All lanes : Anti-CD73 antibody [EPR6114] (ab133582) at 1/1000 dilution

Lane 1 : Wild-type A-431 whole cell lysate
Lane 2 : NT5E knockout A-431 whole cell lysate
Lane 3 : A375 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 63 kDa

Lanes 1 - 3: Merged signal (red and green). Green - ab133582 observed at 63 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab133582 was shown to recognize NT5E in wild-type A-431 cells as signal was lost at the expected MW in NT5E knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and NT5E knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. Ab133582 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human spleen tissue sections labeling CD73 with Purified ab133582 at 1:2000 dilution (1.12 µg/ml).

Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0).

ImmunoHistoProbe one step HRP Polymer (ready to use) was used.

Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Immunocytochemistry/ Immunofluorescence analysis of A375 (Human malignant melanoma epithelial cell) cells labeling CD73 with Purified ab133582 at 1:250 dilution (8.9 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung carcinoma tissue sections labeling CD73 with Purified ab133582 at 1:2000 dilution (1.12 µg/ml).

Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0).

ImmunoHistoProbe one step HRP Polymer (ready to use)was used.

Negative control:PBS instead of the primary antibody.

Hematoxylin was used as a counterstain.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue sections labeling CD73 with Purified ab133582 at 1:2000 dilution (1.12 µg/ml).

Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0).

ImmunoHistoProbe one step HRP Polymer (ready to use) was used.

Negative control: PBS instead of the primary antibody.

Hematoxylin was used as a counterstain.

Anti-CD73 antibody [EPR6114] (ab133582) at 1/1000 dilution + A375 ( Human malignant melanoma epithelial cell) whole cell lysates at 15 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 63 kDa

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labelling CD73 with ab133582 at 1/100 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

All lanes : Anti-CD73 antibody [EPR6114] (ab133582) at 1/1000 dilution

Lane 1 : Human fetal liver tissue lysate
Lane 2 : A375 cell lysate
Lane 3 : HepG2 cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled Goat anti-Rabbit IgG at 1/2000 dilution

Predicted band size: 63 kDa