All lanes : Anti-p38 gamma/MAPK12 antibody [EPR6528(N)] (ab205926) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 2 : MAPK12 knockout HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 3 : Human skeletal muscle tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 42 kDa
Observed band size: 42 kDa

Lanes 1-3: Merged signal (red and green). Green - ab205926 observed at 42 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab205926 Anti-MAPK 12 antibody [EPR6528(N)] was shown to specifically react with MAPK 12 in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266280 (knockout cell lysate ab258041) was used. Wild-type and MAPK 12 knockout samples were subjected to SDS-PAGE. ab205926 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. 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 in 20000 dilution for 1 hour at room temperature before imaging.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized A673 (Human muscle Ewing's Sarcoma cell line) cells labeling MAPK 12 with ab205926 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear and cytoplasmic staining on A673 cell line. The nuclear counterstain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).

The negative controls are as follows:-

-ve control 1: ab205926 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.

-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

Lane 1: Wild type HAP1 whole cell lysate (0 µg)
Lane 2: empty knockout HAP1 whole cell lysate (20 µg)
Lane 3: MAPK 12 whole cell lysate (0 µg)
Lanes 1 - 3: Merged signal (red and green). Green - ab205926 observed at 45 kDa. Red - loading control, ab18058, observed at 124 kDa.

All lanes : Anti-p38 gamma/MAPK12 antibody [EPR6528(N)] (ab205926) at 1/10000 dilution

Lane 1 : Human skeletal muscle lysate
Lane 2 : Human fetal kidney lysate
Lane 3 : Human fetal liver lysate
Lane 4 : Human fetal skin lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 42 kDa
Observed band size: 42 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-p38 gamma/MAPK12 antibody [EPR6528(N)] (ab205926) at 1/1000 dilution

Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate
Lane 2 : K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

Predicted band size: 42 kDa
Observed band size: 42 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-p38 gamma/MAPK12 antibody [EPR6528(N)] (ab205926) at 1/1000 dilution

Lane 1 : Rat heart lysate
Lane 2 : Rat spleen lysate
Lane 3 : C6 (Rat glial tumor cells) whole cell lysate
Lane 4 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate
Lane 5 : Rat muscle lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

Predicted band size: 42 kDa
Observed band size: 42 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1 and 2: 3 minutes; Lane 3 and 4: 1 minute; Lane 5: 30 seconds

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized K562 (Human chronic myelogenous leukemia cells from bone marrow) cells labeling MAPK 12 with ab205926 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear and cytoplasmic staining on K562 cell line. The nuclear counterstain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).

The negative controls are as follows:-

-ve control 1: ab205926 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.

-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.