FIGURE 4.

Sp3 is required for ITGB8 expression and αvβ8-mediated TGF-β activation. A, EMSA in adult lung fibroblasts using a biotinylated probe (sequence indicated above) covering the CRE, CCAAT, and SP transcription factor binding sites in the core promoter with or without the indicated unlabeled competitors. Wt is the unmutated competitor. CRE/CCAAT mt, SP, or triple mt indicates mutations in each or all of the indicated sites. CRE wt is an unrelated CRE consensus site competitor (32). The figure is a composite from a single representative gel. B, EMSA supershift analysis using polyclonal antibodies against Sp1, Sp3, or Sp1 and Sp3 combined. Supershifted complexes are indicated by arrows. The figure is a composite from a single representative gel. C, quantitative RT-PCR results for SP1, SP3, and the ITGB8 in adult lung fibroblasts transfected with siRNA against SP1, SP3, or control (± S.E.). The measured transcript is labeled above each respective graph. D, flow cytometry for surface expression of the integrin β8 subunit on adult lung fibroblasts transfected with siRNA against SP3 or control (± S.E.). MFI, mean fluorescence intensity. E, TGF-β activation assays of adult lung fibroblasts treated with siRNA against SP3 or control using control or anti-β8 blocking antibodies (± S.E.). * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001.