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. 2009 Aug 6;106(33):14156–14161. doi: 10.1073/pnas.0904429106

Fig. 3.

Fig. 3.

OsEro1 is a glycoprotein with the N-terminal region targeting to the rER membrane. (A) Extracts from seeds (10 DAF) expressing OsEro1-GFP or spGFP-OsEro1ΔN were incubated in the absence (−) or presence (+) of Endo H or PNGase F and then subjected to SDS/PAGE. Crude extracts of Escherichia coli cells transformed with the OsEro1 expression vector, expressing OsEro1 Met-1 to Ile-474 with a predicted MW of 53 kDa, were also separated by SDS/PAGE (Left, lane C). (B) Extracts from seeds (10 DAF) expressing each of 3 chimeric genes encoding OsEro1-GFP, OsEro1ΔC-GFP, and spGFP-OsEro1ΔN were fractionated by centrifugation under the following conditions: control buffer, high-salt buffer, alkaline buffer, and Triton X-100 buffer. The supernatant (S) and pellet (P) fractions were separated by SDS/PAGE. The endogenous and GFP-fused OsEro1 proteins were analyzed by Western blot analysis with antibodies against OsEro1 (Ero1, Ero1-GFP, and spGFP-Ero1ΔN) and GFP (Ero1ΔC-GFP). As a positive control, extracts from GFP-Sec61β-expressing seeds were fractionated as above, followed by Western blot analysis with anti-GFP antibody.