Phosphorimager plate

HEK293^DAX-CD2 cells seeded on poly-D-lysine-coated plates were treated with Dox at 1 µg/mL to activate XBP1s for 24 h. Cells were then starved for 1 h in DMEM + 10% FBS lacking Cys and Met. Cells were metabolically labeled in pulse medium containing [³⁵S]-Cys/Met (MP Biomedicals, ~0.1 mCi/mL final concentration) for 20 min, and then incubated in pre-warmed chase media for the indicated times. Lactacystin (10 µM) was added as indicated at the beginning of the chase period. Media was collected and cells were lysed in a 1% Triton X-100 buffer. CD2 was immunopurified using M2 anti-FLAG agarose beads (Sigma; A2220) and washed with RIPA. Immunoisolates were eluted by boiling in 6X–Laemmli buffer and separated by SDS-PAGE. The gels were then dried, exposed to phosphorimager plates (GE Healthcare), and imaged with a Typhoon imager. Band intensities were quantified in ImageQuant TL.

HEK293^DAX or HepG2^DA cells plated on poly-D-lysine coated plates were metabolically labeled in, DMEM-Cys/-Met (CellGro) supplemented with glutamine, penicillin/streptomycin, dialyzed fetal bovine serum, and EasyTag EXPRESS³⁵S Protein Labeling Mix (Perkin Elmer) for 30 min. Proteasome and lysosome inhibitors were added at the indicated concentrations 1 hr prior to metabolic labeling and were included in both the metabolic labeling and chase. Cells were washed twice with complete media and incubated in pre-warmed DMEM for the indicated times. Media or lysates were harvested at the indicated times. Lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 50 mM Tris pH 7.5, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS) with fresh protease inhibitor cocktail (Roche). FLAG tagged TTR variants were immunopurified using M1 anti-FLAG agarose beads (Sigma-Aldrich) and washed with RIPA buffer. The immunoisolates were then eluted by boiling in Laemmli buffer and separated on SDS-PAGE. The gels were then dried, exposed to phosphorimager plates (GE Healthcare), and imaged with a Typhoon imager. Band intensities were quantified by densitometry in ImageQuant.

HEK293^HSR cells transiently transfected with FLuc were seeded on poly-D-lysine-coated plates and treated with vehicle, dox at 1 μg/mL to activate dn-cHSF1, or Shield-1 at 1 μM to activate cHSF1 for 48 h. Cells were then starved for 30 min in DMEM + 10% FBS lacking Cys and Met. Cells were metabolically labeled in pulse medium containing [³⁵S]-Cys/Met (MP Biomedical, ~0.1 mCi/mL final concentration) for 15 min prior to lysis. Cells were lysed in a 1% Triton X-100 buffer. Lysates were boiled in 1X-Laemmli buffer and separated by SDS-PAGE. The gels were then dried, exposed to phosphorimager plates (GE Healthcare), and imaged with a Typhoon imager.