Anti-LC3B 抗体 - Autophagosome Marker

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-LC3B antibody - Autophagosome Marker (AB48394)

HeLa (human epithelial cell line from cervix adenocarcinoma) cells (wild type, left; LC3B knockout HeLa, right) stained for LC3B using ab48394 (red) at 0.3 μg/ml in ICC/IF. Primary antibody was incubated for 3 hours at room temperature, followed by NorthernLights™ 557-conjugated Anti-Rabbit IgG Secondary Antibody. Counterstained with DAPI (blue).

LC3 was detected in immersion fixed Cloroquine treated Hela cells (left) but was not detected in LC3 knockout Hela cells (right).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-LC3B antibody - Autophagosome Marker (AB48394)

ab48394 staining LC3B in HeLa cells treated with calmidazolium chloride (ab120658), by ICC/IF. Increase of LC3B expression correlates with increased concentration of calmidazolium chloride, as described in literature.
The cells were incubated at 37°C for 6h in media containing different concentrations of ab120658 (calmidazolium chloride) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab48394 (1 μg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

• IHC-P

PubMed

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LC3B antibody - Autophagosome Marker (AB48394)

Immunostaining of LC3 (green) in the arcuate hypothalamic nucleus of lean and obese mice (A; 8 weeks of a HFD or B; 16 weeks of a HFD). DAPI (blue) was used for nuclear staining.

The brain was excised after the mice were decapitated. Each SNC was fixed in 4% paraformaldehyde and each hypothalamus was processed for paraffin embedding and sectioned into 5.0 μm sections. Samples were incubated with primary antibodies overnight and with secondary antibodies conjugated to FITC or rhodamine for 2 hours (sc2777and sc2092, respectively; Santa Cruz Biotechnology, Santa Cruz, CA). The DAPI stain was used for nuclear staining while the Leica FW 4500 B microscope captured the images. Hypothalamic areas were observed according to the landmarks in the mouse brain atlas. Analysis and documentation of the results were performed using Leica Application Suite V3.6 (Switzerland).

Portovedo M. et al PLoS One. 2015 Mar 18;10(3):e0119850. doi: 10.1371/journal.pone.0119850. eCollection 2015 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

• ICC/IF

AbReview53768****

Immunocytochemistry/ Immunofluorescence - Anti-LC3B antibody - Autophagosome Marker (AB48394)

ab48394 staining LC3B in a Rat hepatocyte by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with formaldehyde, permeabilized with 0.2% Triton X-100 in PBS and blocked with 1% Donkey serum in 0.1% PBST for 60 minutes at 21°C. Samples were incubated with primary antibody (1/50 in PBS + 1% BSA) for 3 hours at 22°C. An Alexa Fluor^® 394-conjugated Donkey anti-rabbit IgG polyclonal was used as the secondary antibody at a dilution of 1/200.

This image is courtesy of an Abreview by Armen Petrosyan.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LC3B antibody - Autophagosome Marker (AB48394)

Formalin-fixed, paraffin-embedded mouse brain tissue stained for LC3B using ab48394 at 1/200 dilution in immunohistochemical analysis. The specific signal of LC3 was detected using HRP-conjugated secondary antibody with DAB reagent, and nuclei of cells were counterstained using hematoxylin. This LC3 antibody generated a low to moderate levels of cytoplasmic staining in the glial cells. The neurons depicted a moderate to strong staining for LC3 in their cytoplasm.

• WB

Supplier Data

Western blot - Anti-LC3B antibody - Autophagosome Marker (AB48394)

Western Blot shows lysates of HeLa (human epithelial cell line from cervix adenocarcinoma) cell line and LC3B knockout HeLa cell line (KO) untreated (-) or treated (+) with 50 uM Chloroquinine for 18 hours. PVDF membrane was probed with 0.5 ug/mL ab48394 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.

All lanes:

Western blot - Anti-LC3B antibody - Autophagosome Marker (ab48394)

Predicted band size: 15 kDa

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• WB

Supplier Data

Western blot - Anti-LC3B antibody - Autophagosome Marker (AB48394)

Western blot shows lysates of mouse NIH/3T3 (mouse embryo fibroblast cell line) and rat PC-12 (rat adrenal gland pheochromocytoma cell line) cell lines untreated (-) or treated (+) with Chloroquine. PVDF membrane was probed with 0.5 ug/mL rabbit anti-LC3B polyclonal Antibody (ab48394), followed by 1 : 2000 dilution of goat anti-rabbit IgG secondary antibody.

All lanes:

Western blot - Anti-LC3B antibody - Autophagosome Marker (ab48394)

Predicted band size: 15 kDa

false

• IHC-P

PubMed

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LC3B antibody - Autophagosome Marker (AB48394)

LC3B immunofluorescence in primary follicles of PD 13 ovaries. The red dots represent LC3b and DAPI (blue) indicated cell nuclei.

For the immunofluorescence analysis, the ovaries were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 5 μm slices. After antigen retrieval, the slides were blocked with goat serum and incubated with primary antibody (rabbit anti-LC3B at 1 : 200) overnight at 4°C. Alexa Fluor 594 (Invitrogen) was used as the secondary antibody in immunofluorescence assays.

Jiang C. et al PLoS Genet. 2017 Jan 10;13(1):e1006535. doi: 10.1371/journal.pgen.1006535. eCollection 2017 Jan. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

• WB

AbReview10746****

Western blot - Anti-LC3B antibody - Autophagosome Marker (AB48394)

All lanes:

Western blot - Anti-LC3B antibody - Autophagosome Marker (ab48394) at 1/2000 dilution

Lane 1:

Rat whole tissue lysate - Normal liver at 30 µg

Lane 2:

Rat whole tissue lysate - liver treated with AEE788 at 50 mg/kg 3 times a week for 1 week at 30 µg

Lane 3:

Rat whole tissue lysate - liver treated with RAD at 2.5 mg/kg daily for 1 week at 30 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab6721'>ab6721</a>) at 1/5000 dilution

Predicted band size: 15 kDa

Observed band size: 17 kDa

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Exposure time: 1min

This image is courtesy of an anonymous Abreview

• WB

CiteAb

Western blot - Anti-LC3B antibody - Autophagosome Marker (AB48394)

Western Blotting using Anti-LC3B antibody - Autophagosome MarkerAnti-LC3B antibody - Autophagosome Marker, ab48394. Publication image from Wirth, M. et al., 2019, Nat Commun, 31053714. Legend direct from paper.

Rendering GABARAP more LC3B-like impairs NDP52 degradation. a Control (wild-type) and hexa ATG8 CRISPR knockout (KO) HeLa cell lines stably expressing indicated MYC-ATG8 constructs or empty vector (EV) in full medium, Earle’s balanced salt solution (EBSS) (SM) or EBSS + bafilomycin A1 (BafA1) (SMB) for 7 h prior to lysis and immunoblot with indicated antibodies. Expression of MYC-ATG8 constructs was induced by 1 µg/ml doxycycline for 6 days. Note, immunoblot of non-induced cells showing equal p62 and NDP52 protein levels in the reconstituted hexa ATG8 CRISPR KO HeLa cell lines is included as Supplementary Fig. 7a. b, c Quantification of p62 (b) and NDP52 (c) protein levels (normalized to actin) shown in a. Statistical analysis of (SM) using one-way analysis of variance (ANOVA) test; mean ±s.d.; data from four (p62) and three (NDP52) independent experiments. ****p ≤ 0.0001; ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05; ns, not significant. d Structure of FYCO1 LIR motif bound to LC3B (PDB ID 5WRD). Non-conserved LIR docking site (LDS) residues of LC3B are displayed in white sticks. LC3B is displayed in white cartoon and the FYCO1 LIR in green transparent cartoon. Hydrophobic pocket (HP) 1 and HP2 are indicated in pink and blue, respectively. e Structure of PCM1 LIR motif bound to GABARAP (P212121). Non-conserved LDS residues of GABARAP are displayed in white sticks. GABARAP is displayed in white cartoon and the PCM1 LIR motif is shown in orange transparent cartoon. f Surface electrostatic potential of FYCO1 : LC3B structure in the same orientation as shown in d. g Surface electrostatic potential of PCM1 : GABARAP (P212121) structure with ULK1 LIR superimposed (pink cartoon). f, g Projections shown are −5 (red) and +5 (blue) kT/e using pymol apbs plugin with red and blue representing negative and positive potential, respectively. Red dashed lines encircle basic patch in LC3B (d, f) and extension of HP2 in GABARAP (e, f)

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