C jun

Protein extraction from cells treated with LJE and UVB irradiation was done by using CytoBuster™ Protein Extraction Reagent (EMD Millipore Corp, Burlington, MA, USA) that contained the Xpert Phosphatase (GenDEPOT, Barker, TX, USA) and Protease Inhibitor (GenDEPOT, Barker, TX, USA). The extracted proteins were separated with NuPAGE™ 4–12% Bis-Tris Protein gels (Thermo Fisher Scientific, Waltham, MA, USA) and transferred to the polyvinylidene fluoride (PVDF). The antibodies used for all the blots were as follows: p38 (phosphorylated) (dilution 1:1000), SAPK/JNK (phosphorylated) (dilution 1:1000), c-Jun (phosphorylated) (dilution 1:1000), ATF-2 (phosphorylated) (dilution 1:1000), p38 (dilution 1:1000), SAPK/JNK (dilution 1:1000), c-Jun (dilution 1:1000), ATF-2 (dilution 1:1000) (Cell Signaling Technology, Danvers, MA, USA), PKR (phosphorylated) (dilution 1:500) (R&D Systems, Minneapolis, MN, USA), PKR (dilution 1:1000) (Thermo Fisher Scientific, Waltham, MA, USA), beta-actin (dilution 1:15,000) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The scanning densitometric values of each band were analyzed with Image J and represented the graph as ratio to loading control.

Immunofluorescence antibodies: c-Jun (Cell Signaling Technology, rabbit 1:800), Sox10 (R and D Systems, goat 1:100), CGRP (Peninsula, rabbit 1:1000), neurofilament (Abcam, rabbit 1:1000), donkey anti-goat IgG (H+L) Alexa Fluor 488 conjugate (Invitrogen, 1:1000), and Cy3 donkey anti-rabbit IgG (H+L) (Jackson Immunoresearch, 1:500).
Antibodies used for western blotting: c-Jun (Cell Signaling Technology, rabbit 1:1000), p75 NTR (Millipore, rabbit 1:1000), serine 63 phosphorylated c-Jun (Cell Signaling Technology, rabbit 1:1000), GAPDH (Sigma-Aldrich, rabbit 1:5000), calnexin (Enzo Life Sciences, rabbit 1:1000), and anti-rabbit IgG, HRP-linked (Cell Signaling Technology, 1:2000).

Total protein was extracted from the liver of experimental fish with Total Protein Extraction Kit (Applygen Technologies Inc, Beijing, China), and nuclear and cytosolic fractions were collected using a nuclear protein extraction kit (Pierce, Nashville, TN, USA), according to the instructions of the manufacturers. Protein concentration was measured using BCA kit (Pierce). Fifty micrograms of protein was loaded onto 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Pall Corporation, Port Washington, NY, USA). The membranes were incubated with 5% skimmed milk for 1 h at room temperature. Immunoblots were obtained using antibodies against the following proteins: ERK1/2, ERK1/2 (pThr202/Tyr204), JNK1/2, JNK1/2(pThr183/Thr185), p38, p38 (pThr180/Thr182), IKKα/β, IKKα/β (pSer176/Ser180), IκBα, IκBα (pSer32/Ser36), c-Jun, c-Jun (pSer73), NF-κB p65 and beta-actin (Cell Signaling Technology, Danvers, MA, USA). Western blots were exposed using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, Waltham, MA, USA) and film images were scanned by Epson Perfection V33 (China).