Nrdp1

5 × 10⁶ YTS-CLEC16A and YTS cells were lysed in ice cold IP-lysis buffer. After centrifugation at 13,000 rpm for 10 min at 4°C, supernatants were pre-cleared with 50 μl of agarose G beads (Invitrogen) and IgG for 45 min at 4°C. The pre-cleared lysates were incubated with CLEC16A, Vps-16A, CD226, Hrs, Actinin-4, Nrdp1, IgG (Millipore) antibodies for 1 h at 4°C and then with 50 μl of agarose G beads for an additional 1 h at 4°C. Immune complexes were washed three times, dissolved in SDS sample buffer, electrophoresed, and transferred onto nitrocellulose membranes (Invitrogen). Western Blot analysis was performed as described above. Membranes were probed for CLEC16A (Abgent), Vps-16A (Santa Cruz), CD226, Hrs, and Nrdp1 (Novus BioLogicals). For CLEC16A in murine splenic cells, spleens were harvested and cell suspensions were prepared by dicing spleens. 250 × 10⁶ splenocytes from control and knockout mice were lysed in ice cold IP-lysis buffer and IP pull down was performed as described above. Membranes were probed for CLEC16A and Hrs. The membranes were washed and incubated with a respective mouse/rabbit secondary antibody and bound antibody was detected with WesternBright ECL kit (Advansta). Membranes were stripped and re-probed for β-actin as a loading control.

Briefly, lysis was performed with NP40 lysis buffer. The lysates were electrophoresed on 4–12% NuPAGE Bis-Tris gels in MOPS SDS running buffer and transferred onto nitrocellulose membranes (Invitrogen) overnight. The membranes were blocked in 3% BSA and incubated with indicated primary antibodies where specified for: mouse CLEC16A, PINK1 (Abgent), GFP, Nrdp1 (Novus Biologicals), TOM20 (ProteinTech), Parkin, p62/SQSTM1, LC3 I/II, ATG16L1, cytochrome c, caspase-9, p-ERK1/2 and EK1/2 (Santa Cruz), cleaved Caspase-3, p-Akt (ser473), p-Akt (Ser308) and total Akt (Cell signaling). The membranes were washed and incubated with a respective mouse/rabbit secondary antibody and bound antibody was detected with WesternBright ECL kit (Advansta). Membranes were stripped and re-probed for β-actin as loading control.