Immunofluorescence analysis of Egl nine homolog 1 was performed using 70% confluent log phase HeLa treated with cobalt chloride (200 micromolar for 48h). The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with PHD2 Polyclonal Antibody (Product # PA5-78511) at 1:100 dilution in 0.1% BSA, incubated at 4-degree Celsius overnight, and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), 1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300 dilution). Panel d represents the merged image showing cytoplasmic localization of PHD2. Panel e represents the untreated cells showing lower PHD2 expression levels. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.