Anti phospho sapk jnk t183 y185

Proteins were electrophoresed through precast 4–20% tris‐glycine gels (Bio‐Rad) and transferred with an iBlotTM Gel Transfer Device (Novex‐Life Technologies) onto nitrocellulose membranes (Invitrogen). Membranes were blocked in blocking buffer for fluorescent western blotting (Rockland MB‐070‐010) for 1 h at room temperature and incubated overnight in primary antibodies at 4°C. The following primary antibodies were used: anti‐actin (Sigma‐Aldrich A2066; 1:3,000), anti‐eEF2 (Cell Signaling Technology 2332; 1:1,000), anti‐Myc (Sigma‐Aldrich C3956; 1:1,000), PSD‐95 (Millipore MAB1598 1:1,000), β‐Actin (Cell Signaling Technology 3700, 1:10,000), anti‐phospho‐eEF2(T56) (Cell Signaling Technology 2331; 1:1,000), anti‐SAPK/JNK (Cell Signaling Technology 9252), anti‐phospho‐SAPK/JNK(T183/Y185) (Cell Signaling Technology 9251), and anti‐PrP^C (Cayman 189720; 1:500). For blotting, AZ59 was used at 100 ng/mL. Appropriate secondary antibodies were applied for 1 hour at room temperature (Odyssey donkey anti‐mouse or donkey anti‐rabbit conjugated to IRDye 680 or IRDye 800, LI‐COR Biosciences) and proteins were visualized with a LI‐COR Odyssey infrared imaging system. Quantification of band intensities was performed within a linear range of exposure.