Figure 4.

Sha does not have reduction of thylakoid-bound NCED3 but does differ from Ler in apparent molecular mass of stromal NCED3. A, Immunoblot detection of NCED3 in total protein compared with isolated chloroplasts of Ler, Sha, and nced3. In each lane, 40 μg of protein was loaded. B, Chloroplast fractionation by Percoll gradient centrifugation and immunoblotting of thylakoid and stromal proteins. Blots were stripped and reprobed with AtOE33 as a marker of the thylakoid fraction and GS2 as a marker of the stromal fraction. In each lane, 40 μg of chloroplast protein, 30 μg of thylakoid membrane protein, or 10 μg of stromal protein was loaded. The experiment was repeated with similar results. Additional replicate assays using an alternative chloroplast fractionation procedure are shown in Supplemental Figure S6.