Protein Extraction and Immunoanalysis Protocol

Proteins were extracted by ristocetin‐induced platelet agglutination buffer (Solarbio), and WB, Co‐IP, and confocal microscopy were performed as previously described.
¹² (link) Anti‐RTN3 primary antibody (1:2000) was self‐produced. Anti‐GAPDH primary antibody (10494‐1‐AP, 1:5000), anti‐flag primary antibody (20543‐1‐AP, 1:5000), and anti‐p62 primary antibody (18420‐1‐AP, 1:1000) were purchased from Proteintech Company (Wuhan, China). Anti‐SPHK2 primary antibody (YT4383, 1:1000) and anti‐CYP27A1 primary antibody (YT1202, 1:1000) were purchased from ImmunoWay Biotechnology Company (Suzhou, China). Anti‐myc primary antibody (sc‐40, 1:100), anti‐CERS2 primary antibody (sc‐390745, 1:100), and anti‐LC3A/B primary antibody (sc‐398822, 1:100) were purchased from Santa Cruz Biotechnology Incorporated (Santa Cruz, USA). Anti‐myc magnetic beads (Beyotime) and anti‐flag magnetic beads (Beyotime) were used for Co‐IP, and self‐produced anti‐RTN3 primary antibody (1:400) and anti‐CD31 primary antibody (66065‐2‐Ig, 1:200) were purchased from Proteintech Company; anti‐LC3 primary antibody (PM036, 1:200) were purchased from MBL International Corporation (Kyushu, Japan); anti‐CERS2 primary antibody (sc‐390745, 1:100) and DAPI (Solarbio) were used for confocal microscopy.