Anti-NEK7 antibody [EPR4900]

• IP

Lab

Immunoprecipitation - Anti-NEK7 antibody [EPR4900] (AB133514)

ab133514 immunoprecipitating NEK7. 10μg of cell lysate was incubated with primary antibody at a dilution of 1/20 and VeriBlot for IP Detection Reagent (HRP) (ab131366) at a dilution of 1/1000.

Lane 1 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate 10ug
Lane 2 : NIH/3T3 whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab133514 in NIH/3T3 whole cell lysate

All lanes:

Immunoprecipitation - Anti-NEK7 antibody [EPR4900] (ab133514)

Predicted band size: 35 kDa

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• WB

Lab

Western blot - Anti-NEK7 antibody [EPR4900] (AB133514)

Lane 1 : Wild-type HAP1 cell lysate (40 μg)
Lane 2 : NEK7 knockout HAP1 cell lysate (40 μg)
Lane 3 : A549 cell lysate (20 μg)
Lane 4 : Jurkat cell lysate (20 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab133514 observed at 35 kDa. Red - loading control, ab18058, observed at 124 kDa.

ab133514 was shown to specifically react with NEK7 when NEK7 knockout samples were used. Wild-type and NEK7 knockout samples were subjected to SDS-PAGE. ab133514 and ab18058 (loading control to Vinculin) were diluted at 1/10000 and 1/10,000 dilution respectively and incubated overnight at 4C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-NEK7 antibody [EPR4900] (ab133514)

Predicted band size: 35 kDa

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• WB

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Western blot - Anti-NEK7 antibody [EPR4900] (AB133514)

All lanes:

Western blot - Anti-NEK7 antibody [EPR4900] (ab133514) at 1/10000 dilution

Lane 1:

Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate at 20 µg

Lane 2:

C6 (Rat glial tumor glial cell) whole cell lysate at 20 µg

Lane 3:

NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 35 kDa

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• WB

Unknown

Western blot - Anti-NEK7 antibody [EPR4900] (AB133514)

All lanes:

Western blot - Anti-NEK7 antibody [EPR4900] (ab133514) at 1/10000 dilution

Lane 1:

Jurkat cell lysate at 10 µg

Lane 2:

A549 cell lysate at 10 µg

Lane 3:

C6 cell lysate at 10 µg

Lane 4:

RAW264.7 cell lysate at 10 µg

Lane 5:

PC12 cell lysate at 10 µg

Lane 6:

NIH/3T3 cell lysate at 10 µg

Secondary

All lanes:

Goat-anti-rabbit HRP at 1/2000 dilution

Predicted band size: 35 kDa

Observed band size: 32 kDa

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• WB

CiteAb

Western blot - Anti-NEK7 antibody [EPR4900] (AB133514)

Western Blotting using Anti-NEK7 antibody [EPR4900], ab133514. Publication image from Li, X. et al., 2017, Nat Commun, 28656979. Legend direct from paper.

Loss of MARK4 affects IL-1β production under NLRP3 inflammasome activation.(a) Mark4 expression under unprimed and LPS primed conditions in mouse bone marrow-derived macrophages (BMDM). Cytoplasm proximity ligation assay (PLA) signal per cell cytoplasm is used for quantification. Mean±s.e.m. for all the cells taken from 5 to 8 different views at x 40 magnification for each group. Experiments have been repeated three times. Scale bar, 10 µm. (b) siRNA of MARK4, NLRP3 or ASC caused reduction of ATP induced IL-1β production in THP-1 cells. (c) shRNA of MARK4 caused reduction of IL-1β induced by NLRP3 stimuli, including monosodium urate (MSU) and cholesterol crystals (CC) in THP-1 cells. Mock represents macrophages without further stimulation. (d) BMDM derived from Mark4 KO mice exhibited selectivity towards NLRP3 activating stimuli. Mean±s.e.m., three to four independent experiments combined (b–d), comparisons of the two different groups were analysed by unpaired t test. NS was considered as not statistically significant. *P<0.05, **P<0.01 and ***P<0.001 were considered as statistically significant (a–d). (e) Representative western blots of caspase-1 (Casp1) and IL-1β in the supernatant (Sup.) in Mark4+/+ and Mark4−/− macrophages activated with indicated stimuli. Cell lysates were used as controls to indicate equal amount of cells for analysis.

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