Figure 4

Western blot analyses to confirm Smad1 interaction with prosequence-containing HsN3 and the detection of Smad1 interaction with HsN3 assembly intermediates. A&B. Immunoprecipitation and Western blot analyses of specific interaction between Smad1 and the prosequence-containing HsN3. COS1 cells were transfected with the indicated full length HsN3 and Smad1 or Smad2 and the interaction was analyzed via immunoprecipitation followed by Western blot, as indicated. Lactacystin treatment (indicated in panel B., lane 4) was 8 hrs. The co-precipitated prosequence-containing HsN3 and the naturally processed mature HsN3 is marked and labeled similarly as in Fig. 3. "Ig.G.L" represents antibody light chain. C. The interaction between Smad1 and prosequence-containing HsN3 is not dependent upon the prosequence of HsN3. T7-ΔN3: T7-tagged deletion mutant of HsN3 that lacks the prosequence. To distinguish this form of mutant HsN3 from the naturally processed HsN3 in panels A & B, T7-ΔN3 is also labeled with a cartoon symbol that matches the assembly defective β subunit shown in Fig. 2B. D.-F. Smad1 and Smad1C (MH2 domain) both bind to HsN3-associated proteasome assembly intermediates accumulated as a result of artificial removal of the prosequence of HsN3. COS1 cells were transiently transfected with the indicated constructs, labeled with ³⁵S-methionine for 4 hrs before cells were harvested and analyzed by immunoprecipitation, as indicated. The eight distinct bands associated with T7-ΔN3 as shown in Fig. 2C is again marked here in lanes 2 and 4 in panel E. An additional four distinct small molecular weight bands are also detected in these two lanes, as marked by "?". The identities of these proteins are unknown, but they were always detected only when Smad1C and ΔN3 were co-expressed.