Western blot analysis was performed on whole cell extracts (30 µg lysate) of NIH/3T3 (Lane 1), NIH/3T3 treated with Anisomycin (10 µg/mL for 30 min) (Lane 2), NIH/3T3 treated with EGF (100 ng/mL for 15 min) (Lane 3), NIH/3T3 treated with TNF alpha (10 ng/mL for 15 min) (Lane 4), A-431 (Lane 5), A-431 treated with EGF (20 ng/mL for 60 min) (Lane 6) and A-431 treated with Afatinib (0.5 µM for 6 h) followed by EGF (20 ng/mL for 60 min) (Lane 7). The blot was probed with Anti-Phospho-c-Jun (Ser73) Polyclonal Antibody (Product # PA5-17819, 1:500 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4,000 dilution). A 42 kDa band corresponding to Phospho-c-Jun (Ser63) was observed across the cell lines tested upon Anisomycin, EGF and TNF alpha treatment and was also observed to downregulate upon Afatinib treatment.