FIGURE 4.

SMPDL3A is secreted from HMDMs, N-glycosylated, and present in human serum. A, HMDM cell lysates and conditioned media from T-0901317-treated cells were incubated ± PNGaseF, separated by SDS-PAGE, and analyzed by Western blotting for human SMPDL3A as described under “Experimental Procedures.” B, SMPDL3A in fasted plasma from five healthy volunteers. Equal volumes of plasma (equivalent to 200 nl of neat plasma) were separated by SDS-PAGE and detected by anti-SMPDL3A Western blot. Area of negative staining above SMPDL3A band is due to human serum albumin. C, human plasma is N-glycosylated. 200 nl of neat plasma from a healthy donor was incubated ± PNGaseF and analyzed by anti-SMPDL3A Western blotting. D, SMPDL3A in mouse plasma (equivalent to 200 nl of neat plasma) and conditioned media from J774 macrophages, analyzed using anti-mouse SMPDL3A Western blotting.