Figure 3. The extracellular domain of PD-L1 binds to TβRII to protect it from degradation.

(A) Upper: HSCs expressing TβRII-HA fusion protein were subjected to IP using anti-HA and co-precipitated PD-L1 was detected by WB. PD-L1 and TβRII-HA were co-precipitated by anti-HA. Lower: HSCs expressing TβRI-FLAG and HA-PD-L1 fusion proteins were subjected to coIP using anti-FLAG. HA-PD-L1 and TβRI-FLAG were not co-precipitated by anti-FLAG. Data are representative of multiple repeats with similar results.

(B) HSCs expressing TβRII-HA fusion protein were transduced with retroviruses encoding full-length PD-L1 (FL), the extracellular domain of PD-L1 (Ex), or the C-terminal portion of PD-L1 (the transmembrane domain + cytoplasmic domain [T + C]). Cells were collected for coIP using anti-FLAG followed by WB for HA to detect PD-L1/TβRII-HA binding. TβRII-HA was co-precipitated with PD-L1 FL or PD-L1 Ex by anti-FLAG.

(C) HSCs co-expressing TβRII-HA and FLAG-PD-L1 FL were collected for double IF for HA (red) and FLAG (green). TβRII-HA and FLAG-PD-L1 FL co-localized at the plasma membrane (arrows) and in the endosomes (arrowheads) of a HSC. Cell nuclei were stained by DAPI. Scale bar, 20 μm.

(D) FLAG-tagged PD-L1 domains were introduced into PD-L1 knockdown HSCs and cells were collected for WB for TβRII. PD-L1 FL and PD-L1 Ex rescued the TβRII protein level of PD-L1 knockdown HSCs. **p < 0.01 by ANOVA, n = 3.

(E) Control and PD-L1 knockdown HSCs, stimulated without or with TGF-β1 (5 ng/mL) for 6 h, were collected for biotinylation of cell surface protein followed by streptavidin agarose pull down to quantitate TβRII at the plasma membrane of HSCs. TGF-β1 stimulation reduced plasma membrane TβRII and at the basal condition PD-L1 knockdown reduced the plasma membrane TβRII level. **p < 0.01, ***p < 0.001 by ANOVA, n = 3. PM, plasma membrane. Data in (D and E) are represented as mean ± SEM.