Ubiquitin lys48

VLX1570 was synthesized by OnTarget Chemistry AB (Uppsala, Sweden). BSO, Thapsigargin, Z-VAD-FMK were obtained from Sigma Aldrich (Darmstadt, Germany). Antibodies were from the following sources: ubiquitin Lys48 (Merck, Kenilworth, NJ, USA), active caspase-3, BIP, and XBP1S obtained from Cell Signaling Technology (Danvers, MA, USA). PARP and antiheme oxygenase-1 were obtained from BD Biosciences (San Jose, CA, USA). β-actin and HSP70B were obtained from Sigma-Aldrich (Darmstadt, Germany). USP14 was obtained from Bethyl Laboratories (Montgomery, TX, USA).

Cells were lysed in Laemmli buffer (62.5 mM TrisHCl, 2% SDS, 10% Glycerol, 0.1% Bromphenolblue, 5 mM β-mercaptoethanol). Whole cell lysates were separated on 10% SDS polyacrylamide gels and electro blotted onto nitrocellulose membranes (Whatman). Membranes were blocked for 1 hour in 5% (w/v) non-fat milk in TBS-T (20 mM Tris base, 137 mM sodium chloride, 0.1% Tween 20, pH 7.6). The membranes were probed with antibodies against p97 (Abcam), HDAC6 (Cell Signaling), ubiquitin Lys48 (Merck), ubiquitin Lys63 (Millipore), LC3B (Cell Signaling) or α-tubulin (Abcam) as an endogenous control. As secondary antibodies, horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse antibodies (Jackson ImmunoResearch) were used. Signals were detected using the ECL Western blotting detection reagents (GE Healthcare) and the Alpha Imager Software system (Alpha Innotech). Expression analysis of specific proteins was performed by pixel quantification of the electronic image.

Antibodies were from the following sources: ubiquitin Lys48 (#05-1307, Merck, Kenilworth, NJ USA), eIF2α (#5324S, Cell Signaling, Danvers, MA, USA), phospho-eIF2α (#9722S, Cell Signaling, Danvers, MA, USA ), CHOP (#5554 Cell Signaling, Danvers, MA, USA ), active caspase-3 (#9661S, Cell Signaling, Danvers, MA, USA ), BIP (#3183S, Cell Signaling, Danvers, MA, USA ), PARP #556494, BD Biosciences, San Jose, CA, USA), and β-actin (#A5316, Sigma-Aldrich, St Louis, MO, USA). Anti-HSC70/HSP70 antibody (clone N27), recognizing both HSC70 and HSP70, was purchased from Enzo Life Sciences, (Farmingdale, NY, USA) and anti-RPL10A (NBP2-47298). antibody was from Novus Biologicals, Centennial, CO, USA.