STAT and MAPK activation by receptor tyrosine mutants.
Nuclear extracts of 3 × 106 bone marrow cells were stimulated with or without EGF (100 ng/mL) and were incubated with 0.2 ng 32P-labeled double-stranded (A) β-cas (derived from the 5′ region of the β-casein gene) or (B) a high-affinity mutant of SIE. As a control, bone marrow cells of a normal mouse were stimulated with G-CSF (25 ng/mL), and STAT complexes were identified as described.37 S1, STAT1; S3, STAT3; S5, STAT5. These figures represent 1 of 3 independent experiments. (C) Bone marrow cells (3 × 106) expressing each receptor construct were stimulated with or without EGF (100 ng/mL), lysed, and electrophoresed on a 10% polyacrylamide gel. Proteins were transferred to a polyvinylidene difluoride membrane and were incubated with antiphospho-p44/42 MAPK antibody, followed by horseradish peroxidase-conjugated anti–rabbit immunoglobulin and enhanced chemiluminescence. The membrane was stripped and reprobed with anti-ERK 1 antibody. The main panel represents 1 of 2 independent experiments. The additional panel represents the repeated results for the Y744 mutant. Infection efficiency (Inf Eff) of bone marrow cells expressing each receptor construct for all experiments is also indicated.
