Anti-NDP52 抗体

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-NDP52 antibody (AB68588)

ab68588 staining NDP52 in HeLa cells +/- Chloroquine (50μM, 24 hours). The cells were fixed with 4% paraformaldheyde (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab68588 at 5μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control, at 1/1000 dilution. The secondary antibodies were ab150081 (colored green) and ab150120 (pseudo-colored red) used at 1/1000 dilution for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 μM for 1 hour at room temperature.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NDP52 antibody (AB68588)

IHC image of NDP52 staining in Human Cerebral Cortex FFPE section, performed on a BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab68588, 5μg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX

• WB

Lab

Western blot - Anti-NDP52 antibody (AB68588)

Western blot : Anti-CALCOCO2 antibody (ab68588) staining at 1 ug/ml, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab68588 was shown to bind specifically to CALCOCO2. A band was observed at 57 kDa in wild-type A549 cell lysates with no signal observed at this size in CALCOCO2 knockout cell line. To generate this image, wild-type and CALCOCO2 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-NDP52 antibody (ab68588) at 1 µg/mL

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

CALCOCO2 knockout A549 cell lysate at 20 µg

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

Jurkat cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 57 kDa

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• WB

Project

Western blot - Anti-NDP52 antibody (AB68588)

All lanes:

Western blot - Anti-NDP52 antibody (ab68588) at 1 µg/mL

Lane 1:

TE 671 (Human Rhabdomyosarcoma) Whole Cell Lysate at 10 µg

Lane 2:

HeLa Whole Cell Lysate - Hydroxyurea Treated (48hr, 2µM) at 10 µg

Lane 3:

Ramos (Human Burkitt's lymphoma cell line) Whole Cell Lysate at 10 µg

Lane 4:

Human placenta tissue lysate - total protein (ab29745) at 10 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Predicted band size: 52 kDa

Observed band size: 55 kDa

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Exposure time: 5min

• WB

CiteAb

Western blot - Anti-NDP52 antibody (AB68588)

Western Blotting using Anti-NDP52 antibody, ab68588. 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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• WB

CiteAb

Western blot - Anti-NDP52 antibody (AB68588)

Western Blotting using Anti-NDP52 antibody, ab68588. Publication image from Johnson, G. V. et al., 2014, Nat Commun, 24667209. Legend direct from paper.

NDP52 plays an important role in the clearance of phosphorylated tau(a) Primary cortical neurons were transduced with lentivirus expressing sh-RNA for Nrf2 or rat NDP52 (rNDP52), or a scrambled sh-RNA at DIV 1, and maintained until DIV 6. The levels of tau phosphorylated at Ser262/Ser356 and Ser396/Ser404 were analyzed by immunoblotting using 12E8- and PHF1-specific antibodies, respectively. Total tau was detected with a polyclonal tau-specific antibody (Tau). The relative molecular masses (kD) are indicated to the left of each blot. (b) Bar graph of the relative optical density of phosphorylated tau normalized to actin. Data shown are mean±SE of three independent experiments and were analyzed using Student’s t test. (*, p<0.05; ***, p<0.001) (c, d) Primary cortical neurons were transduced with a control lentivirus (FIGB) or with one expressing human NDP52 (hNDP52) at DIV 1. To induce autophagy, trehalose (150 mM) was added at DIV 5 and the neurons incubated for 24 h (DIV 6). Primary cortical neurons were fixed with 4% PFA, and stained with the 12E8 or PHF1 antibodies. The optical density of tau phosphorylated at Ser262/Ser356 (12E8). Scale bar = 20 µm. (e) and Ser396/Ser404 (PHF1) (f) in the soma of approximately 30 neurons randomly chosen was analyzed with the ImageJ program. Data were analyzed using Student’s t test (***, p<0.001)

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