Figure 5. Sustained oncogenic Kras signaling is attenuated in the absence of TSP-1.

(A) Western blot of ERK, p19^ARF, and p16^INK4a expression after Kras^G12D activation in Kras^G12D×Tsp-1^+/+ and Kras^G12D×Tsp-1^–/– primary adult lung fibroblasts isolated at the indicated times after oncogenic Kras activation. β-actin served as loading control. (B) Immunofluorescence for p19^ARF in Kras^G12D×Tsp-1^+/+ and Kras^G12D×Tsp-1^–/– lung sections isolated 21 weeks after Kras^G12D activation. Hoechst stain indicates nuclei. Scale bars: 50 μm. (C) Kras^G12D×Tsp-1^+/+ and Kras^G12D×Tsp-1^–/– primary lung fibroblasts were infected with Ad-Cre, harvested at the indicated times, and probed for pMEK and total MEK by immunoblotting. β-actin served as loading control. (D) Tsp-1^+/+ and Tsp-1^–/– primary lung fibroblasts were infected with Hras^V12 or mock retrovirus, harvested at the indicated times, and probed for TSP-1, pERK, total ERK, and p19^ARF expression by immunoblotting. Uninfected lung fibroblasts were used as negative controls; β-actin served as loading control. (E) Tsp-1^+/+ and Tsp-1^–/– primary lung fibroblasts were treated with 10 nM EGF, harvested at the indicated times, and probed for TSP-1, pERK, and total ERK expression by immunoblotting. β-actin served as loading control.