Lanes 1, 5 and 9: Wild-type HAP1 cell lysate (20 µg)
Lanes 2, 6 and 10: PKC delta knockout HAP1 cell lysate (20 µg)
Lanes 3, 7 and 11: A431 cell lysate (20 µg)
Lanes 4, 8 and 12: HeLa cell lysate (20 µg)
Lanes 1, 2, 3 and 4: Green signal from target – ab108972 observed at 78 kDa
Lanes 5, 6, 7 and 8: Red signal from loading control – ab8245 observed at 37 kDa
Lanes 9, 10, 11 and 12: Merged (red and green) signal

ab108972 was shown to specifically react with PKC delta when PKC delta knockout samples were used. Wild-type and PKC delta knockout samples were subjected to SDS-PAGE. ab108972 and ab8245 (loading control to GAPDH) were diluted 1/10000 and 1/2000 respectively and incubated overnight at 4ºC. 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 1/10000 dilution for 1 h at room temperature before imaging.

Immunohistochemical analysis of PKC delta in paraffin embedded Human breast carcinoma tissue, using ab108972 at a 1/50 dilution.

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

Primary ab Dilution 1:100,000 dilution, Secondary ab description and code (ab id) Goat Anti-Rabbit IgG, (H+L), HRP conjugated (ab97051), Secondary ab dilution 1:20,000 dilution, Blocking buffer and concentration 5% NFDM/TBST, Diluting buffer and concentration 5% NFDM/TBST, Lane 1: THP-1 whole cell lysate treated with Calyculin A with no peptides, Lane 2: THP-1 whole cell lysate treated with Calyculin A with PKCd S645Pho peptides, Lane 3: THP-1 whole cell lysate treated with Calyculin A with PKCd unmodified peptides, Lane 4: None, Observed MW   78 kDa, Exposure time   10 seconds

Primary ab Dilution 1:100,000 dilution, Secondary ab description and code (ab id) Goat Anti-Rabbit IgG, (H+L), HRP conjugated (ab97051), Secondary ab dilution 1: 20,000 dilution, Blocking buffer and concentration 5% NFDM/TBST, Diluting buffer and concentration 5% NFDM/TBST, Lane 1: Untreated THP-1 (Human monocytic leukemia cell line) whole cell lysates 15ug, Lane 2: THP-1 (Human monocytic leukemia cell line) treated with 50nM Calyculin A for 60 minutes whole cell lysates 15ug. Lane 3: THP-1 (Human monocytic leukemia cell line) treated with 50nM Calyculin A for 60 minutes’ whole cell lysates 15ug. Then the membrane was incubated with phosphatase. Lane 4: None, Observed MW 78 kDa, Exposure time 5 seconds

Primary ab dilution 1: 1000 dilution (2.355 μg /ml), Secondary ab description and code (ab id) Goat Anti-Rabbit IgG, (H+L), HRP conjugated (ab97051), Secondary ab dilution    1:100,000 dilution, Blocking buffer and concentration 5% NFDM/TBST, Diluting buffer and concentration 5% NFDM /TBST, Lane 1: PKCd S645Pho peptides, Lane 2: PKCd unmodified peptides, Lane 3: None, Lane 4: None, Observed MW   N/A, Exposure time   3 minutes

Antigen PKCd S645Pho peptide, PKCd S645Pho unmodified peptide, Antigen concentration 1000ng/ml, Primary antibody concentration range 0~1000ng/ml, Secondary antibody Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG(H+L), Secondary antibody concentration 1:2500 dilution