Phospho p38 mapk

Western blot (WB) analysis was performed as previously described [22 (link), 23 (link)]. In brief, cells were washed twice with cold PBS and then lysed with fresh RIPA buffer containing a cocktail of protease inhibitors. A total of 30 μg of protein from each sample was separated on 10% sodium dodecyl sulphate (SDS) polyacrylamide gels and then transferred to a PVDF membrane. After incubation with primary antibodies against CD9, CD63, CD81, Flotillin 1, HSP70, CHOP, cleaved Caspase 3, cleaved PARP, Survivin, GRP78, GRP94, phospho-eIF2α, eIF2α, phospho-p38 MAPK, p38 MAPK (Abcam, Cambridge, MA, USA, 1:2000 dilution), or beta actin (Abcam, 1:10,000 dilution) in TBST with 5% BSA, the membranes were washed three times with 5% milk (10 min each time) and then incubated with horseradish peroxidase-conjugated goat anti-rabbit or mouse secondary antibodies. The signals were visualized with enhanced chemiluminescence reagents (ECL, Thermo Fisher Scientific, Rockford, IL, USA).

Protein was isolated from LPA-stimulated splenocytes from NOD mice using Mammalian Protein Extraction Buffer (GE Healthcare, Piscataway, NJ, USA) supplemented with protease inhibitors. Proteins were resolved by SDS-PAGE, and western blotting was performed with antibodies against phospho-p38 MAPK (Abcam, Cambridge, UK), total-p38 MAPK (Abcam), ROCK2 and β-actin (Santa Cruz). β-actin was used as a loading control. Signals were detected using Fujifilm luminescent image analyzer LAS4000 with an ECL detection kit. Three or four separate experiments were performed with different samples.