Rabbit monoclonal anti perk

Cell lysates were mixed with Laemmli buffer plus dithiothreitol (DTT), heated for 5 min at 95 °C, and loaded onto SDS-PAGE (sodium dodecyl sulfate—polyacrylamide gel electrophoresis) gels. The gels were run in Tris–Glycine SDS running buffer and then transferred onto nitrocellulose membranes. Blocking consisted of at 5% skim milk or 2.5% BSA, depending on the primary antibody used. Imaging was performed using the Li-Cor Odyssey fluorescence imaging system. Primary antibodies consisted of: monoclonal mouse anti-6xHis-Tag (Invitrogen, MA1-21315; 1:4000), polyclonal rabbit anti-ATF6 (Novus Biologicals, NBP1-75478; 1:1000), monoclonal rabbit anti-BiP (Cell Signaling Technology, C50B12; 1:1000), monoclonal rabbit anti-Calnexin (Cell Signaling Technology, C5C9; 1:1000), monoclonal rabbit anti-COG5 (Sigma-Aldrich, SAB4200440; 1:600), monoclonal mouse anti-GAPDH (Invitrogen, ZG003; 1:4000), monoclonal mouse anti-GFP (Cell Signaling Technology, 2955S; 1:1000), polyclonal rabbit anti-IRE1a (Cell Signaling Technology, 3294S; 1:1000), monoclonal rabbit anti-PDI (Cell Signaling Technology, 2446S; 1:1000), and monoclonal rabbit anti-PERK (Cell Signaling Technology, D11A8; 1:1000). Secondary antibodies consisted of: Donkey anti-Rabbit IRDye 680, Donkey anti-Rabbit IRDye 800, Donkey anti-Mouse IRDye 680, and Donkey anti-Mouse IRDye 800 (Li-Cor; 1:20,000).

To determine PERK knockdown efficiency in genetic Perk knockout neurons, mouse cerebral cortex was isolated from day 0 Perk-floxed Nestin-Cre pups, and homogenized mechanically in ice-cold buffer (100 mM HEPES, 1 mM EDTA, 2 mM EGTA, 0.5 mM DTT, supplemented with 1X protease inhibitor and 1X phosphatase inhibitor cocktails; pH was adjusted to 7.0 before use) using a polypropylene pestle. Tissue lysates for western blot were prepared using RIPA buffer with 1X protease inhibitor and 1X phosphatase inhibitor cocktails. Samples were denatured by boiling in 2X Laemmli buffer for 5 min. The following primary antibodies were used in western blot analysis: monoclonal rabbit anti-PERK (Cell Signaling), monoclonal mouse anti-β-actin (GenScript).

TMA was used for the analysis of the expression of cancer dormancy markers, including NR2F1, NANOG, MIG6, and PERK. TMAs were generated as described below. Tissue samples from endoscopic biopsy were fixed in 10% buffered formalin for 24–48 h, and then embedded in paraffin. The representative cores (2 mm in diameter) were isolated from the individual paraffin blocks and arranged in new tissue array blocks using a trephine apparatus (Superbiochips Laboratories, Seoul, Korea). Included cases had tumors occupying more than 10% of the core area. The TMA blocks contained up to 60 cores. The 4 μm sections from TMA blocks were stained with the following primary antibodies—rabbit monoclonal anti-NR2F1 (Abcam, Cambridge, MA, USA); rabbit monoclonal anti-NANOG (Abcam, Cambridge, MA, USA); rabbit polyclonal anti-MIG6 (Sigma-Aldrich, St. Louis, MO, USA); and rabbit monoclonal anti-PERK (Cell Signaling Technology, Danvers, MA, USA). Immunostaining was performed using the BenchMark XT platform (Ventana Medical Systems, Tucson, AZ, USA) according to the manufacturer’s instructions. The intensity of expression was interpreted as 0, 1+, 2+, and 3+. Intensity of 2+ or 3+ in at least 10% tumor cells was defined as positive expression; that of 0 or 1+ was defined as negative expression (Figure 3).