Effect of eIF2B5 down-regulation.

<p>A. Identical amounts of total cell protein extracted from DDR1 and sh2B5 cells (stably expressing shRNA against eIF2B5 3′UTR) were subjected to Western blot analysis using antibodies specific for eIF2B5 and p38. B. 5×10⁵ cells were labeled with [³⁵S]-Met/Cys mix for 20 minutes followed by protein extraction, TCA-precipitation and scintillation counting of equal amounts of protein. The data represent average of three independent experiments performed in triplicates+/−SE. C. Control (open bars) or sh2B5 cells (dark bars) were incubated with 10 µg/ml Tunicamycin (Tun) for the indicated times, followed by XTT viability assay. Cell viability is expressed as percentages of viable cells grown in Tun-free medium. The data represent average of three independent experiments performed in triplicates+/−SE.</p

ER-stress response.

<p>A. The indicated cells were treated with 3 µM Thapsigargin (Tg) for the indicated time points followed by Western blot analysis using antibodies specific for eIF2B5, ATF4, GADD34, phosphorylated eIF2α (eIF2α -P), total eIF2α and p38. B. Cells were treated with 1 µg/ml Thapsigargin (Tg) for 24 h followed by Western blot analysis using antibodies specific for PARP and total eIF2α.</p

Proteome profiling of ER proteins at baseline.

<p>The SILAC methodology followed by mass spectrometry of microsomal preparations was processed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003783#s4" target="_blank">Materials and Methods</a>. Untreated labeled DDR1 cells were mixed at a 1∶1 ratio with unlabeled sh2B5 or sh2B5+2B5(R195H) cells. The level of Bip, PDIA1, PDIA3, PDIA4 and PDIA6 relative to DDR1 control cells is shown.</p

Proteome profiling of ER proteins following stress.

<p>The SILAC methodology followed by mass spectrometry of microsomal preparations was processed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003783#s4" target="_blank">Materials and Methods</a>. Labeled DDR1 cells and unlabeled sh2B5 or sh2B5+2B5(R195H) were treated with 1 µM Tg for 0, 12 and 24 h. The unlabeled cells at each time point were mixed at a 1∶1 ratio with the labeled DDR1 controls. The level of Bip, PDIA1, PDIA3, PDIA4 and PDIA6 at each time point (except for PDIA6 at baseline in sh2B5+2B5(R195H) cells) relative to DDR1 control cells is shown.</p