Figure 3. Derlin-1 is dispensable for CT function in zebrafish embryos and retro-translocation of the A1-chain in mammalian cells.

(A) Protein extracts from 5 controls (Ctrl) or Derlin-1 morphants (72-hpf) were immunoblotted for Derlin-1 and β-actin. (B) Control or Derlin-1 morphants were intoxicated or not with 1.5 nM CT at 16- to 18-hpf and imaged at 72 hpf. Insets to scale with top panels. (C) Protein extracts from Cos-1 cells transfected with the indicated siRNA (Der1* indicates combination of 2 siRNAs) were subject to immunoblot as in A. (D) qRT-PCR for DERL1 from control and Der1 siRNA–treated cells (mean ± SD, n = 3). (E) Cytosolic and membrane fractions were prepared from Cos-1 cells transfected with the indicated siRNA and intoxicated with WT or R192G CT. Immunoblots were probed with CTA, CTB, BiP, or β-actin antibodies. Purified toxin standards, A1-chain, and uncleaved A1/A2-chain complex are indicated. (F) MFI of GFP expression in Cos-1 cells transfected with CFTRΔF508-GFP and control or Der1 siRNA. Maximal CFTRΔF508-GFP accumulation is achieved by treatment with the proteasome inhibitor MG132 (10 μM; Ctrl + MG132), and these values were set to 100% in each experiment. Blue bars indicate means, and error bar indicates variance of Ctrl + MG132 samples. (G) Immunoblot of crude protein extracts from samples in F probed with mAbs against CFTR and β-actin. Filled and open arrowheads represent the immature and mature glycosylated forms of CFTR, respectively. (H) Cells transfected with control, Derlin-2 (Der2), or a combination of Derlin-1 and Derlin-2 siRNA were analyzed for Derlin-1 or -2 by immunoblot. (I) Same as E, except cells were transfected with control, Derlin-2, or both Derlin-1 and -2 siRNA.