Mass spectrometric analysis of immunoprecipitates of anti-CIB1 and EphA2 antibodies with lysates from KSHV infected HMVEC-d cells.

<p>(A) Serum-starved (8 h) HMVEC-d cells were either mock or KSHV infected (20 DNA copies/cell) for 10 min and immunoprecipitated either with anti-CIB1 or anti-EphA2 antibodies. Immunoprecipitated proteins were separated by SDS-PAGE gel and gel slices (as indicated by the band #) were analyzed by mass spectrometry. (B) Serum starved (8 h) HMVEC-d cells were either left uninfected or KSHV infected (20 DNA copies/cell) for the indicated time points and immunoprecipitated either with anti-CIB1 or anti-EphA2 antibodies. Immunoprecipitates were subjected to Western blot analysis for the indicated molecules identified by mass spectrometry. Blots were then stripped and probed for total EphA2 and CIB1 levels. 20 µg of whole cell lysate (WCL) protein was also analyzed by Western blot to check for all indicated signal molecules at the total protein level. (C) Serum starved (8 h) control or CIB1-shRNA transduced HMVEC-d cells were either left uninfected or KSHV infected (20 DNA copies/cell) for 10 min, immunoprecipitated with anti-EphA2 antibodies and immunoprecipitates were subjected to Western blot analysis for total EphA2 and the indicated molecules identified by mass spectrometry. As negative control, cells were infected for 10 min with KSHV pre-incubated with heparin (50 µg/ml) for 1 h at 37°C. 20 µg of whole cell lysate protein was analyzed by Western blot to check for the indicated molecules at the total protein level with tubulin as a loading control.