Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: non-muscle Myosin IIA knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: HEK-293 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab75590 observed at 230 kDa. Red - loading control, ab18058, observed at 124 kDa.

ab75590 was shown to recognize non-muscle Myosin IIA in wild-type HAP1 cells along with additonal cross-reactive bands. No band was observed when non-muscle Myosin IIA knockout samples examined. Wild-type and non-muscle Myosin IIA knockout samples were subjected to SDS-PAGE. ab75590 at a concentration of 1 µg/ml and ab18058 (loading control to Vinculin) at a dilution of 1/1,000 were 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 hour at room temperature before imaging.

ICC/IF image of ab75590 stained HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.

The cells were 4% formaldehyde fixed (10 minutes) and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1 hour to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab75590 at 5 µg/ml overnight at +4°C. The secondary antibody (green) was Alexa Fluor^® 488 goat anti- rabbit IgG (H+L) used at a 1/1,000 dilution for 1 hour. Alexa Fluor^® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1 hour. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43 µM.

All lanes : Anti-non-muscle Myosin IIA antibody (ab75590) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) whole cell lysate
Lane 2 : HL-60 (Human promyelocytic leukemia cell line) whole cell lysate
Lane 3 : Jurkat (Human T cell lymphoblast-like cell line) whole cell lysate
Lane 4 : HEK-293 (Human embryonic kidney cell line) whole cell lysate
Lane 5 : Human kidney tissue lysate - total protein (ab30203)

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 227 kDa
Observed band size: 227 kDa
Additional bands at: 100 kDa, 35 kDa, 45 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 30 seconds

All lanes : Anti-non-muscle Myosin IIA antibody (ab75590) at 1 µg/ml

Lane 1 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate
Lane 2 : Mouse kidney tissue lysate
Lane 3 : Mouse lung tissue lysate
Lane 4 : PC-12 (Rat adrenal pheochromocytoma cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 227 kDa
Observed band size: 227 kDa
Additional bands at: 35 kDa, 43 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 1 minute