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Figure 4 | Virology Journal

Figure 4

From: Interaction of human dipeptidyl peptidase IV and human immunodeficiency virus type-1 transcription transactivator in Sf9 cells

Figure 4

HIV1-Tat induces tyrosine phosphorylation of human-DPPIV in Sf9 cells. (A) Sf9 cells were infected with the indicated recombinant baculoviruses. After 50 h a portion of the DPPIV-expressing cells were treated with 2.7 nM final concentration of purified recombinant Tat protein (GTH: GST-Tat-His), and cultured for further 22 h. The resulting cell lysates were subsequently subjected to IP with anti-phospho-tyrosine mAb (upper panel) or anti-DPPIV pAb (lower panel). Each immunoprecipitate was separated by SDS-PAGE, blotted and probed for with anti-DPPIV pAb. DPPIV/vector: cells co-infected with DPPIV- and a control pFASTBAC1-vector recombinant virus. (B) The band intensities of phospho-DPPIV protein (in B, upper panel) were quantified with the QuantityOne software (Bio-Rad) and calibrated against the total DPPIV protein. The value of DPPIV being expressed alone for 72 h was set as standard (= 1) and the relative phosphorylation fold deduced as quotients of the values got for the other samples and the standard. (C) Sf9 cells were infected with DPPIV recombinant baculovirus and cultured for 68 h then treated with GST-Tat-His or GST and cultured for further 4 h. After solubilisation, each lysate was divided into two equal portions and either treated with alkaline phosphatase (lanes 1 and 3) or left untreated (lanes 2 and 4). The lysates were subjected to IP with anti-phospho-tyrosine mAb (upper panel) or anti-DPPIV pAb (lower panel). Immunoprecipitates were separated by SDS-PAGE, blotted and subsequently probed for with anti-DPPIV pAb. The figure represents one of three independent experimental results.

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