HUVEC were transfected with pcDNA3-FLAG-FoxO1-3A21 constitutively active mutant, pcDNA3-FLAG-FoxO1-3A-S218D mutant or empty vector control and 24 h after transfection 10 × 106 cells were fixed for 30 seconds with 3% formaldehyde and cross-linking was stopped with 2.5 M glycine.
(E) CGNs transfected with GFP vector plus wild type MCU, MCUD260A mutant, MCUE263A mutant or empty vector were treated with 70 μmol/L H2O2 for 24 h.
To assess the ability of the triple mutant to induce a myeloproliferative disease in vivo, lethally irradiated Balb/c mice were transplanted with bone marrow transduced with wild type, triple mutant or empty vector.
Figure 3B shows that all six FGFR3 mutants caused significant growth arrest when compared to cells transfected with wild-type FGFR3, kinase-inactive K508M mutant or empty vector, suggesting each has this capacity.
However, PRAP1 mRNA could not be induced in mutant or empty vector (V -transfected p53−/− cells.
Adipocytes expressing an inactive A2myr-PKB/Akt mutant or empty expression vector were used as negative controls.
In order to test whether SUMO modification of DRIL1 can modulate its transcriptional activity, we expressed wt, K398R, Kx4R mutants or empty vector in 293T cells and performed genome-wide expression microarray analysis (the raw data of the microarray analyses are available at http://www.ebi.ac.uk/microarray-as/aer/ ae-main[0], accession E-TABM-681).ac.uk/microarray-as/aer/ ae-main[0]
One of these plasmids (HA-cmv-USP28, pRC-cmv-Myc wild type, Myc-T58A mutant vector, or empty vector), or all three plasmids (pRC-cmv-Myc, pFlag-Fbw7α and HA-cmv-USP28) were transiently transfected into A549 cells in a 6-well plate (for Western blot) or in a 100 mm-dish (for IP) with lipofectamine LTX reagent (Invitrogen).
Wild-type SMC1 or SMC3, hinge mutant alleles, or empty vector (YIplac211) alone were integrated at the ura3 loci of appropriate td-smc1 or td-Smc3 strains.
Transfection of HCT116 human colorectal cancer cells with lipofectamine 2000 (Invitrogen) was performed using TCF4 luciferase reporter plasmids, modified Topflash and Fopflash, transfection control pCMV β (Promega, Madison, WI, USA), and WT ADAR1, editing defective mutant (H910K/A912E) ADAR1, or empty vector.
To confirm interaction between miR-194 and the COUP-TFII-3′-UTR region, cells were transfected with 100 ng of pmirGLO wild type (or 100 ng mutant type) or empty pmirGLO vector (100 ng) in the presence of 10 nM miR-194 precursor (or 10 nM miR-CTL) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).
