Pdcd4

β‐mercaptoethanol, aprotinin, ACh, Histopaque‐1077 and 1119 were obtained from Sigma‐Aldrich. Collagenase V was provided by Roche Diagnostics (Indianapolis, IN, USA). L‐Norepinephrine was purchased from J&K Scientific (Beijing, China). Lipofectamine 3000 was obtained from Invitrogen (California, USA). The antibodies to TH, NET and c‐Fos were from Cell Signaling Technology (Boston, USA). The antibodies to Mlxipl and Pdcd4 were purchased from Proteintech (Chicago, IL, USA). The antibodies to Synapsin, AChE, VAChT, Cadps, Sik2, TH (phospho S31), muscarinic receptors (M1‐M5) and adrenergic receptors (α1a‐α2c) were provided by Abcam (Cambridge, UK). Anti‐Cadps (phospho S259) monoclonal antibody was synthesized and produced by SGE Biotech (Suzhou, China). The phosphorylated antibodies including Sik2 (Phos S358), Mlxipl (Phos S196) and Pdcd4 (Phos S313) were synthesized by PTM BioLab (Hangzhou, China). The information on antibodies used in this study is listed in Table 1.

After being thoroughly grounded in liquid nitrogen, the tissues were collected for western blotting (WB). The total protein of tissues or cells was extracted in RIPA lysis buffer with a cocktail of protease inhibitors (Bimake), and then boiled for 10 min with SDS loading buffer. Equal amounts of protein were electrophoresed on SDS-PAGE in 10% Tris‐glycine gels and transferred to PVDF membranes (Millipore, MA, USA). Membranes were blocked with 5% non-fat milk in tris buffered solution containing 0.05% Tween-20 at room temperature for 1 h, followed by overnight incubation with primary antibodies against β-actin (Beyotime, Beijing, China), PDCD4 (Proteintech, Wuhan, China), phosphorylated NF-κB P65 (p-P65) and NF-κB P65 (P65) (Cell Signaling Technology, Danvers, USA). After washing thrice at room temperature, the membranes were incubated with secondary antibody (Zen Bioscience, Chengdu, China) and signals were visualized by using ECL Plus Western Blotting Reagent Pack (Bio-Red, Hercules, USA). The band intensities were quantified by Fusion Solo Imaging System (VIBER LOURMAT, FRANCE).