Er stress sampler kit

Protein from the treated cell cultures was extracted and Western blot analysis was performed as described previously [37 (link)]. Expression of EBV lytic proteins was detected with anti-Zta (1:200; gift from Prof. P. Farrell, Imperial College, London, UK) and anti-BMRF1 (1:1000; gift from Dr.KH Chan, Department of Microbiology, HKU, Hong Kong SAR, China). Expression of phosphorylated ERK1/2 was detected with p-ERK1/2 rabbit polyclonal antibodies, (1:1000; #9101, Cell Signaling Technology Danvers, MA, USA). Expressions of ER stress markers PERK. IRE-1α, BiP, CHOP, XBP-1s was detected with ER stress sampler kit (1:1000; #9956, Cell Signaling Technology). Expression of autophagic proteins was detected with anti-ATG5 (1:1000; #12994, Cell Signaling Technology), anti-ATG7 (1:1000; #8558, Cell Signaling Technology,), anti-beclin-1 (1:1000; #3738, Cell Signaling Technology), anti-LC3B (1:1000; #2775, Cell Signaling Technology), anti-ATG10 (1:1000; #PA5-78593, Invitrogen), anti-ATG3 (1:1000; #3415, Cell Signaling Technology,), anti-ATG12 (1:1000; #4180, Cell Signaling Technology,) and anti-rab9 (1:1000; #5133. Cell Signaling Technology). Expression of human cellular α-tubulin and β-actin was detected with anti-α-tubulin and anti-β-actin antibody (1:5000; MilliporeSigma, Burlington, MA, USA), respectively, as loading controls.

The following primary antibodies were used (dilution 1:1,000): mouse monoclonal ubiquitin antibody (P4D1) (cat. no. sc-8017, Santa Cruz); rabbit monoclonal β-actin antibody (D6A8) (cat. no. 8457, Cell Signalling); ER stress sampler kit (cat. no. 9956, Cell Signalling) containing BiP (C50B12) (cat. no. 3177), calnexin (C5C9) (cat. no. 2679), Ero1-α (cat. no. 3264), IRE1α (cat. no. 3294), CHOP (L63F7) (cat. no. 2895), PERK (D11A8) (cat. no. 5683), and PDI antibody (C81H6) (cat. no. 3501). The following secondary antibodies were used (dilution 1:10,000): peroxidase AffiniPure F(ab’)₂ fragment goat anti-rabbit IgG (H+L) and peroxidase AffiniPure F(ab’)₂ fragment goat anti-mouse IgG (H+L) (cat. no. 111-036-003 and 115-036-003, Jackson ImmunoResearch Laboratories).
For flow cytometry, dye coupled antibodies CD11b-APC (cat. no. 130-098-088, BD), CD34-PE (8G12) (cat. no. 345802, BD), and CD45-V450 (cat. no. 642275, BD) were used for direct detection (dilution 1:100).