Gefitinib-induced differentiation is EGFR independent. (A) RT-PCR (40 cycles) was performed to evaluate for EGFR and ERBB2 transcript presence in HL-60 and Kasumi-1 cells. GAPD was amplified as a control to demonstrate intact cDNA. Negative water controls and a positive cDNA control (uRNA) synthesized from universal Stratagene RNA were also included. (B) Western immunoblotting was performed to evaluate for the presence of EGFR protein in HL-60 and Kasumi-1 whole-cell lysates (50 μg) with anti-EGFR antibody. An EGFR-expressing positive control, A431 epidermoid carcinoma cells, was included. (C) Immunoprecipitation (IP) was performed with anti-ERBB2 and a mouse IgG control antibody to evaluate for the presence of ERBB2 protein in HL-60 cells with and without 10 μM gefitinib stimulation. Total lysate from SKBR3, a breast cancer cell line known to overexpress ERBB2, was used as a positive control. Western immunoblotting was performed with ERBB2 or a cocktail of 4G10 and pY100 antiphosphotyrosine (PTyr) antibodies.