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. 2008 Sep 15;28(23):6989–7000. doi: 10.1128/MCB.00724-08

FIG. 8.

FIG. 8.

Stabilized GADD34 facilitates protein aggregation. (A) GFP-CFTRΔF508 was coexpressed in HEK293T cells with either cytosolic (RFP) or ER (DsRed2-RFP) proteins. Cells were untreated (control), exposed to 5 μM MG262 for 8 h, or cotransfected with FLAG-GADD34. Cells were analyzed by fluorescence microscopy. (B) GFP-CFTRΔF508 or GFP-GRP94 were expressed in HEK293T cells that were either untreated (control), exposed to 5 μM MG262 for 8 h, or cotransfected with FLAG-GADD34. At 24 h posttransfection, cells were lysed in RIPA buffer. After removing an aliquot (T), the lysates were subjected to centrifugation to separate soluble (S) and insoluble (I) fractions. Proteins were solubilized by heating and sonication in RIPA containing 1% SDS. T, S, and I fractions were subjected to SDS-PAGE and analyzed by immunoblotting. Double asterisks (**) indicate faster-migrating band glycosylation intermediates of CFTR, and a single asterisk (*) indicates HMW aggregated forms of CFTR. (C) Distribution of FLAG-GADD34 in HEK293T cells cotransfected with GFP-GRP94, CFTRΔF508, or GFP-250 was analyzed in the T, S, and I fractions by immunoblotting. Tubulin and GM130 immunoblots served as controls.