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. 2018 Aug 2;69(21):5013–5027. doi: 10.1093/jxb/ery287

Fig. 3.

Fig. 3.

Recombinant 16γz and 27γz are retained intracellularly but have different solubility. Protoplasts were isolated from tobacco leaves and transiently transformed either with plasmids encoding the indicated constructs or with the empty vector (Co) and analysed after incubation for 20 h. (A) Protoplasts (in) or incubation medium (out) were homogenized in the absence (–) of 2-ME. After centrifugation, soluble (S1) and insoluble fractions were collected. The insoluble material was resuspended in the presence (+) of 2-ME and subjected to a second centrifugation, to obtain the new soluble (S2) and insoluble (I) fractions. (B) Protoplasts were homogenized in the absence of 2-ME. After centrifugation, soluble (S1) and insoluble fractions were collected. The insoluble material was resuspended with 70% ethanol and subjected to a second centrifugation, to obtain the new soluble (SE) and insoluble (I) fractions. (C) As in (B), but the first homogenization was performed in the presence of 4% 2-ME. In (A–C), the upper images show analysis of each fraction by SDS-PAGE and protein blotting with anti-FLAG antibody, whilst the lower images show Ponceau S staining. The positions of molecular mass markers are shown to the left, in kD. In (B, C) the positions of dimers (dim) and monomers (mon) of 27γzf (27) and 16γzf (16) are indicated.