Ngfr5

PCI-13 cells were incubated with serum-free DMEM medium for 24 hrs. The cells were incubated with either monoclonal antibody to CD271 (NGFR5, Thermo Scientific) for 30 min and then, incubated with recombinant human NGF (Shenandoah Biotechnology, Inc.) for 1 hr. Whole cell lysates were collected, and proteins were separated by gel electrophoresis. Western analysis was performed with phospho-Erk, total Erk, and actin antibodies, described above.

The following antibodies were used for the flow cytometry analysis and cell sorting: CD4 (1:400, RM4-5, #553051), CD8 (1:400, 53-6.7, #553033), CD44 (1:400, IM7, #553134), CD25 (1:200, PC61, #562606), IL-2 (1:200, JES6-5H4, #554428), IL-4 (1:200, 11B11, #554435), IL-10 (1:200, JES5-16E3, #554467), IL-17 (1:200, TC11-18H10, #560184), GM-CSF (1:200, MP1-22E9, #564747), PD-1 (1:200, J43, #562584), CTLA-4 (1:200, UC10-4F10-11, #553720), and GITR (1:200, DTA-1, #558140) antibodies were purchased from BD Biosciences. Ki67 (1:100, anti-human, clone B56, #556027), CD103 (1:100, M290, #557495), and ICOS (1:200, 7E.17G9, #552146) antibodies were purchased from BD Pharmingen. IFN-γ (1:200, XMG1.2, #25-7311-41), Foxp3 (1:200, FJK-16s, #12-5773-82), LAG3 (1:200, C9B7W, #17-2231-82), Tim3 (1:200, RMT3-23, #12-5870-82), and KLRG1 (1:200, 2F1, #25-5893-82) antibodies were purchased from eBioscience. TIGIT (1:200, 1G9, #142103) and Bcl2 (1:100, BCL/10C4, #633503) antibodies were purchased from Biolegend. NGFR (1:800, NGFR5, #MS-394-B1) and Live/Dead cell stain kit (#L34955) were purchased from Thermo Fisher Scientific.