All lanes : Anti-PPP2R5E antibody [EPR17146] (ab198290) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : PPP2R5E knockout HeLa cell lysate
Lane 3 : HEK-293 cell lysate
Lane 4 : Daudi cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 55 kDa

Lanes 1-4: Merged signal (red and green). Green - ab198290 observed at 55 kDa. Red - loading control ab8245 observed at 37 kDa.

 ab198290 Anti-PPP2R5E antibody [EPR17146] was shown to specifically react with PPP2R5E in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265637 (knockout cell lysate ab258135) was used. Wild-type and PPP2R5E knockout samples were subjected to SDS-PAGE. ab198290 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.

 

Anti-PPP2R5E antibody [EPR17146] (ab198290) at 1/1000 dilution + 293 (Human epithelial cells from embryonic kidney) cell lysate at 20 µg

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

Predicted band size: 55 kDa
Observed band size: 55 kDa

Blocking/dilution buffer: 5% NFDM/TBST.

Anti-PPP2R5E antibody [EPR17146] (ab198290) at 1/1000 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: 55 kDa
Observed band size: 55 kDa

Blocking/dilution buffer: 5% NFDM/TBST.

Anti-PPP2R5E antibody [EPR17146] (ab198290) at 1/1000 dilution + Human pancreas lysate at 10 µg

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

Predicted band size: 55 kDa
Observed band size: 55 kDa

Blocking/dilution buffer: 5% NFDM/TBST.

All lanes : Anti-PPP2R5E antibody [EPR17146] (ab198290) at 1/1000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Rat brain lysate
Lane 3 : C6 (Rat glial tumor cell) lysate
Lane 4 : NIH/3T3 (Mouse embryo fibroblast cells) 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: 55 kDa
Observed band size: 55 kDa

Blocking/dilution buffer: 5% NFDM/TBST.

Immunofluorescent analysis of 100% methanol-fixed, 0.1% Triton X-100 permeabilized C6 (Rat glial tumor) cells labeling PPP2R5E with ab198290 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Cytoplasm staining on C6 cell line is observed. 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 1/500 dilution (red).
The negative controls are as follows:-
-ve control 1 - ab198290 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 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 1/500 dilution.