Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: JNK1 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: MCF7 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab199380 observed at 46 and 54 kDa. Red - loading control, ab8226, observed at 42 kDa.

ab199380 was shown to recognize JNK1 when JNK1 knockout samples were used, along with additional cross-reactive bands. Wild-type and JNK1 knockout samples were subjected to SDS-PAGE. ab199380 and ab8226 (loading control to beta Actin) were diluted to 1/2500 and 1/1000 respectively and incubated overnight at 4°C. 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 h at room temperature before imaging.

All lanes : Anti-JNK1 antibody [EPR17557] (ab199380) at 1/5000 dilution

Lane 1 : Full length Human JNK1 recombinant protein
Lane 2 : Full length Human JNK2 recombinant protein
Lane 3 : Full length Human JNK3 recombinant protein

Lysates/proteins at 0.01 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 44 kDa
Observed band size: 48 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Recombinant full length JNK1 protein contains aa1-427 with His-Tag®. Recombinant full length JNK2 protein contains aa1-424 with GST-tag and JNK3 protein contains aa1-464 with GST-tag.

All lanes : Anti-JNK1 antibody [EPR17557] (ab199380) at 1/10000 dilution

Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate
Lane 2 : U937 (Human histiocytic lymphoma cells) whole cell lysate
Lane 3 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate
Lane 4 : Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 44 kDa
Observed band size: 46,54 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Anti-JNK1 antibody [EPR17557] (ab199380) at 1/2500 dilution + Human fetal brain lysate at 10 µg

Secondary
Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 44 kDa
Observed band size: 46,54 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-JNK1 antibody [EPR17557] (ab199380) at 1/2500 dilution

Lane 1 : Human fetal heart lysate
Lane 2 : Human fetal kidney lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 44 kDa
Observed band size: 46,54 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-JNK1 antibody [EPR17557] (ab199380) at 1/5000 dilution

Lane 1 : Rat brain lysate
Lane 2 : Rat heart lysate
Lane 3 : Rat spleen lysate
Lane 4 : C6 (Rat glial tumor cells) whole cell lysate
Lane 5 : RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysate
Lane 6 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate
Lane 7 : NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 44 kDa
Observed band size: 46,54 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-JNK1 antibody [EPR17557] (ab199380) at 1/2500 dilution

Lane 1 : MDCK (Canine kidney cell line) whole cell lysates
Lane 2 : COS-1 (African green monkey kidney fibroblast-like cell line) whole cell lysates
Lane 3 : UMNSAH/DF-1 (Transformed chicken embyronic fibroblast cells) whole cell lysates

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 44 kDa
Observed band size: 46,54 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-JNK1 antibody [EPR17557] (ab199380) at 1/2500 dilution

Lane 1 : Xenopus (X. tropicalis) muscle lysates
Lane 2 : BHK (Hamster kidney fibroblast cells) whole cell lysates

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 44 kDa
Observed band size: 46,54 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.