Fig 5. MARCH8 interacts with PD-L1 (A) with its N-terminal region (B and C) in a phosphorylation-independent manner (D) and ubiquitinates PD-L1 in cells (E and F).

A and E, HEK293T cells were co-transfected with the indicated plasmids for 36 h followed by addition of MG132 (10 μM) for 9 h (A) or 2 h (E) and then harvested for preparation of whole cell lysates (WCL) for subsequent IP/Immunoblotting (IB) to detect different proteins as indicated. B, Schematic illustration of the MARCH8 domains, in which the RING domain was separated and indicated as RING and ΔC-tail. C, HEK293T cells were co-transfected with HA-PD-L1 construct and an indicated plasmid carrying a given MARCH8 gene as illustrated in B for 48 h. Cells were treated with 10 μM MG132 for 12 h before harvested for preparation of whole cell lysates and subsequent IP/IB (C). D, HEK293T cells were co-transfected with HA-PD-L1 and MARCH8 constructs for 48 h including treatment with 10 μM MG132 for 12 h before harvesting. The cells were harvested and lysed with non-phosphatase inhibitors-containing EBC buffer and treated with or without γ-phosphatase for 30 min at 37°C before being subject to IP/IB assay. F, HEK293T cells were co-transfected with different plasmids as indicated for 36 h followed by treatment with 10 μM MG132 for additional 12 h. Cells were then harvested for preparation of whole cell lysates and subsequent IP/IB.