Anti sumo 1 antibody

The samples were lysed in RIPA buffer supplemented with protease inhibitors at 4°C for 30 min. The protein suspensions were incubated with an anti-SUMO-1 antibody (#4930; 1:50 dilution, Cell Signaling Technology) or control IgG (A7016, Beyotime, Shanghai, China) with gentle shaking at 4°C overnight, and then Protein A/G (P2012, Beyotime) was added and incubated with gentle shaking at 4°C for 1–3 h. Each mixture was centrifuged at 2,500 rpm for 5 min at 4°C. Subsequently, the supernatant was removed, and the beads were washed for four times using PBS. The immunoprecipitated protein were removed from the beads by heating in 1× sample loading buffer at 100°C for 10 min, and the samples were subjected to Western blot analyses using anti-α-syn antibody (#2642; 1:1,000 dilution, Cell Signaling Technology).

The cells were lysed with the appropriate volume of Pierce™ IP Lysis Buffer (Thermo Fisher Scientific) supplemented with 1X PIC and 1X PMSF and 25 mM of N-ethylmaleimide (NEM) (Thermo Fisher Scientific). Lysates were placed on ice for 10 min and centrifuged at 4 °C for 15 min at 15,000× g to collect protein lysates. For immunoprecipitation, 500 µL of cell lysate (1 mg/mL) was diluted with the Pierce™ IP Lysis Buffer supplemented with PIC, PMSF, and NEM and incubated with PROX1 antibody (Cell Signaling Technology, Danvers, MA, USA) and as the control normal rabbit IgG (Cell Signaling Technology) overnight at 4 °C with rotation. After overnight incubation, 50 mL of protein A/G agarose beads were washed twice with the IP lysis buffer, were added to each sample, and incubated at room temperature for 2 h with gentle rotation, followed by elution with IgG Elution Buffer (ThermoScientific). As described above, the eluted samples were run in 12%, Bis-Tris, 1.0 mm, Mini Protein Gel. The PROX1 SUMOylation was detected by probing the blot with an anti-SUMO1 antibody (Cell Signaling Technology) [17 (link)].