Immun blot pvdf membrane for protein blotting

RIPA buffer (50 mM HEPES KOH pH 7.9, 150 mM NaCl, 1.5 mM MgCl₂, and 1.0% v/v NP-40) was added to cells or tumors for protein extraction. Next, 20 µg of protein were separated on 4–12% Bis-Tris Gels (Life Technologies) and transferred to an Immun-Blot PVDF Membrane for Protein Blotting (BIO-RAD, Hercules, CA, USA). Membranes were incubated with the following primary antibodies: anti-beta-actin (Chemicon, Temecula, CA, USA) and anti-PKR (product number: 3210; Cell Signaling, Danvers, MA, USA). Anti-phosphorylated PKR was purchased from Life Technologies (product number: 44–668 G, Figs. 1 and 2), Abcam (Cambridge, England, product number: ab32036; Figs. S2 and S3). Species-specific secondary antibody kits were purchased from General Electric (Charles Coffin, NY, USA). The signal was visualized with an ECL Prime Kit (General Electric). The density of the bands was quantified by normalization to β-actin using Image J Software (National Institutes of Health, Bethesda, MD, USA).

For separation by molecular weight, protein samples (5 μg) were separated by SDS-PAGE using 10% polyacrylamide gels (ATTO, Tokyo, Japan). For separation according to the isoelectric focusing point and molecular weight, protein samples (50 μg) prepared for 2D Western blotting experiments were separated using IPG dry-strip gels (pI range: 3–10; length: 18 cm; GE Healthcare) for the first dimension and 10% SDS-PAGE (ATTO) for the second dimension. 2D separation was performed as mentioned above. Following electrophoresis, proteins were transferred to polyvinylidene difluoride membranes (Immun-Blot PVDF Membrane for Protein Blotting, Bio-Rad, Hercules, CA) and reacted with anti-calreticulin antibodies (rabbit polyclonal antibody, anti-Calreticulin (#06-661) Upstate, Merck Millipore, Burlington, MA) at a dilution of 1:1000. After incubation with secondary antibodies (ECL Anti-rabbit IgG, Horseradish Peroxidase-Linked Species-Specific F(ab’)2 Fragment, GE Healthcare) at a dilution of 1:2000, immunoreactive spots were detected by enhanced chemiluminescence (ECL PRIME; GE Healthcare) and LAS-3000 (FujiFilm, Tokyo, Japan). The intensity of each protein spot was quantified (ImageQuant; GE Healthcare).

Protein sample preparation and immunoblotting were performed as described previously5 (link)17 (link). In brief, tissue samples (i.e., embryos/larvae or eye tissues) were collected in chilled PBS and dissolved in lysis buffer [25 mM Tris, 150 mM NaCl, 1 mM EDTA·2 Na, 1% Igepal CA-630, 1% proteinase inhibitor cocktail (P-8340, Sigma, MO 63103, U.S.A), 1% sodium deoxycholate, and 0.1% SDS, pH 7.5] at a concentration of one tissue sample/7 µl lysis buffer. The supernatant was mixed with an equal volume of 2× sample buffer [100 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 0.01% (w/v) bromophenol blue (Wako, Osaka, Japan), and 10% β-mercaptoethanol (Sigma)], boiled for 5 min, and then stored at −20°C until use. Proteins were separated on a 10% gel (456–1033; Mini-Protean TGX Gels; Bio-Rad, CA 94547, U.S.A) by SDS-PAGE and transferred onto a PVDF membrane (Immun-Blot PVDF membrane for protein blotting, Bio-Rad). The blots were conventionally labelled with the primary antibody and visualised with the biotinylated secondary antibody and an ABC/DAB system (Vectastain ABC Elite kit, DAB substrate kit, Vector Laboratories).