When VPS33B (c.1225+5G>C) mutant was cotransfected with VIPAR, the two proteins colocalized mainly in electrondense cytoplasmic clusters that contained no membranes (Fig. 4C).
To determined whether histone H3 phosphorylation at Ser10 might directly regulate LMP1-induced AP-1 activation, mock, H3 WT or H3 S10A mutant was cotransfected with AP-1 reporter plasmid into LMP1 expressing CNE1 cells.
Next, to determine whether the Id2 mutants are repressors of transcriptional activity of E-cadherin gene, the promoter construct of E-cadherin, Id2 or either of its mutants were cotransfected into MCF-7 and SKOV-3 cells.
An amount of 1 μg of E-cadherin promoter gene construct (E-cadK1 Luc) or the E-box mutant construct and 1 μg of Id2 or its mutants were cotransfected.
In order to support the potential interaction between eEF1A isoforms in vivo, recombinant GST-eEF1A1 wt and mutants were cotransfected with eEF1A2-HIS wt in COS 7 cells and then analyzed by western blot after GST pull-down.
Flag-tagged Fes or Myc-tagged p130Gag-Fps and mutants thereof were cotransfected with GST-cortactin into HEK293T cells.
Related wild-type or mutant luciferase constructs were cotransfected with pCMV-Myc-AP-2γ or pCMV-Myc into MDA-MB-453, MDA-MB-231, and HBL-100 cells; and the reporter gene luciferase activity was assayed.
Finally, WT or mutant SF1 vectors (10ng/well) were cotransfected with Cyp11a and MIS reporters into TM3 (mouse Leydig) and TM4 (mouse Sertoli) cells, respectively.
Wild-type (wt) or mutant (mut) 3′-UTR vectors were cotransfected with miR-302c and miR-520c mimics or negative controls into 293T cells using Lipofectamine 2000 (Invitrogen).
For reporter assays, wide-type or mutant reporter constructs (15 ng) were cotransfected into 293T cells in twelve-well plates with miR-21 or miR-control (50 nM; GenePharma, Shanghai, China) and Renilla plasmid (5 ng) using lipofectamine 2000 (Invitrogen).
