FIGURE 5.

Western blot analysis of immunoprecipitates. Immunoprecipitates were generated using two monoclonal antibodies, clone 3G8 (left) and clone 245536 (right). A, blots were developed using anti-CD16 polyclonal showed broad-band between 20 to 40 kDa. Additional band at 75 kDa was observed. In CD4⁺ cells, additional band at ∼60 kDa was observed in immunoprecipitates with clone 245536. Low molecular weight proteins were observed prominently in IPs with 245536 in CD4⁺ T-cells. A 53-kDa band was observed in all cells. Lanes left to right, Jurkat 2 (un-stimulated); P116 (un-stimulated); THP-1 (un-stimulated); naïve CD4⁺ T-cells (stimulated with anti-CD3+ICs+C5b9); Jurkat (stimulated with anti-CD3+ICs+C5b-9); Magic Mark XP molecular weight markers; Jurkat 1 (stimulated with anti-CD3+ICs+C5b-9); naïve CD4⁺ T-cells (stimulated with anti-CD3+ICs+C5b-9); THP-1 (un-stimulated) and P116 (stimulated with anti-CD3+ICs+C5b-9). B, in CD4+ T-cells and THP-1, a broad protein band at 60–80 kDa and 20–37 kDa along with minor bands at 40 and 50 kDa are observed. C, in non-denaturing gels, anti-CD16 polyclonal antibody recognized a protein at ∼58–60 kDa in Jurkat, P116 and two monocyte preparations. THP-1 cells showed multiple bands. HFF and 293T cells did not react to 58–60 kDa protein but showed faint bands. HFF showed a low MW reactive protein. D, reduction and alkylation of IPs from Jurkat, P116, THP-1, and monocytes showed major multiple bands between 20 to37 kDa and a band at 53 kDa. 293T cells only showed two low intensity bands at 20–25 kDa. E, N-glycanase digestion of IPs from both monocytes and CD4⁺ T-cells demonstrated a major protein band at 21 kDa. A protein band at 53 kDa with a faint band at 40 kDa was also present.