FIGURE 3. Thrombin promotes p66Shc-Ser³⁶ phosphorylation via EGFR transactivation of the MEK-ERK pathway in cardiac fibroblasts.

Cardiac fibroblasts were treated as described in the legend to Fig. 2. Immunoblots (IB) are of cell lysates, post-immunoprecipitation lysates (cleared of Shc proteins) (A), and immunoprecipitated (IP) p66Shc protein (A–C). Equal amounts of immunoprecipitated protein (derived from 600 μg of starting cell extract) were probed for p66Shc-pSer³⁶ immunoreactivity and p66Shc protein (A). In panels B and C (and all subsequent figures in this article), immunoblotting for Shc (to verify equal protein recovery and loading) was performed on protein immunoprecipitated from 100 μg of starting cell extract due to limiting amounts of sample material from primary cultures. At this lower level of protein loading, only p52Shc is consistently detected.