Macroautophagy: Novus Biologicals

Autophagy Detection Methods

Traditionally, the process of autophagy has been studied using electron microscopy. The discovery and characterization of ATG proteins facilitated the development of molecular tools and approaches to identify and quantitate autophagic activity. Ideally, a combination of approaches should be implemented including steady-state and flux measurements for the assessment of autophagic activity in different systems.

Steady-State Assay: Autophagosome Number

Assays to determine the amount or number of autophagosomes generally focus on the LC3 protein. LC3 may be found in the cytosolic and nuclear compartments. The lipidated form or LC3-II is the only protein known to associate with the autophagosome’s inner membrane. LC3-II levels, detected by immunofluorescence (ICC/IHC/IF) and immunoassay (WB) provide a good estimation of autophagosome number.

Monitoring Autophagic Flux

Autophagic flux refers to the complete processing of cargo, from sequestration to its degradation and recycling of basic components back to the cytosol. Increased LC3-II signal in immunofluorescence or immunoblot assays may result from increased autophagy. However, increased LC3-II or autophagosome number may also result from a blockade in autophagic flux. Therefore, to clearly distinguish between these mechanisms, assays that measure autophagic flux should be combined with steady-state assays.

Additional Methods to Monitor Autophagy

Assay	Method
GFP-LC3, monitors number of puncta as a measure of autophagic flux.	IF
Tandem mRFP/mCherry-GFP-LC3, monitors the change in fluorescence (yellow to red) as a measure of autophagic flux.	IF
GFP-LC3 Lysosomal delivery and proteolysis, monitors free GFP as a measure of autophagic flux.	WB

LC3-II Puncta (ICC/IHC/IF): This method combines the use of antibodies to LC3 and fluorescence microscopy to quantify fluorescent puncta, which correlate with the number of autophagosomes. This assay reports the number of puncta per cell or number of cells with puncta. Generally, basal autophagic activity is present and therefore most cells will have puncta.

LC3-II Puncta (ICC/IHC/IF) Immunocytochemistry/Immunofluorescence: Confocal analysis of HeLa cells using LC3B antibody ([NB600-1384], 1:5,000). An Alexa Fluor 488-conjugated Goat anti-rabbit IgG was used as secondary antibody (Green). Actin filaments were labeled with Alexa Fluor 568 Phalloidin (Red). DAPI was used to stain the cell nuclei (Blue).

Protocols

Inhibition of Autophagy and LC3B Antibody (NB100-2220) Immunocytochemistry (ICC)

Immunohistochemistry-Paraffin Protocol for LC3B/MAP1LC3B Antibody (NB100-2220)

LC3-Conversion (WB): This method measures the conversion of LC3, from unlipidated LC3-I to the lipidated form LC3-II. An increased conversion ratio correlates with increased autophagosome number. Quantification should be based on the comparison of LC3-II levels to the levels of a housekeeping protein (e.g., actin). In some systems, the levels of housekeeping proteins are affected by autophagy and LC3-I may be used for comparison.

LC3/MAP1LC3A Antibody Pack [NB910-40752] - Detection of LC3B in untreated and treated U87-MG (human glioblastoma astrocytoma) lysates using [NB600-1384]. Arsenic Trioxide induces autophagy by downregulating survivin.

The most cited LC3B antibody

Caveats to the use of LC3 for the Assessment of Autophagic Activity	ICC/IHC	WB
1. Tissues and cells differ in the magnitude of LC3 turnover.	√	√
2. LC3-II level alone is not enough evidence of autophagy.	√	√
3. LC3-II may associate with other membranous structures.	√	√
4. Some antibodies may not sufficiently bind to LC3-I.	√	√
5. LC3-I is more sensitive to degradation than LC3-II.		√
6. LC3 protein levels may be heterogeneous in a cell population or tissue.		√
7. To different LC3 family members may be involved in autophagy requiring the use of multiple antibodies.	√	√

Explore Autophagy Signaling Pathway

LC3 Turnover Assay (WB, ICC/IF): This assay monitors the accumulation of autophagosomes to determine autophagic flux. Reagents that block autophagic flux and prevent LC3-II degradation including lysosomotropic agents and protease inhibitors are commonly used. The difference in the amount of LC3-II in the presence and absence of these agents provides a measure of autophagic flux. This assay allows researchers to distinguish between true autophagy induction and blockade of autophagic flux.

HeLa human cervical epithelial carcinoma parental cell line and LC3B knockout HeLa cell line (KO) were untreated (-) or treated (+) with 50 μM Chloroquine for 18 hours. Whole cell protein lysates were prepared in 1x Laemmli sample buffer and approximately 10 μg of each lysate was separated on a 4-15% gel by SDS-PAGE, transferred to 0.2 μm PVDF membrane and blocked in 5% non-fat milk in TBST. PVDF membranes were probed with (A) rabbit anti- LC3B polyclonal [NB100-2220] and (B) rabbit anti-LC3B monoclonal [NBP2-46892] followed by HRP conjugated anti-rabbit IgG secondary antibody. A specific band was detected for LC3B at approximately 15 kDa (as indicated) in the parental HeLa cell line, but is not detectable in the knockout HeLa cell line. GAPDH is shown as a loading control. This experiment was conducted under reducing conditions.

The most cited LC3B antibody

Reagents for Autophagy Turnover Assay

Autophagy Inhibitors	Inhibitory Mechanism
Chloroquine Bafilomycin A1 Ammonium chloride Azithromycin	Inhibitors of Lysosome Acidification
E64d Pepstatin A1	Protease Inhibitors
Xanthohumol ML 240	Inhibitors of Autophagosome Maturation
Nocodazole Vinblastine	Inhibitors of Lysosome-Autophagosome Fusion

LAMP2/CD107b RNAi	Inhibitors of Autolysosome Maturation
LAMP2 CRISPR Knockout	Inhibitors of Autolysosome Maturation

Available at Tocris: www.tocris.com

Protocols

Inhibition of Autophagy and LC3B Antibody (NB100-2220) Western Blot

Western Blot Protocol for p62/SQSMT1 Antibody (NBP1-48320)

Western Blot Protocol for Atg5 Antibody (NB110-53818)

Western Blot Protocol for Atg16L1 Antibody (NB110-60928)

Explore Autophagy Signaling Pathway

p62/SQSTM1 Degradation Assay (WB, FC, ELISA, ICC/IF): Removal of cytosolic p62/SQSTM1 occurs primarily by autophagy. Therefore, the level of p62/SQSTM1 is inversely correlated to autophagic activity. Similar to LC3-II, blocking degradation of cytosolic components with Chloroquine, inhibits autophagic flux and leads to the accumulation of p62/SQSTM1. The levels of p62/SQSTM1 may be quantitated by flow cytometry, immunoblot and ELISA and serves as a biomarker for the analysis of autophagic flux.

p62/SQSTM1 Degradation Assay

HeLa cells mock (left) and treated with 50 μM Chloroquine for 24 hours (right) were fixed in 4% paraformaldehyde at room temperature for 15 minutes. (Green): SQSTM1 protein stained by SQSTM1 antibody (1478) [NBP2-43663] diluted at 1:1000. (Red): Actin stained with Phalloidin diluted at 1:200. (Blue): Hoechst 33342 staining.

Additional Targets to Monitor Autophagy

Target	Method	Considerations
mTOR, AMPK and ATG1/ULK1*	WB, IP, Kinase Assay	Levels of ULK1 in some systems are too low to detect phosphorylated forms. TOR activity inversely correlates with autophagic activity, although TOR independent pathways also control autophagy.
ATG12, ATG5 and ATG16L1	ICC/IF	Conjugation of ATG12-ATG5 may reflect autophagic activity, but in some cells these proteins mainly exist in the conjugated form.
WIPI (endogenous WIPI1 or WIPI2)	ICC/IF	WIPI positive puncta may not be present in all cells.

*ULK1 serves as a switch for autophagy regulation, mTOR and AMPK directly phosphorylate ULK1 inhibiting or activating its function, respectively.

Explore Autophagy Signaling Pathway