Figure 3

WB analysis of reduction in PD-L1 on the cell surface. (A) PD-L1 levels in plasma membrane isolated from MDA-MB231 GFP. WB signal from samples treated with durvalumab, GS-4224, or INCY-11 is compared to the one obtained for the DMSO control. Na⁺/K⁺ ATPase is used as the plasma membrane marker, and calreticulin as the ER marker. T stands for total protein fraction, PM stands for plasma membrane fraction. (B) Titration of GS-4224 compound in MDA-MB-231 GFP cells. Tenfold serial dilution was used with a concentration range: 1 nM–1 µM and with samples collected at 24 h and 48 h. Annotation is as in (A), and MC stands for macrocycle, BMS-10034. (C) Bar graph comparison of WB signal intensity for PD-L1 plasma membrane protein in MDA-MB-231 GFP cells treated with either 1 µM GS-4224 or 1 µM BMS-10034. Na⁺/K⁺ ATPase is used as a control for general plasma membrane protein levels. Cropped and grouped blots here and in all the following figures are explicitly delineated either with dividing lines or white space. All uncropped blots are included in the Supplemental Information.