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. 2005 Oct 24;171(2):281–289. doi: 10.1083/jcb.200502067

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Copyright © 2005, The Rockefeller University Press

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Figure 8. — Thylakoid-associated GFPΔTPP intermediate is partially protected from proteolysis and buried within the membrane. (A) Tobacco protoplasts transfected with a plasmid encoding pre-GFPΔTPP were pulsed for 3 h with ³⁵S-Met and ³⁵S-Cys. Chloroplasts were isolated and fractionated, and purified thylakoids were split into eight identical aliquots and incubated either in the absence or in the presence of different proteases and detergent (Triton X-100), as indicated. TL, thermolysin; PK, proteinase K; TRP, trypsin. (B) Protoplasts expressing pre-GFP were pulsed with ³⁵S-Met and ³⁵S-Cys as in A, after which the stromal fraction was prepared and analyzed directly after immunoprecipitation with GFP antibodies (−TL) or after incubation with thermolysin as in A (+TL). Proteins were immunoprecipitated using anti-GFP antisera and analyzed by SDS-PAGE and fluorography.