Lane 1: Wild-type HAP1 whole cell lysate (40 µg)
Lane 2: CD146 knockout HAP1 whole cell lysate (40 µg)
Lane 3: A375 whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab75769 observed at 120-72 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab75769 was shown to specifically react with CD146  in wild-type HAP1 cells as signal was lost in CD146 knockout cells. Wild-type and CD146 knockout samples were subjected to SDS-PAGE. ab75769 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/10000 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 1/10000 dilution for 1 hour at room temperature before imaging.

Immunohistochemistry experiments were used to compare symptomatic carotid plaques (SC) and asymptomatic carotid plaques (AsC)

Asymptomatic lesions presented higher CD146⁺ pericyte infiltration, p

ab75769 used at 1/200 dilution.

(After Figure 2 of Davaine et al)

Immunocytochemistry/Immunofluorescence analysis of A375 (human malignant melanoma) cells labelling CD146 with purified ab75769 at 1/250. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

Control 1: primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human spleen tissue labelling CD146 with purified ab75769 at 1/250. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a goat anti-rabbit IgG H&L (HRP) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Flow Cytometry analysis of A375 (human malignant melanoma) cells labeling CD146 with unpurified ab75769 at 1/20 dilution (10ug/mL) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^®488) at 1/2000 dilution was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) was used as the unlabeled control.

All lanes : Anti-CD146 antibody [EPR3208] (ab75769) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : MCAM knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 72 kDa
Observed band size: 120 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab75769 observed at 120 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab75769 was shown to react with CD146 in wild-type HeLa cells in western blot. The band observed in knockout cell line ab261790 (knockout cell lysate ab256985) lane below 120kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HeLa and MCAM knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab75769 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 in 1000 dilution and a 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.

All lanes : Anti-CD146 antibody [EPR3208] (ab75769) at 1/10000 dilution (purified)

Lane 1 : A375 cell lysate
Lane 2 : Human fetal artery lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 72 kDa

Blocking buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM /TBST.

Anti-CD146 antibody [EPR3208] (ab75769) at 1/10000 dilution (purified) + HUVEC cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 72 kDa

Blocking buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM /TBST.

Anti-CD146 antibody [EPR3208] (ab75769) at 1/10000 dilution (purified) + B16-F0 cell lysate at 20 µg/ml

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 72 kDa

Blocking buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM /TBST.

Anti-CD146 antibody [EPR3208] (ab75769) at 1/10000 dilution (purified) + Rat placenta lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 72 kDa

Blocking buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM /TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of melanoma tissue labelling CD146 with unpurified ab75769 at 1/250. A HRP/AP polymerized secondary antibody was used.

Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of breast carcinoma vessels tissue labelling CD146 with unpurified ab75769.

Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of urinary bladder transitional carcinoma vessels tissue labelling CD146 with unpurified ab75769.

Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of glioma vessels tissue labelling CD146 with unpurified ab75769.

Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of normal tonsil tissue labelling CD146 with unpurified ab75769.

Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of normal spleen tissue labelling CD146 with unpurified ab75769.

Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

Flow Cytometry analysis of HUVEC cells labelling CD146 with purified ab75769 at 1/50 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.