Cross-linking of two α subunits through one β1 subunit. (A and B) The single-Cys mutants of FLAG-pWT α and pWT β1 were coexpressed, and the extent of α-β1 cross-linking (indicated at the bottom) was determined. The effect of DTT reduction of the samples is shown in the second lane. (C) Cells expressing the double-Cys mutants of FLAG-HRV-pWT α and pWT β1 were either untreated (lane 1), treated with 20 mM DTT (lane 2), or treated with 20 mM DTT and then, after washing, with 40 µM quaternary piperazinium diamide (QPD; lane 3). (D) Whole lysate of surface-labeled cells expressing the indicated α and β constructs. (E) FLAG-pWT β1 and its Cys mutants in the same cell extracts as in D were captured by anti-FLAG antibody conjugated to Sepharose (Sigma-Aldrich) and were eluted with FLAG peptide. Biotinylated complexes in this extract were captured on Neutravidin beads, released in SDS, and electrophoresed, immunoblotted, and visualized with anti-α antibody and HRP-tagged secondary antibody.