Anti-JNK1 + JNK2 (phospho T183 + Y185) antibody

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-JNK1 + JNK2 (phospho T183 + Y185) antibody (AB4821)

ab4821 staining JNK1 + JNK2 (phospho T183 + Y185) in A549 cells (green, panel a) by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% paraformaldehyde, permeabilized with 0.25% Triton X-100 and blocked with 5% BSA for 1 hour at room temperature. Samples were incubated with primary antibody (2ug/ml in 1% BSA) for 3 hours at room temperature. An Alexa Fluor® 488-conjugated Goat anti-rabbit IgG polyclonal was used as the secondary antibody (1/400). Nuclei stained with DAPI (blue, panel b), F-actin stained with Alexa Fluor® 594 Phalloidin (red, panel b) and merged images (panel d).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-JNK1 + JNK2 (phospho T183 + Y185) antibody (AB4821)

ab4821 staining JNK1+JNK2 (phospho T183 + Y185) in human foreskin fibroblasts by ICC/IF. The cells were fixed in cytoskeletal fixative, permeabilized in 0.5% Triton X-100 and blocked in 2% dillution buffer (2%BSA + 0.1% Triton X-100) for 1 hour at 25°C. The primary antibody was diluted, 1/100 and incubated with sample for 12 hours. An Alexa Fluor® 594 conjugated goat polyclonal to rabbit IgG, diluted 1/250 was used as secondary.

This image is courtesy of an Abreview submitted by Mr George Chennell

• FuncS

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Functional Studies - Anti-JNK1 + JNK2 (phospho T183 + Y185) antibody (AB4821)

MCF7cells were incubated at 37°C for 4h with vehicle control (0 μM) and different concentrations of cryptotanshinone (ab120666). Increased expression of JNK1+JNK2 (phospho T183 + Y185) in MCF7 cells correlates with an increase in cryptotanshinone concentration, as described in literature.

Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 10μg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 5% BSA before being incubated with ab4821 at 1/1000 dilution and ab8227 at 1 μg/ml overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (ab97051) at 1/10000 dilution and visualised using ECL development solution.

• FuncS

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Functional Studies - Anti-JNK1 + JNK2 (phospho T183 + Y185) antibody (AB4821)

MEF1 cells were incubated at 37°C for 48h with vehicle control (0 μM) and 5 μM of glibenclamide (ab120267) in DMSO. Increased expression of of JNK1+JNK2 (phospho T183 + Y185) (ab4821) correlates with an increase in glibenclamide concentration, as described in literature.

Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 10μg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 3% milk before being incubated with ab4821 at 1/1000 dilution and ab85139 at 1 μg /ml overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (ab97051) at 1/10000 dilution and visualised using ECL development solution.

• WB

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Western blot - Anti-JNK1 + JNK2 (phospho T183 + Y185) antibody (AB4821)

To demonstrate the phosphorylation of JNK 1 & 2 in a cell based assay, 293 cells were treated with ultraviolet irradiation (UV). Proteins from cell extracts were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to nitrocellulose. Membranes were incubated with either 1 μ g/mL ab4821 or 1 μg/mL anti-JNK1 pan. After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG alkaline phosphatase and bands were detected using the Tropix WesternStar detection method.
To demonstrate the phosphorylation of JNK 1 & 2 in a cell based assay, 293 cells were treated with ultraviolet irradiation (UV). Proteins from cell extracts were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to nitrocellulose. Membranes were incubated with either 1 µ g/mL ab4821 or 1 µg/mL anti-JNK1 pan. After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG alkaline phosphatase and bands were detected using the Tropix We

All lanes:

Western blot - Anti-JNK1 + JNK2 (phospho T183 + Y185) antibody (ab4821)

Predicted band size: 48 kDa

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• WB

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Western blot - Anti-JNK1 + JNK2 (phospho T183 + Y185) antibody (AB4821)

Proteins were transferred to a nitrocellulose membrane and blocked with 5% skim milk for 1 hour at room temperature.

All lanes:

Western blot - Anti-JNK1 + JNK2 (phospho T183 + Y185) antibody (ab4821) at 1/1000 dilution

Lane 1:

HEK-293 cell line at 20 µg

Lane 2:

HEK-293 treated for 5 minutes with 200 mM of Anisomycin at 20 µg

Lane 3:

HEK-293 treated for 20 minutes with UV at 20 µg

Lane 4:

MCF7 cell line at 20 µg

Lane 5:

MCF7 treated for 5 minutes with 200 mM of Anisomycin at 20 µg

Lane 6:

K562 cell line at 20 µg

Lane 7:

K562 treated for 20 minutes with UV at 20 µg

Lane 8:

HeLa cell line at 20 µg

Lane 9:

HeLa treated for 20 minutes with UV at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1/5000 dilution

Predicted band size: 48 kDa

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