Anti-BTK (phospho Y223) antibody [EP420Y]

• IP

Unknown

Immunoprecipitation - Anti-BTK (phospho Y223) antibody [EP420Y] (AB68217)

ab68217 (purified) at 1/50 immunoprecipitating BTK (phospho Y223) in Ramos cells (Lane 1) treated with 1 mM Pervanadate for 30 min. For western blotting, a HRP-conjugated anti-rabbit IgG (H+L) was used as the secondary antibody (1/1000).

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Immunoprecipitation - Anti-BTK (phospho Y223) antibody [EP420Y] (ab68217)

Predicted band size: 76 kDa

Observed band size: 76 kDa

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• WB

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Western blot - Anti-BTK (phospho Y223) antibody [EP420Y] (AB68217)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-BTK (phospho Y223) antibody [EP420Y] (ab68217) at 1/1000 dilution

Lane 1:

Ramos (human Burkitt's lymphoma B lymphocyte) whole cell lysate at 10 µg

Lane 2:

Ramos treated with 1mM pervanadate for 30 min whole cell lysate at 10 µg

Lane 3:

Ramos treated with 1mM pervanadate for 30 min whole cell lysate. Then the membrane was incubated with alkaline phosphatase. at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 76 kDa

Observed band size: 76 kDa

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• WB

Lab

Western blot - Anti-BTK (phospho Y223) antibody [EP420Y] (AB68217)

All lanes:

Western blot - Anti-BTK (phospho Y223) antibody [EP420Y] (ab68217) at 1/10000 dilution

Lane 1:

Untreated K562 cell lysate at 10 µg

Lane 2:

K562 cells treated with pervanadate at 10 µg

Secondary

All lanes:

HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 76 kDa

Observed band size: 76 kDa

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• Dot

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Dot Blot - Anti-BTK (phospho Y223) antibody [EP420Y] (AB68217)

Dot blot analysis of BTK (phospho Y223) phospho peptide (Lane 1) and BTK phospho peptide (Lane 2) labeling BTK (phospho Y223) with ab68217 at a dilution of 1/1000. A Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) was used as the secondary antibody at a dilution of 1/20,000. Blocking buffer : 5% NFDM/TBST. Dilution buffer : 5% NFDM /TBST.

• WB

CiteAb

Western blot - Anti-BTK (phospho Y223) antibody [EP420Y] (AB68217)

Western Blotting using Recombinant Anti-BTK (phospho Y223) antibody [EP420Y], ab68217. Publication image from Yoshimura, A. et al., 2015, Nat Commun, 26059659. Legend direct from paper.

BTK promotes ASC aggregation and interacts with ASC and NLRP3.(a) Immunoblot analysis of ASC in BS3-treated or -untreated cell lysates of LPS-primed murine peritoneal macrophages that were pretreated with LFM-A13 and then stimulated with nigericin for 30 min. (b) Immunostaining of ASC of LPS-primed murine peritoneal macrophages that were pretreated with LFM-A13 and then stimulated with nigericin for 30 min. Nuclei were counterstained with Hoechst 33342. ASC, green; nuclei, blue. Scale bars, 100 µm. (c) Immunoblot analysis of ASC in the Triton X-soluble and -insoluble fractions of LPS-primed murine peritoneal macrophages that were pretreated with LFM-A13 and then stimulated with ATP or poly(dA : dT) for 2 h. (d) Immunoblot analysis of human NLRP3, BTK and ASC in the Triton X-soluble and -insoluble fractions of HEK 293T cells transfected with Flag-ASCWT or -ASCY146F, T7-NLRP3, or HA-BTK. Six hours after the transfection, LFM-A13 was added to the cell culture. Data are representative of three independent experiments. (e) Co-immunoprecipitation and immunoblot assays of HA-BTK and NLRP3 or ASC from PMA-primed and -unprimed THP-1 cells that stably expressed HA-BTK. Thirty minutes after the pretreatment with LFM-A13, cells were stimulated with alum for 3 h. (f) In-situ proximity-ligation assay of BTK–NLRP3 complexes in LPS-primed murine peritoneal macrophages pretreated with LFM-A13 and then stimulated with nigericin for 30 min. BTK–NLRP3 complexes, red; nuclei, blue. Data are representative of three independent experiments. Scale bars, 10 µm.

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