Fig. 9.
Short hairpin RNA (shRNA) silencing of syntaxin 3 reduces surface CFTR in cAMP-stimulated Caco-2BBe cells. A: immunoblot analysis of Caco-2BBe cell lysates from control [nontransduced (NT) or scrambled shRNA (SC)] or after transduction with lentiviral particles containing syntaxin 3 (S3). Cells were treated with PBS or 1 mM DbcAMP (cAMP) for 30 min. Lysates (20 μg) were separated on SDS-PAGE gels and analyzed by Western blot using CFTR, syntaxin 3, or β-actin antibodies. Syntaxin 3 shRNA reduced syntaxin 3 expression by ∼100%. β-Actin loading control bands are shown. B: surface-biotinylated CFTR in PBS- or cAMP-stimulated cells transduced with syntaxin 3 shRNA. After PBS or 1 mM DbcAMP treatment, surface proteins were labeled with sulfo-NHS-SS-biotin. Cells were lysed, and proteins were bound to streptavidin-agarose. Equivalent biotinylated protein samples were resolved by SDS-PAGE and immunoblotted to detect CFTR. C: quantification (mean pixel values) of surface CFTR in control and syntaxin 3 shRNA (S3)-transduced Caco-2BBe cells. Data represent means + SE (n > 3).*P = 0.05. Molecular mass standards (kDa) are indicated.