Figure 2

Fhit interacts with activated Gα _q subunits but not with Gβ, small GTPases or RGS proteins. A, HEK293 cells were co-transfected with either pFlag-CMV2-Fhit (F) or pFlag-CMV2 vector (V) and in combination with individual construct encoding wild-type or the constitutively active mutant (RC for G_q, QL for the others) of different Gα proteins: G_q, G₁₁, G₁₄, G₁₆, G_s, G_i2 and G₁₃. After 24 h overexpression, cell lysates were prepared and subjected to immunoprecipitation (IP) with anti-Flag agarose affinity gel. Total cell lysates (TCL) and the immunoprecipitates were analyzed by Western blotting (WB). B, HEK293 cells were transfected with pcDNA3 (Vector) or pFlag-CMV2-Fhit together with Gα₁₆ or Gα₁₆QL. Transfectants were subjected to IP with anti-Gα₁₆ antiserum and protein G agarose. C, DLD-1 colon carcinoma cell lysates were incubated without or with GDPβS or GTPγS for 30 min at 4°C and then subjected to IP with anti-Fhit antiserum and protein A agarose. D, HEK293 cells were co-transfected with either Flag-Gβ₁ or pFlag-CMV2 vector with HA-Gγ₂ and pcDNA3-Fhit. Transfectants were immunoprecipitated with anti-Flag agarose affinity gel. E , HEK293 cells were co-transfected with either pFlag-CMV2-Fhit or pFlag-CMV2 vector and in combination with individual HA-tagged construct encoding Ras, RGS19, Rap1A, or RGS16. Cell lysates were immunoprecipitated with anti-Flag agarose affinity gel. Data shown represent one of three or more sets of immunoblots; other sets yielded similar results.