Horseradish peroxidase coupled anti rabbit immunoglobulin

Cell lysates were collected from lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing phosphatase inhibitors, protease inhibitors and phenylmethylsulfonyl fluoride. The proteins (15-20 μg) were subjected to 30% Acrylamide-Bis gel (8%-12%), and transferred onto the polyvinylidene fluoride membrane (0.22 μm) (Merck Millipore, Billerica, MA, USA). The membrane was incubated with the following primary antibodies: calnexin (Cat.No. 10427-2-AP, Proteintech Group Inc., IL, USA), β-actin (ab8226, 1: 1000, Abcam, UK), CD63 rabbit antibody (1: 1000, ab134045, Abcam, Cambridge, UK), TSG101 rabbit antibody (1: 1000, ab125011, Abcam), CD81 rabbit polyclonal antibody (1: 1000, ab109201, Abcam), Alix rabbit antibody (1 μg/ml, ab76608, Abcam) (1: 1000), smad7 (R&D systems, MAB2029, 1: 1000), SMURF1 (ab236081, 1:1000), SMURF2 [EP629Y3] (ab53316, 1:1000), TGF beta 1 (ab92486, 1:1000), phospho (p)-JNKThr183/Tyr185 (cst4668, 1:1000), p-p38 MAPK (Thr180/Tyr182) (cst4511, 1:1000), p38 (cst8690, 1:1000), and JNK (cst9252, 1: 1000). β-actin was used as the internal reference. Results were visualized with horseradish peroxidase-coupled anti-rabbit immunoglobulin (Dako) using enhanced chemilumine-scence detection reagents. Band densities of targeted proteins were analyzed by Quantity One (Bio-Rad) analysis software and normalized to that of β-actin.

Macrophages (1 × 10⁶ cells) and L3-L4-L5 DRGs were lysed in RIPA buffer (Sigma-Aldrich) supplemented with antiphosphatase (Phostop, Roche) and protease inhibitor (Roche). Protein concentration was determined by BCA assay (Bio-Rad) prior to denaturation. Samples were loaded into 10% SDS-PAGE gels and transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were probed with the following primary antibodies: rabbit anti-SMAD4 (1:1000; Cell Signaling Technology, 46535), rabbit anti–p-ERK (1:1000; Cell Signaling Technology, 9101), rabbit anti–p-p38 (1:1000; Cell Signaling Technology, 9211), rabbit anti-ERK (1:1000; Cell Signaling Technology, 4695), rabbit anti-p38 (1:1000; Cell Signaling Technology, 9212), and rabbit anti-CCL2 (1:1000; Invitrogen, MAS-17040). GAPDH (1:2000; Abcam, ab8245) and β-actin (1:1000; Cell Signaling Technology, 4967) were used as loading controls. Results were visualized with horseradish peroxidase–coupled anti–rabbit immunoglobulin (Dako, Agilent) using enhanced chemiluminescence detection reagents. Protein abundances were analyzed by densitometry scanning using Fiji (ImageJ 1.52i, NIH)