Fig. 4. In vitro adhesion to Ln-332 is rescued by 6 integrins in the absence of 3β1 integrin. Keratinocytes were isolated from newborn Itga3^flox/flox mice and designated MK3⁺. MK3^– cells were then obtained by in vitro deletion of Itga3. (A) Immunoblot depicting the expression of 3 in MK3⁺ and MK3^– cells. Murine mammary cell line Rac-11P and murine fibroblast cell line NIH3T3 were included as a positive and negative control, respectively. (B) Cell surface expression of integrin subunits 5, 2, 6, v, β4 and β1 in MK3⁺ and MK3^– was determined by FACS analysis. The negative control (only secondary antibody) is indicated by the black graph. (C) MK3⁺ and MK3^– cells were seeded onto Col-1 or Ln-332 in K-SFM without supplements in the absence or the presence of the 6-blocking antibody GoH3 (10 µg/ml). After 30 minutes, non-adherent cells were washed away. Values shown represent the average percentages of adherent cells from three experiments performed in triplicate (^*P<0.05, ^***P<0.0005). (D) Immunoprecipitation of integrin subunits 6 and β1 was performed on lysates of MK3⁺ and MK3^– cells. The precipitates were resolved by SDS-PAGE, and analyzed for the presence of the integrins β1 and β4, and the light chains (L) of 3 and 6 by western blotting.