Immunofluorescence analysis of EGFR (pY1173) was done on 70% confluent log phase HeLa cells treated with EGF (200 ng/mL for 10 min). The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with EGFR (pY1173) Rabbit polyclonal Antibody (Product # 44-794G) at 2 µg/mL in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor 488 Goat Anti-Rabbit IgG Secondary Antibody (Product # A-11008) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor 594 Phalloidin (Product # A12381). Panel d is a merged image showing cytoplasmic localization of EGFR (pY1173). Panel e shows untreated cells. Panel f shows no primary antibody control. The images were captured at 20X magnification.