Figure 4. β1 interacts with p27^Kip1 in vivo and in vitro.

(A) Immunoprecipitation analysis of the interactions between p27^Kip1 and β1. Cell lysates from synchronized G0 HeLa cells were divided into three parts and each part was incubated with antibody (β1, GFP and p27^Kip1 mouse monoclonal antibody) overnight and then with protein G-plus-agarose beads at 4°C for 4 h. The beads were washed three times with precipitation buffer and then treated, as above. For detection of interactions, the blot was probed with anti-β1/p27^Kip1 rabbit polyclonal antibodies, followed by ECL. (B) Immunoprecipitation analysis of the interactions between β1 and the α7 subunit. For detection of this interaction, the blot was probed with anti-β1/α7 antibodies, followed by ECL. (C) GST-pull-down analysis of the interactions between p27^Kip1 and β1. p27^Kip1–GST fusion proteins were expressed by the addition of IPTG to 0.2 mM at 25°C for 4 h. Bacterial cells were lysed and the supernatants were incubated with the glutathione resin overnight and then with purified β1–His₆ for 4 h at 4°C, followed by washing and elution. To detect interactions, the blots were probed with anti-β1/His₆ antibodies, followed by ECL. The lower panel shows the amounts of bound GST and GST-tagged proteins by CBB (Coomassie Brilliant Blue) staining. (D) β1-GST fusion proteins were incubated with purified p27^Kip1-His₆ at 4°C for 4 h and then detected with anti-p27^Kip1/His₆ antibody, followed by ECL. The lower panel shows the amounts of bounded GST and GST-tagged proteins by CBB staining.