Western blot was performed using anti-PERK Polyclonal Antibody (Product # PA5-79193) and 125 kDa band corresponding to PERK was observed across the cell lines tested, also, the protein expression was increased upon Thapsigargin treatment in MCF7 and U-87 MG along with uncharacterized bands (*). Whole cell extracts (30 µg lysate) of MCF7 (Lane 1), MCF7 treated with Thapsigargin (1umol/L for 24 hrs) (Lane 2), U-87 MG (Lane 3), U-87 MG treated with Thapsigargin (1umol/L for 24 hrs) (Lane 4), HeLa (Lane 5), T-47D (Lane 6), Hep G2 (Lane 7) and U-2 OS (Lane 8) were electrophoresed using Novex® NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (0.5 µg/mL) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Western blot analysis of PERK in Lane 1: human HeLa whole cell lysate, Lane 2: human COLO-320 whole cell lysate, Lane 3: human A549 whole cell lysate, Lane 4: human SK-OV-3 whole cell lysate, Lane 5: human A431 whole cell lysate, Lane 6: rat brain tissue lysate, Lane 7: mouse brain tissue lysate using 50 µg (reducing conditions) per well. Electrophoresis was performed on 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours and protein was transferred to a nitrocellulose membrane at 150mA for 50-90 minutes. Sample was blocked with 5% Non-fat Milk/TBS for 1.5 hours at room temperature, incubated with PERK polyclonal antibody (Product # PA5-79193) at a dilution of 0.5 µg/mL (overnight at 4°C), followed by goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:10,000. Signal development was performed using a chemiluminescence (ECL) kit.