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. 2000 Feb;11(2):765–772. doi: 10.1091/mbc.11.2.765

Figure 3.

Figure 3

Effect of ribosome and the translocon complex on nascent chain oligomerization. BHK cells infected by recombinant SFV expressing HA-RAP (A, C, and G) or HA-RAP (TM−) (B, D–F, and H) were labeled for 1 min followed by a 20-min chase with either 1 mM puromycin or 1 mM cycloheximide. The lysate was immunoprecipitated with α-NHA, trimer-specific monoclonal antibody α-N2, or α-FLAG (M2) and analyzed by 5.5% two-dimensional gels. The markers are wild-type HA (80 kDa), muscle phosphorylase (97 kDa), and E. coli β-galactosidase (115 kDa). (A) HA-RAP: 1-min pulse followed by 20-min chase with puromycin, immunoprecipitated with α-N2. (B) Same as A, except HA-RAP (Tm−) virus was used. (C) HA-RAP: 1-min pulse followed by 20-min chase with cycloheximide, immunoprecipitated with α-N2. (D) Same as C, except HA-RAP (Tm−) virus was used. (E) HA-RAP (Tm−): 1-min pulse followed by 20-min chase with puromycin, immunoprecipitated with α-NHA, (F) HA-RAP (Tm−): 1-min pulse followed by 20-min chase with cycloheximide, immunoprecipitated with α-NHA. (G) HA-RAP: 1-min pulse, followed by 20-min chase with cycloheximide, immunoprecipitated with α-FLAG. (H) Same as G, except HA-RAP (Tm−) virus was used.