Immunofluorescence was performed on fixed and permeabilized A-375 (human malignant melanoma cell line) cells which were serum starved (19 hours) and serum released (24 hours) for detection of Cdk1 (pTpY14/15) using Anti-Cdk1 (pTpY14/15) Recombinant Rabbit Multiclonal Antibody (ab277772, 1-2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor^® 488 conjugate (1/2000). Panel a) shows representative cells that were stained for detection and localization of Cdk1 (pTpY14/15) protein (green), Panel b) is stained for nuclei (blue) with DAPI. Panel c) represents cytoskeletal F-actin staining using Alexa Fluor^® 555 Rhodamine Phalloidin (1/300). Panel d) is a composite image of Panels a, b and c clearly demonstrating cytoplasmic localization of Cdk1 (pTpY14/15) Panel e) shows no signal which demonstrates antibody specificity against Cdk1 (pTpY14/15) phosphorylated peptide (Antibody was incubated with phosphorylated peptide for 1 hour/37C). Panel f) represents control cells with no primary Antibody to assess background.

Western blot analysis was performed on whole cell extracts (30 μg lysate) of A375 (1), A375 (Serum starved24 hours and released for 19 hours) (2), PC3 (human prostate adenocarcinoma cell line) (3), PC3 (human prostate adenocarcinoma cell line) (Serum starved24 hours and released for 19 hours) (4), Caco-2 (human colorectal adenocarcinoma cell line) (5), Caco-2 (human colorectal adenocarcinoma cell line) (Serum starved24 hours and released for 19 hours) (6), K562 (human chronic myelogenous leukemia lymphoblast cell line) (7), K562 (human chronic myelogenous leukemia lymphoblast cell line) (Serum starved24 hours and released for 19 hours) (8), COLO 205 (human colon adenocarcinoma cell line) (9) and COLO 205 (human colon adenocarcinoma cell line) (Serum starved24 hours and released for 19 hours) (10). The blots were probed with Anti-Cdk1 (pTpY14/15) Recombinant Rabbit Multiclonal Antibody (ab277772, 1-2 μg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP conjugate (0.4 μg/mL, 1/2500 dilution). A 34 kDa band corresponding to Cdk1 (pTpY14/15) was observed across cell lines tested. Treatment response is more evident in cell lines where basal expression is less. Resolved proteins were transferred onto a nitrocellulose membrane with iBlot Dry Blotting System. The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed usingECL Chemiluminescent Substrate.