provides an economical means of detecting target proteins related to autophagy vesicle elongation pathway. The kit contains enough antibody to perform two western blots per primary.

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Background

Autophagy is a catabolic process for the autophagosomic-lysosomal degradation of bulk cytoplasmic contents (1,2). Autophagy is generally activated by conditions of nutrient deprivation, but it has also been associated with a number of physiological processes including development, differentiation, neurodegenerative diseases, infection, and cancer (3). Autophagy marker Light Chain 3 (LC3) was originally identified as a subunit of microtubule-associated proteins 1A and 1B (termed MAP1LC3) (4) and subsequently found to contain similarity to the yeast protein Apg8/Aut7/Cvt5 critical for autophagy (5). Three human LC3 isoforms (LC3A, LC3B, and LC3C) undergo post-translational modifications during autophagy (6-8). Cleavage of LC3 at the carboxy terminus immediately following synthesis yields the cytosolic LC3-I form. During autophagy, LC3-I is converted to LC3-II through lipidation by a ubiquitin-like system involving Atg7 and Atg3 that allows for LC3 to become associated with autophagic vesicles (6-9). The presence of LC3 in autophagosomes and the conversion of LC3 to the lower migrating form, LC3-II, have been used as indicators of autophagy (10). Numerous mammalian counterparts to yeast Atg proteins have been described, including three Atg8 proteins (GATE-16, GABARAP, and LC3) and four Atg4 homologs (Atg4A/autophagin-2, Atg4B/autophagin-1, Atg4C/autophagin-3, and Atg4D/autophagin-4) (10-12). The cysteine protease Atg4 is pivotal to autophagosome membrane generation and regulation (13). GABAA receptor associated protein (GABARAP) is an Atg8 family protein with a key role in autophagy, which was originally discovered as a protein associated with the GABAA receptor regulating receptor trafficking to the plasma membrane (14). Processing of GABARAP involves cleavage by Atg4 family members (15,16) followed by conjugation by the E1 and E2 like enzymes Atg7 and Atg3 (17,18).

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 2

Cell Signaling Technology Logo
Western blot analysis of extracts from RD cells, untreated (-) or Torin 1-treated (250 nM, 4 hr; +), using LC3A/B (D3U4C) XP® Rabbit mAb.
undefined Image 1: Autophagy Vesicle Elongation (LC3 Conjugation) Antibody Sampler Kit
Western blot analysis of extracts from various cell lines using Atg4B (D1G2R) Rabbit mAb.
undefined Image 2: Autophagy Vesicle Elongation (LC3 Conjugation) Antibody Sampler Kit
Western blot analysis of A172 and C2C12 cells, untreated (-) or chloroquine-treated (50 μM, overnight; +), using GABARAP (E1J4E) Rabbit mAb.
undefined Image 3: Autophagy Vesicle Elongation (LC3 Conjugation) Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 2

Cell Signaling Technology Logo
Western blot analysis of extracts from various cell lines using Atg3 Antibody.
undefined Image 4: Autophagy Vesicle Elongation (LC3 Conjugation) Antibody Sampler Kit
After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
undefined Image 5: Autophagy Vesicle Elongation (LC3 Conjugation) Antibody Sampler Kit
Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-) or SignalSilence® Atg4A siRNA I #6427 (+), using Atg4A (D62C10) Rabbit mAb (upper) or α-Tubulin (11H10) Rabbit mAb #2125 (lower). The Atg4A (D62C10) Rabbit mAb confirms silencing of Atg4A expression, while the α-Tubulin (11H10) Rabbit mAb is used as a loading control.
undefined Image 6: Autophagy Vesicle Elongation (LC3 Conjugation) Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 2

Cell Signaling Technology Logo
Western blot analysis of extracts from various cell lines using Atg7 (D12B11) Rabbit mAb.
undefined Image 7: Autophagy Vesicle Elongation (LC3 Conjugation) Antibody Sampler Kit
Western blot analysis of extracts from HeLa, NIH/3T3, and KNRK cells, untreated (-) or chloroquine-treated (50 μM, overnight; +), using LC3A/B (D3U4C) XP® Rabbit mAb.
undefined Image 8: Autophagy Vesicle Elongation (LC3 Conjugation) Antibody Sampler Kit
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing full-length mouse Atg4B (mAtg4B; +), using Atg4B (D1G2R) Rabbit mAb.
undefined Image 9: Autophagy Vesicle Elongation (LC3 Conjugation) Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 2

Cell Signaling Technology Logo
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with constructs expressing Myc/DDK-tagged full-length human GABARAP (hGABARAP-Myc/DDK; +), human GABARAPL1 (hGABARAPL1-Myc/DDK; +), or human GABARAPL2 (hGABARAPL2-Myc/DDK; +), using GABARAP (E1J4E) Rabbit mAb.
undefined Image 10: Autophagy Vesicle Elongation (LC3 Conjugation) Antibody Sampler Kit
Western blot analysis of extracts from HeLa cells, mock transfected or transfected with mouse Atg3, using Atg3 Antibody.
undefined Image 11: Autophagy Vesicle Elongation (LC3 Conjugation) Antibody Sampler Kit
Western blot analysis of extracts from various cell lines using Atg4A (D62C10) Rabbit mAb.
undefined Image 12: Autophagy Vesicle Elongation (LC3 Conjugation) Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 2

Cell Signaling Technology Logo
Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-) or SignalSilence® Atg7 siRNA I #6604 (+), using Atg7 (D12B11) Rabbit mAb (upper) or α-Tubulin (11Η10) Rabbit mAb #2125 (lower). The Atg7 (D12B11) Rabbit mAb confirms silencing of Atg7 expression, while the α-Tubulin (11H10) Rabbit mAb is used as a loading control.
undefined Image 13: Autophagy Vesicle Elongation (LC3 Conjugation) Antibody Sampler Kit
Immunohistochemical analysis of paraffin-embedded mouse prostate using LC3A/B (D3U4C) XP® Rabbit mAb.
undefined Image 14: Autophagy Vesicle Elongation (LC3 Conjugation) Antibody Sampler Kit
Western blot analysis of extracts from RD cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-) or SignalSilence® Atg4B siRNA I #6336 (+), using Atg4B (D1G2R) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). The Atg4B (D1G2R) Rabbit mAb confirms silencing of Atg4B expression, while the β-Actin (D6A8) Rabbit mAb is used as a loading control.
undefined Image 15: Autophagy Vesicle Elongation (LC3 Conjugation) Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 2

Cell Signaling Technology Logo
Confocal immunofluorescent analysis of A172 cells, untreated (left) and chloroquine-treated (50 μM, overnight; right), using GABARAP (E1J4E) Rabbit mAb (green) and β-Actin (8H10D10) Mouse mAb #3700 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
undefined Image 16: Autophagy Vesicle Elongation (LC3 Conjugation) Antibody Sampler Kit
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a DYKDDDDK-tagged human Atg4A construct (+), using Atg4A (D62C10) Rabbit mAb (upper) and DYKDDDDK Tag Antibody #2368 (lower).
undefined Image 17: Autophagy Vesicle Elongation (LC3 Conjugation) Antibody Sampler Kit
Immunohistochemical analysis of paraffin-embedded human squamous cell lung carcinoma using LC3A/B (D3U4C) XP® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).
undefined Image 18: Autophagy Vesicle Elongation (LC3 Conjugation) Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 2

Cell Signaling Technology Logo
Immunoprecipitation of Atg4B from RD cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype control #3900 (lane 2) or Atg4B (D1G2R) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using Atg4B (D1G2R) Rabbit mAb.
undefined Image 19: Autophagy Vesicle Elongation (LC3 Conjugation) Antibody Sampler Kit
Immunohistochemical analysis of paraffin-embedded NIH/3T3 cell pellets, control (left) or chloroquine-treated (right), using LC3A/B (D3U4C) XP® Rabbit mAb.
undefined Image 20: Autophagy Vesicle Elongation (LC3 Conjugation) Antibody Sampler Kit
Confocal immunofluorescent analysis of HeLa (upper) and C2C12 (lower) cells, chloroquine-treated (50 μM, overnight; left), nutrient-starved with EBSS (3 hr, middle) or untreated (right) using LC3A/B (D3U4C) XP® Rabbit mAb (green) and β-Actin (13E5) Rabbit mAb (Alexa Fluor® 555 Conjugate) #8046 (red). Blue pseudocolor= DRAQ5® #4084 (fluorescent DNA dye).
undefined Image 21: Autophagy Vesicle Elongation (LC3 Conjugation) Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 2

Cell Signaling Technology Logo
Flow cytometric analysis of HeLa cells, untreated (blue) or treated with chloroquine (50 µM, 16 hr; green) using LC3A/B (D3U4C) Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414 was used as a secondary antibody.
undefined Image 22: Autophagy Vesicle Elongation (LC3 Conjugation) Antibody Sampler Kit
Simple Western™ analysis of lysates (0.1 mg/mL) from Ba/F3 cells using Atg7 (D12B11) Rabbit mAb #8558. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ ​​​​​​​Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
undefined Image 23: Autophagy Vesicle Elongation (LC3 Conjugation) Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.