Figure 5.

Immunoblot Analyses of Protein Extracts from Transgenic arg1-2 Carrying the cMyc-ARG1 Construct, Showing the Membrane and Cytoskeleton Association of ARG1.

(A) Fractionation of cMyc-ARG1, AtSEC12 (an integral membrane protein control), α-tubulin, and actin (integral/peripheral membrane protein controls) in the nucleus-free total protein extracts (S1), the microsomal membrane fraction (P150), and the soluble fraction (S150). A total of 50 μg was loaded per lane. The results shown are representative of five experiments.

(B) Fractionation of cMyc-ARG1, AtSEC12, and actin in microsomes treated with buffer containing the reagents specified at top. The pellet (P) and supernatant (S) of each treatment were derived from 90 μg of P150 starting materials, except those of the Na₂CO₃ treatment, which were derived from 110 μg of P150. The results shown are representative of three experiments.

(C) Fractionation of cMyc-ARG1 and actin in the microsomes, the cytoskeleton-enriched pellet, and the supernatant after treatment with 1% (v/v) Triton X-100, the pellet after depolymerization of the cytoskeleton with 0.5 M KI, and the cytoskeleton-enriched pellet and the supernatant after cytoskeleton repolymerization upon dialysis of the KI supernatant. The pellet and supernatant of Triton X-100 treatment were derived from 85 μg of P150 starting materials. A total of 30 μg of KI and postdialysis pellets and the volume equivalent of postdialysis supernatant were loaded.