Ab75749

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).

PRKRA and TARBP2 protein levels were assayed from the tissues and MEFs using standard western blotting protocols. A rabbit monoclonal antibody raised against a synthetic peptide corresponding to the C-terminal end of human PRKRA (#ab75749; Abcam) and a TARBP2 antibody from a previous study (Zhong et al. 1999 (link)) were used to quantify protein levels. GAPDH was used as a loading control.