Transfected HEK 293 cells

FIGURE 5.4 Microphysiometry responses of HEK 293 cells transfected with human calcitonin receptor, (a) Use of microphysiometry to detect receptor expression. Before transfection with human calcitonin receptor cDNA, HEK cells do not respond to human calcitonin. After transfection, calcitonin produces a metabolic response, thereby indicating successful membrane expression of receptors, (b) Cumulative concentration-response curve to human calcitonin shown in real time. Calcitonin added at the arrows in concentrations of 0.01, 0.1, 1.10, and lOOnM. Dose-response curve for the effects seen in panel B. 

Tong, J., McCarthy, T. V., and MacLennan, D. H. (1999). Measurement of Resting Cytosolic Ca2+ Concentrations and Ca2+ Store Size in HEK-293 Cells Transfected with Malignant Hyperthermia or Central Core Disease Mutant Ca2+ Release Channels. J Biol Chem 274(2) 693—702. 

Western blots of microsomal fractions from transfected HEK-293 cells with the PLN-L39stop mutant, indicated that the PLN-L39stop protein could not be detected. In addition, confocal microscopy in HEK-293 cells transfected with PLN-L39stop revealed detectable immunoreactive protein signals in a small percent of cells and the PLN-mutant was mainly localized to the cell membrane, compared with PLN-WT, which localized to the endoplasmic reticulum. Consistent with these findings, human PLN-L39stop homozygous ventricles had no detectable PLN. 

Data were obtained in HEK-293 cells transfected with hSERT or hNET (Owens et al., 1997). 

Furthermore, we have similarly detected full-length p52 over-expressed in mammahan cells, using total protein extracts in conjimction with the detection system described above. CodeHnk protein-DNA microarrays were probed with total protein obtained from HEK 293 cells transfected with 4 jig eukaryotic expression vector encoding full-length human NF-kB p52... 

The downstream consequences of 3-IB-PPl- and PrINZ-induced Akt hyperphosphoiyladon were assessed in HEK 293 cells transfected with die constitutively activated myi-HA-osAktl. Both inhibitors decreased the phosphoiylation level of Ser9 on GSK3P in an inverse dose-dependent manner to the induction of Akt hyperphoqjhoryla-tion, which suggests diat PriNZ and 3-IB-PPl block downstream signaling of Akt while concomitandy inducing Akt hyperphosphorylation (Supplementary Fig. 2 online). 

Figure 6 Hyperphosphorylated Akt is hyperactive in vitro after dissociation of Akt inhibitor. HEK 293 cells transfected with HA-asAktl were cultured either with or without serum overnight and then treated with 2.5 piM PriDZ for 30 min before stimulation with lGF-1 (50 ng ml-i) for 10 min. HA-asAktl was immunoprecipitated from cell lysates and assayed for Akt activity. Data are represented as mean s.e.m. (n = 8) and as relative values to iGF-l-stimulated HA-asAktl activity. The Immunoprecipitates were analyzed for Akt (Thr308, Ser473) phosphorylation by Immunoblotting.

FIGURE 5.6 Calcitonin receptor responses, (a) Real-time melanin dispersion (reduced light transmittance) caused by agonist activation (with human calcitonin) of transfected human calcitonin receptors type II in melanophores. Responses to 0.1 nM (filled circles) and lOnM (open circles) human calcitonin, (c) Dose-response curves to calcitonin in melanophores (open circles) and HEK 293 cells, indicating calcium transient responses (filled circles). 

FIGURE 5.11 Microphysiometry responses to 1 nM human calcitonin, (a) Responses obtained from HEK 293 cells stably transfected with low levels of human calcitonin receptor (68 pM/mg protein). Response is sustained, (b) Response from HEK 293 cells stably transfected with high levels of receptor (30,000 pM/mg protein). Data redrawn from [8],... 

Expression and Recording Systems. HEK-293 cells have been transfected with cDNA for HERG-1 to produce a stable expression system. The cell line has been obtained under license for the laboratory of Craig January at the University of Wisconsin (Mohammad et al., 1997). 

Whether the two SPHK isoforms play similar or distinct roles is yet to be determined. Transfection of HEK 293 cells with a dominant negative form of SPHKl abolished subsequent TNFa-stimulated SIP formation although basal levels were unaffected (Pitson et al, 2000). Therefore, distinct isoforms of SPHK may regulate separate pools of SIP. 

Transfection ofmouse SlPphosphatase into HEK 293 cells, resulting in a 3-fold increase in membrane SIP phosphatase activity, caused a 50% deaease in SIP levels (reduced by 0.6 pmol/nmol phospholipid) and a 2 fold inaease in ceramide (inaeased by 23 pmol/nmol phospholipid) whereas sphingosine levels were similar to vector controls (0.8 pnnol/nmol phosphohpid). SIP phosphatase transfected cells underwent apoptosis in response to serum withdrawal, C2-ceramide, peroxide or doxorubicin with 2-3 fold higher frequency compared to vector-transfected control cells. Surprisingly, exogenously added SIP, which normally confers protechon, inaeased apoptosis. This may be due to its metabolism to ceramide (Mandala et al, 2000) although other factors may also be involved. 

TABLE 1. Transfection Efficiencies for Selected Carriers Using EAhy 926, HSVEC 1, and HEK 293 Cell Lines and the Step 2 and Step 5 Products... 

Fig. 12 Transfection efficiency of the complexes of protonated poly(TMPTAl-AEPZ2)/DNA (pCMV-Luc) complexes in COS-7 and HEK 293 cells compared to that of PEI (25 kDa). The transfection efficiency of PEI (25 K) was obtained under an optimal N/P ratio of 10 1. Values were presented as mean standard deviation (n = 4). Reproduced with permission from [127]. Copyright 2005 ACS...

To examine the phosphorylation of the myc-tagged wild-type 5-HT3A receptor subunits from the guinea pig, stably transfected HEK 293 cells were meta-bolically labeled with [32P]phosphoric acid (158). It was demonstrated that both splice variants of the 5-HT3A receptor subunit were phosphorylated. Moreover, site-specific mutagenesis revealed that phosphorylation occurs at Ser409, a potential target of PKA. 

HEK-293 cells, derived from human embryo kidney, were transformed with human type 5 adenovirus (Graham et al., 1977). These cells exhibit epithelial morphology and can be adapted to suspension growth in serum-free media. In addition, this cell line is easily transfected and has been explored for viral vector production for gene therapy and for obtaining human recombinant proteins with normal glycosylation profiles. 

Fig. 12.14 Left Structures of mono-substituted alkylpolyamines (AKla-5a), two bri-substituted analogues (AK6b, 7b) were also synthesized and examined for SAR confirmation. Right Inhibition of LPS (100 ng/mL)-induced NF-kB induction in HEK-293 cells stably transfected with Tlr4, MD-2, CD 14, and an NF-icB-secreted alkaline phosphatase reporter gene construct. Polymyxin B was used as the positive control...

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