HCAECs were maintained in EBM-2 alone (Control) or EBM-2 containing the ER stressor thapsigargin (“Thaps”, 1 μM) for 24 hours in the presence or absence of SKF96,365 (“SKF”, 30 μM); treatment with SKF alone was included as a control. In A), following treatments total RNA and cDNA were prepared as described in Methods. Expression levels of the UPR components IRE1α, PERK, BiP, CHOP and ERO1α were examined by qRT-PCR. Graphs represent mean ± SEM of at least three independent experiments performed in triplicates. B) After the treatments described above, cells were processed for immunodetection of IRE1α (~130 kDa), BiP (~75 kDa) or CHOP (~27 kDa) in whole cell lysates. Membranes were reprobed for GAPDH (~37 kDa) to control for protein loading. Blots are representative of three independent experiments.