Molecular cloning and characterization of rat LC3A and LC3B—Two novel markers of autophagosome

Wu, Jiaxue; Dang, Yongjun; Su, Wei; Liu, Chao; Ma, Hongshen; Shan, Yuxi; Pan, Yuan; Wan, Bo; Guo, Jinhu; Liu, Yu

doi:10.1016/j.bbrc.2005.10.211

Cited by 161 publications

(122 citation statements)

References 22 publications

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“…Because the presence of autophagic vacuoles is by far the most important morphological feature of cells with autophagic activity, demonstration of these structures by transmission electron Alb-Cre + mice were stained for LC3A and LC3B, two LC3 isoforms known to be associated with autophagic membranes. 22 For this purpose, we used a highly sensitive immunohistochemical detection method (Vectastain ABC), which is based on the formation of large macromolecular complexes containing avidin and biotinylated horseradish peroxidase. Western blot experiments revealed that rabbit polyclonal LC3A raised against the C-terminal cleavage site of LC3A as well as mouse monoclonal LC3B antibody (clone 5F10) and rabbit monoclonal LC3B antibody (clone D11) raised against the N-terminus of LC3B, were highly Furthermore, TEM revealed autophagic vacuoles in starved hepatocytes, but not in control liver (Fig.…”

Section: Resultsmentioning

confidence: 99%

Immunohistochemical analysis of macroautophagy

et al. 2013

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T ransmission electron microscopy (TEM) is an indispensable standard method to monitor macroautophagy in tissue samples. Because TEM is time consuming and not suitable for daily routine, many groups try to identify macroautophagy in tissue by conventional immunohistochemistry. The aim of the present study was to evaluate whether immunohistochemical assessment of macroautophagy-related marker proteins such as LC3, ATG5, CTSD/cathepsin D, BECN1/Beclin 1 or SQSTM1/p62 is feasible and autophagy-specific. For this purpose, livers from starved mice were used as a model because hepatocytes are highly sensitive to autophagy induction. ATG7-deficient mouse livers served as negative control. Our findings indicate that unambiguous immunodetection of LC3 in paraffin-embedded tissue specimens was hampered due to low in situ levels of this protein. Maximum sensitivity could only be obtained using high-quality, isoform-specific antibodies, such as antibody 5F10, in combination with Envision+ signal amplification. Moreover, LC3 stains were optimal in neutral-buffered formalin-fixed tissue, immersed in citrate buffer during antigen retrieval. However, even when using this methodology, LC3 monitoring required overexpression of the protein, e.g., in GFP-LC3 transgenic mice. This was not only the case for the liver but also for other organs including heart, skeletal muscle, kidney and gut.

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Section: Resultsmentioning

confidence: 99%

Immunohistochemical analysis of macroautophagy

et al. 2013

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“…Among the three isoforms MAP1LC3A (or simply LC3A) is a well-defined autophagosomal marker with a conserved post-translational modification at the C-terminal Gly120. 7,8 In contrast, the post-translational modification of the MAP1LC3B isoform seems not to be conserved. [7][8][9] Immunohistochemical analysis.…”

Section: Resultsmentioning

confidence: 99%

“…7,8 In contrast, the post-translational modification of the MAP1LC3B isoform seems not to be conserved. [7][8][9] Immunohistochemical analysis. The LC3A protein was detectable in the cytoplasm of normal mice tissues in the liver (both hepatocytes and hepatic sinusoids), kidney (tubular cells) and lung (bronchial and alveolar epithelium) (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…7 In contrast, Wu and coworkers reported that MAP1LC3A is abundant in heart, brain, skeletal muscle, lung and testis and undetectable in liver, pancreas, spleen and small intestine of rat tissues, whereas MAP1LC3B is expressed highly in liver, brain, skeletal muscle and testis but is undetectable in lung. 8 Wu and coworkers revealed that both LC3A and LC3B are associated with the autophagic membranes in rats. 8 Furthermore, MAP1LC3C is poorly expressed in tissues, showing a relative prevalence in placenta, lung and ovaries.…”

Section: Resultsmentioning

confidence: 99%

“…8 Wu and coworkers revealed that both LC3A and LC3B are associated with the autophagic membranes in rats. 8 Furthermore, MAP1LC3C is poorly expressed in tissues, showing a relative prevalence in placenta, lung and ovaries. 7 In our study we were able to detect the mRNA levels of LC3A in liver, kidney and lung mouse tissues and, furthermore, imunohistochemistry confirmed cytoplasmic localization of LC3A in these tissues.…”

Section: Resultsmentioning

confidence: 99%

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"Autophagic flux" in normal mouse tissues: Focus on endogenous LC3A processing

Zois¹,

Giatromanolaki²,

Sivridis³

et al. 2011

Autophagy

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A utophagy is a major intracellular pathway for the degradation and recycling of long-lived proteins, mature ribosomes and even entire organelles. The best-studied autophagic marker is the LC3B protein and it is thought that the amount of LC3B-II correlates with the amount of autophagic membranes. Whether LC3A processing, in addition to LC3B, is a valuable endogenous "autophagic flux'' marker is far less clear. The specificity of rabbit polyclonal antibodies to LC3A and LC3B was tested against the commercial available human recombinant proteins LC3A and LC3B. In order to measure "autophagic flux'' in mouse liver, lung, kidney and heart we used: (1) a lysosomotropic reagent, chloroquine, which inhibits intralysosomal acidification and ultimately their fusion with autophagosomes, (2) nutrient starvation as an autophagic stimulus and (3) ionizing radiation, which destabilizes lysosomes. According to the immunoblotting work, the LC3A protein follows discrete patterns of LC3A-I and LC3A-II changes in liver, lung, kidney and heart tissues of mice, whereas the LC3B protein did not follow the same pattern under stressor conditions. We conclude that the endogenous LC3A processing is a major marker of autophagy flux in the mouse model. Fractionated samples (soluble vs membrane fractions) should be used in immunoblotting to allow discrimination between the soluble LC3-I and the membrane-associated LC3-II proteins and the kinetics of modification. Furthermore, "Autophagic flux" in normal mouse tissues

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Inhibition of Autophagy by a Small Molecule through Covalent Modification of the LC3 Protein

Luo

Fan

et al. 2021

Angewandte Chemie

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The autophagic ubiquitin‐like protein LC3 functions through interactions with LC3‐interaction regions (LIRs) of other autophagy proteins, including autophagy receptors, which stands out as a promising protein–protein interaction (PPI) target for the intervention of autophagy. Post‐translational modifications like acetylation of Lys49 on the LIR‐interacting surface could disrupt the interaction, offering an opportunity to design covalent small molecules interfering with the interface. Through screening covalent compounds, we discovered a small molecule modulator of LC3A/B that covalently modifies LC3A/B protein at Lys49. Activity‐based protein profiling (ABPP) based evaluations reveal that a derivative molecule DC‐LC3in‐D5 exhibits a potent covalent reactivity and selectivity to LC3A/B in HeLa cells. DC‐LC3in‐D5 compromises LC3B lipidation in vitro and in HeLa cells, leading to deficiency in the formation of autophagic structures and autophagic substrate degradation. DC‐LC3in‐D5 could serve as a powerful tool for autophagy research as well as for therapeutic interventions.

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Molecular cloning and characterization of rat LC3A and LC3B—Two novel markers of autophagosome

Cited by 161 publications

References 22 publications

Immunohistochemical analysis of macroautophagy

Immunohistochemical analysis of macroautophagy

"Autophagic flux" in normal mouse tissues: Focus on endogenous LC3A processing

Inhibition of Autophagy by a Small Molecule through Covalent Modification of the LC3 Protein

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