Figure 2
From: Folding of newly translated membrane protein CCR5 is assisted by the chaperonin GroEL-GroES
Soluble translation of CCR5 in a cell-free system.
(a) Soluble CCR5 in the supernatant of the cell-free reaction mixture was probed via dot blot analysis. Cell-free reactions were also performed with template DNA encoding another his-tagged receptor, with CCR3, (+) and with no template DNA (−) as controls. (b) Surfactant screening for optimal cell-free translation of CCR5. After performing the cell-free reaction in the presence of different surfactants, samples from the reaction mixtures were first centrifuged and supernatant protein fractions from each sample were then analyzed by dot blot, followed by spot densitometry analyses to compare the amounts of soluble CCR5. The full names of each surfactant are presented in the Materials and Methods. (c) CCR5 protein bands were analyzed by western blotting with the same negative control used in (a) (left) or by fluorescence imaging after the incorporation of fluorescently labeled lysine residues during the translation of CCR5 nascent chains (right). As controls, cell-free reactions were also performed without fluorescent labeling (−).
