Clarity chemiluminescence kit

5r219 cells (1 × 10⁶) were harvested 72 h post-induction. Cell pellets were lysed in 2X Laemmli buffer and 10% of this sample was resolved using NuPAGE 4–12% Bis-Tris gels (Thermo Fisher) and transferred to nitrocellulose membranes using the iBlot2 system (Thermo Fisher), as described by Luitweiler et al. [32 (link)]. Rabbit antibodies to KSHV proteins were used at a dilution of 1:500. Mouse antibody to ORF26 was purchased from Novus and Mouse monoclonal CHOP antibody from Cell Signaling Technology and used at similar dilutions. Rabbit polyclonal to GAPDH (Invitrogen) was used at 1:2500 dilution (Table S1). Blots were processed using the Clarity chemiluminescence kit (Bio-Rad) according the manufacturer’s protocol and imaged using the iBright Imager (Invitrogen).

Western blotting was performed as previously described [76 (link)]. PERK, IRE1, ATF4, eIF4E-BP1, eIF2α and p-eIF2α were immunodetected using rabbit anti-human monoclonal antibodies (Cell Signaling, Danvers, MA, USA, dilution 1:1000) as the primary antibody, and peroxidase-conjugated sheep anti-rabbit (Cell Signaling Technology (Leiden, The Netherlands), dilution 1:5000) as secondary antibody. HA-tagged proteins and HIF-1α were detected using the mouse monoclonal antibodies clone HA-7 (Sigma Aldrich) and Clone 54/HIF-1α (BD Biosciences, Le Pont de Claix, France) respectively, and peroxidase-conjugated horse anti-mouse (Cell Signaling Technology, dilution 1:5000) as secondary antibody.
Protein signals were normalized using either total eIF2α or an anti-β-actin monoclonal antibody (AC-15, Sigma-Aldrich (Saint-Quentin Fallavier France), dilution 1:10,000). Signals were detected using the Clarity chemiluminescence kit (Bio Rad, Marnes-la-Coquette, France). Nuclear and cytoplasmic extracts were obtained by using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Dardilly, France) according to the manufacturer’s protocol.

Cellular fractionation was performed with the nuclear protein extraction kit (Cayman Chemical, Hamburg, Germany) according to the manufacturers’ instructions. Proteins extracted with the RNA NucleoSpin kit separated on 8% bis-acrylamide gel were transferred onto nitrocellulose membranes (Bio-Rad). Membranes were blocked for 1 h at room temperature in Tris-buffered saline (TBS) (100 mM NaCl, 10 mM Tris, pH 7.5) with 5% non-fat dry milk. Membranes were incubated overnight at 4 °C with primary antibodies against the following proteins: fibronectin (Santa Cruz, Heidelberg, Germany), β-actin (Sigma-Aldrich), active (#8814) and total (#8480) β-catenin (both from Cell Signaling, Leiden, The Netherlands). Detection was performed with horseradish peroxydase-labelled secondary antibodies (Rockland Immunochemicals, Limerick, USA) and a clarity chemiluminescence kit (Bio-Rad). Chemidoc MP imaging (Bio-Rad) was used for analysis.