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. 2005 Nov;17(11):3066–3080. doi: 10.1105/tpc.105.035212

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Copyright © 2005, American Society of Plant Biologists

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Figure 8. — Proteinase K Protection Assay.

Nontransformed (WT) and (At)SRC2- or (At)VAP-transformed protoplasts were separated into vacuole (V) and pellet (P) fractions.

(A) Vacuoles were digested (+) or not digested (−) with proteinase K (PK) in the presence (+) or absence (−) of Nonidet P-40 (NP40) detergent. Proteins were detected on immunoblots using anti-(At)SRC2 or anti-(At)VAP recombinant protein antibodies.

(B) Vacuole and pellet fractions were separately treated (+) or not (−) with proteinase K and blotted for the detection of (At)SRC2 (top panel), subunit A of the peripheral complex of V-ATPase (bottom panel to the left), and BiP (bottom panel to the right). Closed and open arrows show the positions of (At)SRC2 or (At)VAP and cross-reacting endogenous proteins, respectively. Positions of molecular mass standards are indicated in kilodaltons at the side.

(C) Protoplasts transfected to express (At)SRC2 (top) or Re-F-B-B (bottom) were labeled with [³⁵S]Met + Cys for 1 h and then fractionated into pellet and vacuole fractions. Aliquots were separately treated (+) or not (−) with proteinase K, immunoprecipitated with anti-(At)SRC2 recombinant protein antibodies (top) or with anti-aleurain antibodies (bottom), and then separated by SDS-PAGE and visualized by fluorography.