Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Butyrophilin 3A/CD277-Dependent Activation of Human γδ T Cells: Accessory Cell Capacity of Distinct Leukocyte Populations.

doi: 10.4049/jimmunol.1600913

Figure Lengend Snippet: FIGURE 6. Expression of FPPS and Rap1 in monocytes and neutro- phils. Highly purified monocytes and neutrophils were incubated in me- dium alone or in the presence of 2, 10, or 50 mM of ZOL. After 3 h, the cells were washed with culture medium and incubated overnight in fresh medium. Cells were collected, washed with PBS, and lysed. Five micro- grams of cellular lysates were separated by SDS-PAGE. Proteins were transferred to nitrocellulose followed by immunoblot with anti-Rap1A/B mAb (clone 26B4) to detect total Rap1, with polyclonal Ab C-17 to detect unprenylated Rap1A (uRap1A), or anti-FPPS polyclonal Ab and appro- priate HRP-conjugated secondary Ab. Blots were stripped and GAPDH served as a loading control. Comparable results were obtained in a second independent experiment.

Article Snippet: Western blots were separately analyzed using rabbit polyclonal Ab against FPPS (PA5-28228; Thermo Fisher Scientific), goat polyclonal Ab against unprenylated Rap 1A (C-17, sc-1482; Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit mAb against total Rap1 (2399; Cell Signaling Technology, Danvers, MA) and processed as described previously (37) with corresponding HRP-coupled secondary Ab and chemiluminescence reagents from GE Healthcare (Freiburg, Germany).

Techniques: Expressing, Incubation, SDS Page, Western Blot, Control