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. 2013 Nov;87(21):11704–11720. doi: 10.1128/JVI.01474-13

Fig 2.

Fig 2

Interaction and validation of YB-1 partner hits. (A, B) Huh7.5 cells stably expressing JFH-1 were transfected with the indicated Flag vectors. At 48 h posttransfection, cell extracts were prepared and subjected to immunoprecipitation (IP) with anti-Flag antibody-coupled resin. Resulting eluates, as well as cell extracts, were analyzed by Western blotting with anti-Flag, anti-NS3, anti-DDX3, anti-DDX6, anti-C1QBP, anti-IGF2BP2, anti-LARP1, anti-ILF2, anti-ILF3, anti-PABPN1, anti-DHX9, and anti-actin antibodies. Stau1 and IGF2BP3 MS hits were also detected in the Flag–YB-1 immunoprecipitate (data not shown). (C) Immunoprecipitation of endogenous proteins YB-1, DDX3, DDX6, IGF2BP2, LARP-1, ILF2, RHA, PABPN1, and ILF3, followed by Western blotting detection of endogenous YB-1 (Reciprocal Co-Ip) and Western blotting detection of other endogenous proteins of the YB-1 RNP complex, i.e., DDX3, DDX6, IGF2BP2, LARP-1, ILF2, RHA, PABPN1, and ILF3. Immunoprecipitation of FLAG protein and endogenous polymerase II (POLII) was used as a negative control. Ctrl −, negative control; Ab, antibody; arrow, specific protein; *, heavy chain of immunoglobulin; **, nonspecific protein.