For immunofluorescence analysis, T98G cells were fixed and permeabilized for detection of endogenous CDK4 (pT172) using Anti- CDK4 (pT172) Recombinant Rabbit Multiclonal Antibody (ab277788, 2 μg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal^™ Secondary Antibody, Alexa Fluor^® 488 conjugate (1/2000). Nuclei (blue) were stained using SlowFade^® Gold Antifade Mountant with DAPI, and Alexa Fluor^® 555 Rhodamine Phalloidin (1/300) was used for cytoskeletal F-actin (red) staining. Detection and localization of CDK4 pT172 (green) in the nucleus can be clearly observed in cell cycle synchronised cells (serum starved overnight and serum released for 10 hours) as compared to untreated cells. Antibody specificity was demonstrated by competition with the CDK4 pT172 peptide, which results in loss of signal. No competition was observed with the non-phospho peptide. The images were captured at 60X magnification.