Perk c33e10

Protein lysates were collected using Blue Lysis Buffer and analyzed by Western blot as previously described [22 (link)]. The recipe for Blue Lysis Buffer consists of 25 mL 2x Novex Tris-Glycine Sample Loading Buffer SDS (ThermoFisher Scientific, Waltham, MA USA, Cat# LC2676), 20 mL T-PER Tissue Protein Extraction Reagent (ThermoFisher Scientific, Waltham, MA USA, Cat# 78510), 200 µL 0.5 M EDTA pH 8.0, 2–3 complete Protease Cocktail tablets for 50 mL, 80 µL 0.1 M Na₃VO₄, 400 µL 0.1 M NaF, 1.3 mL 1 M dithiothreitol. Briefly, primary antibodies against capsid of Venezuelan equine encephalitis virus, TC-83 (Subtype IA/B) Capsid (antiserum, Goat) (BEI resources, NR-9403), VEEV GP (antiserum, Goat), VEEV nsP2 (Kerafast, Boston, MA, USA, Cat# 8A4B3), PERK (C33E10) (Cell Signaling, Danvers, MA, USA, Cat# 3192S), or horse radish peroxidase (HRP)-conjugated β-actin antibody (Abcam, Cambridge, MA, USA, Cat# ab49900) were diluted in 3% milk solution per the manufacturer’s recommended dilutions followed by the addition of the appropriate secondary antibody either anti-rabbit HRP-conjugated (Cell Signaling, Danvers, MA, USA, Cat# 7074), or anti-goat HRP-conjugated antibody. PDVF membranes were imaged on a Chemidoc XRS molecular imager (Bio-Rad, Hercules, CA, USA) using the SuperSignal West Femto Maximum Sensitivity Substrate kit (ThermoFisher, Scientific, Waltham, MA, USA, Cat# 34095).

Whole-cell protein extracts were obtained by lysing cells with Laemmli sample buffer. Protein concentration was measured with a Micro BCA Protein Assay Reagent kit (Pierce, Rockford, IL, USA). Protein extracts (20–40 µg) were subjected to reducing conditions, loaded onto a polyacrylamide gel, and then transferred to Immobilon-P membranes from Millipore (Billerica, MA, USA). One hour after blocking with 5% (w/v) non-fatty milk in Tris-buffered saline solution with Tween^® 20, membranes were incubated with the following specific primary antibodies: β-Actin (AC-15, Sigma-Aldrich), ATF4 (D4B8, Cell Signaling), CHOP (2895, Cell Signaling), NOXA (114C307, Abcam), HRI (MBS2538144, MyBioSource), PKR (3072S, Cell Signaling), PERK (C33E10, Cell Signaling), p-PERK (Thr982) (PA5-102853, Invitrogen), and GCN2 (3302S, Cell Signaling). Antibody binding was detected using a secondary antibody conjugated to horseradish peroxidase, and an enhanced chemiluminescence detection system (Amersham, Little Chalfont, UK). Raw quantification of band intensities was performed using Multi Gauge software V3.0 (Fujifilm Corporation).