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. 2015 Jun 15;26(12):2181–2189. doi: 10.1091/mbc.E15-01-0014

FIGURE 1:

FIGURE 1:

Grp170 and Sil1 knockdown decreases CTA1 retrotranslocation. (A) Lanes 1–5, WCEs derived from 293T cells transfected with the indicated siRNA were analyzed by SDS–PAGE and immunoblotted with the indicated antibodies and the film developed by using ECL. All immunoblots were developed using this method. Lanes 6–11, reverse-transcription PCR analysis of the unspliced (u) and spliced (s) forms of the XBP1 mRNA derived from cells transfected with the indicated siRNA and treated with or without DTT as indicated. (B) Cells were semipermeabilized with digitonin and subjected to centrifugation, and the generated supernatant and pellet fractions were analyzed for the presence of the cytosolic Hsp90 and the ER-resident PDI markers. This fractionation method is used in the retrotranslocation assay. (C) Cells transfected with the indicated siRNA were incubated with CT (10 nM) for 90 min and subjected to the retrotranslocation assay as presented in B. Both the supernatant and pellet fractions were analyzed by SDS–PAGE and immunoblotted with the indicated antibodies. (D) The intensity of the supernatant CTA1 band in C was quantified by ImageJ (National Institutes of Health, Bethesda, MD) using scans of films after ECL. Mean of three independent experiments. A two-tailed t test was used. Error bars, ± SD. (E) In the rescue experiment, cells transfected with scrambled or Grp170 siRNA #1 were transfected with either Grp170-FLAG* or FLAG-Sil1, as indicated. After intoxication with CT (10 nM) for 90 min, cells were processed and analyzed as in C. (F) In a rescue experiment similar to E, cells transfected with scrambled or Sil1 siRNA #2 were transfected with either Grp170-FLAG or FLAG-Sil1*, as indicated. After intoxication with CT (10 nM) for 90 min, cells were processed and analyzed as in C. (G) Cells were cotransfected with Δ F508 CFTR and a scrambled siRNA, Grp170 siRNA #2, or Sil1 siRNA #1. Δ F508 CFTR was immunoprecipitated from the resulting WCE, and samples were subjected to immunoblotting with an antibody against CFTR. The black line indicates that an intervening lane from the same immunoblot has been spliced out.