Revision 1

#43566Store at -20C

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:

WB, IF-IC

REACTIVITY:

H M R

SENSITIVITY:

Endogenous

MW (kDa):

14, 16

Source/Isotype:

Rabbit IgG

UniProt ID:

#Q9CQV6

Entrez-Gene Id:

67443

Product Information

Product Usage Information

Application Dilution
Western Blotting 1:1000
Immunofluorescence (Immunocytochemistry) 1:6400 - 1:12800

Specificity / Sensitivity

LC3B (E7X4S) XP® Rabbit mAb recognizes endogenous levels of total LC3B protein. This antibody detects both type I and type II forms of LC3B. It weakly cross-reacts with LC3A by overexpression.

Species Reactivity:

Human, Mouse, Rat

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human LC3B protein.

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 posttranslational modifications during autophagy (6-9). 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 (6-10). 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 (11).

  1. Reggiori, F. and Klionsky, D.J. (2002) Eukaryot. Cell 1, 11-21.
  2. Codogno, P. and Meijer, A.J. (2005) Cell Death Differ. 12 Suppl 2, 1509-18.
  3. Levine, B. and Yuan, J. (2005) J. Clin. Invest. 115, 2679-88.
  4. Mann, S.S. and Hammarback, J.A. (1994) J. Biol. Chem. 269, 11492-97.
  5. Lang, T. et al. (1998) EMBO J. 17, 3597-607.
  6. Kabeya, Y. et al. (2000) EMBO J. 19, 5720-28.
  7. He, H. et al. (2003) J. Biol. Chem. 278, 29278-87.
  8. Tanida, I. et al. (2004) J. Biol. Chem. 279, 47704-10.
  9. Wu, J. et al. (2006) Biochem. Biophys. Res. Commun. 339, 437-42.
  10. Ichimura, Y. et al. (2000) Nature 408, 488-92.
  11. Kabeya, Y. et al. (2004) J. Cell Sci. 117, 2805-12.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting IF-IC: Immunofluorescence (Immunocytochemistry)

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
KARPAS cell line source: Dr. Abraham Karpas at the University of Cambridge.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

使用に関する制限

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Revision 1
#43566

LC3B (E7X4S) XP® Rabbit mAb

Western Blotting Image 1: LC3B (E7X4S) XP® Rabbit mAb Expand Image
Western blot analysis of extracts from HCT 116 and HCT 116 LC3B knockout cells, untreated (-) or treated with Chloroquine #14774 (50 μM, 18 hr) using LC3B (E7X4S) XP® Rabbit mAb #43566 (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The absence of signal in the HCT 116 knockout cells confirms the specificity of the antibody for LC3B.
Western Blotting Image 2: LC3B (E7X4S) XP® Rabbit mAb Expand Image
Western blot analysis of extracts from HeLa, C2C12, and KNRK cells, untreated (-) or treated with Chloroquine #14774 (50 μM, 18 hr; +), using LC3B (E7X4S) XP® Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western Blotting Image 3: LC3B (E7X4S) XP® Rabbit mAb Expand Image
Western blot analysis of extracts from control HeLa cells (lane 1) or LC3B knockout HeLa cells (lane 2) using LC3B (E7X4S) XP® Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The absence of signal in the LC3B knockout HeLa cells confirms specificity of the antibody for LC3B.
Western Blotting Image 4: LC3B (E7X4S) XP® Rabbit mAb Expand Image
Western blot analysis from KARPAS 299 and VCap cells using LC3B (E7X4S) XP® Rabbit mAb #43566 (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The low signal of LC3B in VCap cells is predicted from RNAseq data and confirms the specificity of the antibody for LC3B.
Immunofluorescence Image 1: LC3B (E7X4S) XP® Rabbit mAb Expand Image
Confocal immunofluorescent analysis of HCT 116 cells either untreated (left) or treated with Chloroquine #14774 (50 µM, overnight) (center) or LC3B HCT 116 knockout cells treated with Chloroquine #14774 (50 µM, overnight) (right) using LC3B (E7X4S) XP® Rabbit mAb (green). Actin filaments were labeled with β-Actin (8H10D10) Mouse mAb (red) and nuclei were labeled with DAPI #4083 (blue).
Immunofluorescence Image 2: LC3B (E7X4S) XP® Rabbit mAb Expand Image
Confocal immunofluorescent analysis of MIA PaCa-2 cells untreated (left), nutrient-starved with EBSS (2 hr; middle), or treated with Chloroquine #14774 (50 µM, 24 hr; right) using LC3B (E7X4S) XP® Rabbit mAb (green) and β-Actin (8H10D10) Mouse mAb #3700 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
Immunofluorescence Image 3: LC3B (E7X4S) XP® Rabbit mAb Expand Image
Confocal immunofluorescent analysis of C2C12 cells untreated (left), nutrient-starved with EBSS (2 hr; middle), or treated with Chloroquine #14774 (50 µM, 24 hr; right) using LC3B (E7X4S) XP® Rabbit mAb (green) and β-Actin (8H10D10) Mouse mAb #3700 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).