Pvdf blocking agent

Protein samples were reduced with DTT and loaded onto NuPAGE Bis-Tris gels to detect total and phosphorylated JNK, p-38, and p-65, HSP90A, PKR, and PACT and onto NuPAGE Tris-Acetate gels to detect AHNAK. For immunodetection, gels were transferred onto PVDF membranes. The membranes were blocked with PVDF blocking agent (Toyobo, Osaka, Japan), and then incubated with anti-JNK antibody (#9252; Cell Signaling Technology, Danvers, MA), anti-phospho-JNK antibody (#4668; Cell Signaling Technology), anti-total p-38 antibody (#8690; Cell Signaling Technology), anti-phospho-p-38 antibody (#4511; Cell Signaling Technology), anti-total p65 antibody (#8242; Cell Signaling Technology), anti-phospho-p65 antibody (#3033, Cell Signaling Technology), anti-HSP90α antibody (GTX109753; GeneTex, Irvine, CA), anti-AHNAK antibody (ab178317; Abcam, Cambridge, MA), anti-PKR antibody (ab32506; Abcam), or anti-PACT antibody (ab75749; Abcam). The immunoreactive proteins were identified using a Westernbright Quantum Kit (K-12042-D20; Advansta, Menlo Park, CA).

All protein samples were reduced with DTT and loaded onto NuPAGE Bis-Tris (Life Technologies) in MOPS buffer. Separated proteins were transferred onto a PVDF membrane for immunodetection. The membrane was blocked with a PVDF blocking agent (TOYOBO, Tokyo, Japan) for 1 hr at room temperature, and was then incubated with the anti-PP antibody (1∶2500) for 1 hr at room temperature. After washing the membrane with T-DPBS, it was incubated with the HRP-conjugated goat anti-rabbit antibody (1∶20,000) as the secondary antibody for 1 hr at room temperature. Immunoreactive proteins were identified using a chemiluminescent substrate (WesternBright ECL). To efficiently detect acidic proteins, the gel was washed and fixed with 25% isopropanol for 3 hr. The fixative solution was replaced every 20 min. The gel was stained overnight with 0.025% Stains-All (Sigma) containing 30 mM Tris-HCl, 7.5% formamide, and 25% isopropanol at pH 8.8 and then washed with deionized water until the protein bands were visible. Coomassie brilliant blue staining was performed with Coomassie brilliant blue R (Sigma) following a general protocol.