Image 1: Structure of LC3B target protein.

Introduction to LC3B

Protein Function

• LC3B is a widely used autophagy marker.  Ubiquitin-like modifier involved in formation of autophagosomal vacuoles (autophagosomes).
• Plays a role in mitophagy which contributes to regulate mitochondrial quantity and quality by eliminating the mitochondria to a basal level to fulfill cellular energy requirements and preventing excess ROS production.
• While LC3s are involved in elongation of the phagophore membrane, the GABARAP/GATE-16 subfamily is essential for a later stage in autophagosome maturation.
• Promotes primary ciliogenesis by removing OFD1 from centriolar satellites via the autophagic pathway.
• Through its interaction with the reticulophagy receptor TEX264, participates in the remodeling of subdomains of the endoplasmic reticulum into autophagosomes upon nutrient stress, which then fuse with lysosomes for endoplasmic reticulum turnover.

Protein Characteristics

• The precursor molecule is cleaved by APG4B/ATG4B to form LC3-I. This is activated by APG7L/ATG7, transferred to ATG3 and conjugated to phospholipid to form LC3-II.

Protein Expression

• Most abundant in heart, brain, skeletal muscle and testis.
• Little expression observed in liver.

Protein Localization

• Can localize to the cytoplasm, cytoskeleton, endomembrane system, lipid vesicles, and autophagosome membranes.
• LC3B-I is predominantly localized in the cytoplasm, LC3-II binds to the autophagic membranes.

Figure 2:  LC3B ICC experimental results using Anti-LC3B [EPR18709] – Autophagosome Marker (ab192890).
ab192890 staining LC3B in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells +/- Chloroquine (50μM, 24 hours).
Green: LC3B, Red: Tubulin, Blue: DAPI.

Isoforms & Post-Translational Modifications

• Human (Q9GZQ8): Predicted molecular weight of 15 kDa.
• Mouse (Q9CQV6): Predicted molecular weight of 15 kDa.
• Rat (Q62625): Predicted molecular weight of 16 kDa.
• LC3B can undergo phosphorylation and lipidation.

WB Experiment Tips

Precautions

• LC3B signal may be weak in some untreated samples; treatment with Bafilomycin A1 / Rapamycin / Hydroxychloroquine / Chloroquine can enhance signal intensity.
• The ratio of detected LC3B-I and II may vary across different samples.

Positive Controls

• Cell lines: BMDM, U-87 MG, C6, and RAW 264.7 whole cell lysates.
• Tissues: Human brain, mouse heart, rat heart, mouse brain, and rat brain lysates.

Example Results

Figure 3: WB - Anti-LC3B Antibody [EPR18709] – Autophagosome Marker (ab192890)

Lane 1 : Human brain lysate
Lane 2 : U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) whole cell lysate
Lane 3 : C6 (Rat glial tumor cell line) whole cell lysate
Lane 4 : RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 5 : Mouse heart lysate
Lane 6 : Rat heart lysate
Lane 7 : Mouse brain lysate
Lane 8 : Rat brain lysate

Primary Antibody:  Anti-LC3B Antibody [EPR18709] – Autophagosome Marker (ab192890), at 1/2000 dilution.
Secondary Antibody: Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution.

Predicted Band Size: 15 kDa
Observed Band Sizes: 14 kDa, 16 kDa

Key control points

In the experiment, in addition to paying attention to routine issues, special attention should be paid to the following key control points:

Sample preparation:

1. Add a complex proteinase inhibitor to avoid degradation of the target protein.
2. Keep the sample on ice throughout the sample preparation process.
3. Determine the total protein concentration of the sample through Bradford analysis, Lowry analysis, or BCA analysis.

Electrophoresis:

1. For target proteins with smaller molecular weights, use a higher concentration of separation gel for electrophoresis.
2. Load at least 20μg total protein for electrophoresis.

Transfer:

1. For target proteins with smaller molecular weights, it is recommended to use a 0.22μm PVDF membrane.
2. It is recommended to use Coomassie Brilliant Blue staining after transfer to confirm the success of the transfer.

References

1. Yongjie Wei, Wei-Chung Chiang, Rhea Sumpter Jr, Prashant Mishra, Beth Levine. Prohibitin 2 Is an Inner Mitochondrial Membrane Mitophagy Receptor. Cell. 2017 Jan 12;168(1-2):224-238.e10. doi: 10.1016/j.cell.2016.11.042. Epub 2016 Dec 22.
2. Haruka Chino, Tomohisa Hatta, Tohru Natsume, Noboru Mizushima. Intrinsically Disordered Protein TEX264 Mediates ER-phagy. Mol Cell. 2019 Jun 6;74(5):909-921.e6. doi: 10.1016/j.molcel.2019.03.033. Epub 2019 Apr 18.
3. Heeseon An, Alban Ordureau, Joao A Paulo, Christopher J Shoemaker, Vladimir Denic, J Wade Harper. TEX264 Is an Endoplasmic Reticulum-Resident ATG8-Interacting Protein Critical for ER Remodeling during Nutrient Stress. Mol Cell. 2019 Jun 6;74(5):891-908.e10. doi: 10.1016/j.molcel.2019.03.034. Epub 2019 Apr 18.
4. Y Kabeya , N Mizushima, T Ueno, A Yamamoto, T Kirisako, T Noda, E Kominami, Y Ohsumi, T Yoshimori. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 2000 Nov 1;19(21):5720-8. doi: 10.1093/emboj/19.21.5720.