Figure 2.

NPC efficiently captures the interaction proteins of CD38 in vitro and in cellulo. A, the proteins, including Nb-551₂, a known CD38 binder serving as a positive control, and BSA, a negative control, were used to test the efficiency and specificity of NPL in vitro. The indicated proteins were incubated at 4 °C for 1 h, UV-irradiated for 8 min on ice, treated with 100 mm DTT for 15 min, and analyzed by Western blotting and Coomassie Blue staining. The molar ratio of NPC probe:reCD38:Nb-551₂:BSA is 5:1:1:2. All protein markers are indicated on the left of the gels or blots in kilodaltons. B, the indicated proteins were incubated with LP-1 cells on ice for 1 h, followed by exposure to UV light for 20 min on ice. The lysates were treated with 100 mm DTT, and the biotinylated proteins were precipitated by the Strep beads and analyzed by Western blotting. IB, immunoblot. C, schematic of the semiquantitative chemical proteomics workflow. D, dot plots of the enriched proteins in NPC-labeled LP-1 cells versus those in CD38-KO LP-1 cells identified by MS. The numbers (≥2) of unique peptides of candidate proteins were plotted. CD71, MHCs, and CD38 are highlighted in red, purple, and green, respectively. The MS experiments were done twice.