Figure 5.

hSH3BGRL is a tumor suppressor, but its mutation can reverse it back to metastatic diver. (a) Lysates from MCF-7 and MDA-MB-231 cells were stably transfected with two individual hSH3BGRL-specific shRNAs as cell pools and immunoblotted with the indicated antibodies, respectively. *p-ERK was detected after serum-free starvation of the cells for 24 h. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was served as a loading control. The indicated relative protein expression level is quantified and shown under the immediate panel. (b) In total, 1x10⁶ MDA-MB-231 Vec or MDA-MB-231 SH3BGRL knockdown cell pools were injected into the tail vein of nude mice. After 13 days, mice were killed and their lungs were photographed and scored for metastatic tumor nodules. (c) Effect of amino-acid alteration of hSH3BGRL to lung metastasis in CHO cells. In total, 1x10⁶ stably transfected cells were injected into nude mice through the tail vein. After 19 days injection, the assayed mice were killed and the dissected lungs were analyzed. The metastatic nodules in each lung in (b) and (c) were counted. Mean±s.d., n=4 or 5, *P<0.05; **P<0.001; ***P<0.0001, unpaired Student's t-test. (d) Lysates from CHO cells stably transfected with hSH3BGRL and its mutant V108A and immunoblotted with the indicated antibodies. The averaged protein relative expression level is quantified and shown under the immediate panel. (e) Co-immunoprecipitation of hSH3BGRL V108 mutant with p-c-Src 527, rather than wild-type hSH3BGRL were analyzed by immunoblotted by the indicated antibodies.