Sc 46661

The treated DRGn and SC-EXOs were collected. Cellular protein was extracted by RIPA lysis buffer. The equal proteins were separated on an SDS-PAGE, and then they are transferred to the PVDF membrane. The membrane was blocked with 5% nonfat dry milk for 1 hour. The PVDF membrane was incubated overnight at 4°C with primary antibodies, including Alix (1 : 2000, ab275377, Abcam), Hsp70 (1 : 2000, ab2787, Abcam), CD63 (1 : 2000, ab134045, Abcam), TSG101 (1 : 2000, ab125011, Abcam), GRP78 (1 : 2000, ab108613, Abcam), IRE1α (1 : 500, sc-390960, Santa cruz), p-IRE1α (1 : 2000, ab124945, Abcam), CHOP (1 : 500, sc-46661, Santa cruz), JNK (1 : 2000, ab208035, Abcam), p-JNK (1 : 2000, ab124956, Abcam), XBP1 (1 : 500, sc-8015, Santa cruz), Bcl-2 (1 : 2000, ab182858, Abcam), Bax (1 : 2000, ab32503, Abcam), Caspase-3 (1 : 2000, ab32351, Abcam), Caspase-12 (1 : 2000, ab62484, Abcam), and β-actin (1 : 2000, ab8226, Abcam). The membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies. The specific bands were observed by using the hypersensitive ECL chemiluminescence kit (New Cell & Molecular Biotech Co., Ltd, P10200).

The SWFs were fully lysed by an ice-cold RIPA solution, and the SWF-lysate was centrifuged at 12000 rpm for 20 min at 4°C. Protein concentration in the supernatants was measured by a BCA protein assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Samples of 20 μg of protein were applied to SDS-PAGE glue, and the protein was transferred to a methanol-activated polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA) after running for 30 min at 80 V and 90 min at 120 V. After blocking PVDF with 5% skim milk, the immunoassay was detected with the corresponding primary antibodies and then incubated with the secondary antibodies. The bands were visualized by an enhanced chemiluminescence (ECL) kit (Bio-Rad, Hercules, USA). A ChemiScope 3400 Mini machine (Clinx, Shanghai, China) was used to detect the signal intensity. The antibodies used for Western blot were as follows: mouse anti-GRP78 (1 : 1000, sc-376768), CHOP (1 : 1000, sc-46661, Santa Cruz Biotechnology, Santa Cruz, USA), rabbit anti-caspase12 (1 : 1000, NBP1-76801), PERK (1 : 1000, NBP1-80930, Novus, USA), phospho-PERK (1 : 1000, DF7576, Affinity Biosciences, OH, USA), anti-ATF4 (1 : 1000, ET1612-37), anti-caspase3 (1 : 100, ET1602-39), and mouse anti-calreticulin (1 : 100, EM1701-61, HuaBio, Hangzhou, China).