Jnk2 n 18

Total liver lysates were analyzed by immunoblotting using antibodies against HBsAg (20-HR20, Fitzgerald), GADD153 (F-168, Santa Cruz), phospho-PERK (16F8, Cell Signaling), PERK (H-300, Santa Cruz), phospho-eIF2α (119A11, Cell Signaling), eIF2α (L57A5, Cell Signaling), β-actin (13E5, Cell Signaling), JNK2 (N-18, Santa Cruz), c-Jun (60A8, Cell Signaling), phospho-c-Jun (D47G9, Cell Signaling), phospho-SAPK/JNK (81E11, Cell Signaling), STAT3 (79D7, Cell Signaling), phospho-STAT3 (D3A7, Cell Signaling).

Midguts were dissected from 4^th instar A. aegypti larvae and pools of 50 entire midguts in 100 μl of rehydration buffer (7 M urea, 2 M thiourea, 4 % CHAPS, 40 mM dithiothreitol, 0.5 % pharmalyte or IPG buffer [GE Life Sciences], 0.002 % bromophenol blue, 2.5 ml) containing protease inhibitors (Complete, Roche Diagnostics) and were kept at −80 °C until processed. Midgut pools were homogenized with a motorized pellet pestle (Motor Sigma-Aldrich Z359971.1EA) on ice. Ten μg of midgut protein was fractionated by 10 % SDS-PAGE and transferred to nitrocellulose membrane. Western blot was performed as described by Aranda et al., (1996) [59 (link)]. JNK protein was detected after incubation of 1 h with polyclonal anti-JNK (1/5,000; JNK2 N-18: sc-827 Santacruz Biotech. Inc). Visualization was performed with goat anti-rabbit secondary antibody coupled to horseradish peroxidase (Amersham) (1/10,000; 1 h), followed by Super Signal chemiluminescence Luminol substrate (Pierce), according to the manufacturer’s instructions.