Western blot - Anti-NLRP3 antibody [EPR23094-1] (ab263899)

False colour image of Western blot: Anti-NLRP3 antibody [EPR23094-1] staining at 1/500 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab263899 was shown to bind specifically to NLRP3. A band was observed at 118 kDa in wild-type THP-1 cell lysates with no signal observed at this size in NLRP3 knockout cell line ab280063 (knockout cell lysate ab280122). To generate this image, wild-type and NLRP3 knockout THP-1 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution 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 (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed (ab216772) at 1/20000 dilution.

All lanes:

Western blot - Anti-NLRP3 antibody [EPR23094-1] (ab263899) at 1/500 dilution

Lane 1:

Wild-type THP-1 cell lysate at 20 µg

Lane 2:

NLRP3 knockout THP-1 cell lysate at 20 µg

Performed under reducing conditions.

Predicted band size: 118 kDa

Observed band size: 118 kDa

Immunoprecipitation - Anti-NLRP3 antibody [EPR23094-1] (ab263899)

NLRP3 was immunoprecipitated from 0.35 mg RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate with ab263899 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab263899 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/1000 dilution.

Lane 1: RAW 264.7 whole cell lysate 10μg

Lane 2: ab263899 IP in RAW 264.7 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab263899 in RAW 264.7 whole cell lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 10 seconds.

All lanes:

Immunoprecipitation - Anti-NLRP3 antibody [EPR23094-1] (ab263899)

Predicted band size: 118 kDa

Western blot - Anti-NLRP3 antibody [EPR23094-1] (ab263899)

The molecular weight/expression profile observed is consistent with what has been described in the literature (PMID: 26939933, 30315268).

The band around 75kDa is the short form of NLRP3 (NLRP3s).

Low expression cell line : Jurkat (PMID: 19767079)

Exposure times:Lane 1: 48 secs;Lane 2: 3 min;Lanes 3-4: 1 sec;Lane 5: 6 secs.

All lanes:

Western blot - Anti-NLRP3 antibody [EPR23094-1] (ab263899) at 1/1000 dilution

Lane 1:

THP-1 (human monocytic leukemia monocyte) whole cell lysate at 20 µg

Lane 2:

Jurkat (human t cell leukemia t lymphocyte) whole cell lysate at 20 µg

Lane 3:

Untreated RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 10 µg

Lane 4:

RAW 264.7 treated with 10 μg/ml LPS for 8 hours, whole cell lysate at 10 µg

Lane 5:

J774A.1 (mouse reticulum cell sarcoma monocyte macrophage) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 118 kDa

Immunoprecipitation - Anti-NLRP3 antibody [EPR23094-1] (ab263899)

NLRP3 was immunoprecipitated from 0.35 mg THP-1 (Human monocytic leukemia monocyte) whole cell lysate with ab263899 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab263899 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/1000 dilution.

Lane 1: THP-1 whole cell lysate 10μg

Lane 2: ab263899 IP in THP-1 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab263899 in THP-1 whole cell lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 30 seconds.

All lanes:

Immunoprecipitation - Anti-NLRP3 antibody [EPR23094-1] (ab263899)

Predicted band size: 118 kDa

Western blot - Anti-NLRP3 antibody [EPR23094-1] (ab263899)

Stimulation is required to allow detection of the NLRP3 protein in C6, PC-12 and NIH/3T3 cell lines.

All lanes:

Western blot - Anti-NLRP3 antibody [EPR23094-1] (ab263899) at 1/1000 dilution

Lane 1:

C6 (Rat glial tumor glial cell) whole cell lysate at 10 µg

Lane 2:

RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 10 µg

Lane 3:

PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate at 10 µg

Lane 4:

NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

Predicted band size: 118 kDa

Observed band size: 118 kDa

Exposure time: 15s