rat kangaroo ptk2 cells  (ATCC)

Journal: Journal of Investigative Dermatology

Article Title: Androgen Regulation of the Human Hair Follicle: The Type I Hair Keratin hHa7 Is a Direct Target Gene in Trichocytes

doi: 10.1111/j.0022-202x.2004.22336.x

Figure Lengend Snippet: Figure 5 The androgen receptor binds to the three hHa7 AREs. (A) Double-stranded, end-labeled oligonucleotides containing the individual hHa7 ARE-1, ARE-2, and ARE-3 sequences were incubated with nuclear protein extracts of untransfected (first lanes) or pSG5-AR-transfected PtK2 cells (second lanes). The indicated oligo- nucleotides were further incubated with nuclear protein extracts of pSG5-AR-trans- fected PtK2 cells, to which a polyclonal antibody against the human AR was added (third lanes). (B) End-labeled hHa7 ARE oligonucleotides, incubated with nuclear extracts of pSG5-AR-transfected PtK2 cells (first lanes), were competed with a 100-fold excess of the corresponding unlabeled oligonucleotides (second lanes) or a random oligonucleotide (third lanes). DNA/protein binding complexes are mar- ked by arrowheads; the two supershifted complexes are denoted by asterisks.

Article Snippet: Cell lines Rat kangaroo kidney epithelial cells PtK2 (ATCC number, CCL-56); PC3-ARwt cells (derived from the androgenindependent prostate cell line PC3 by stable transfection of the human AR), kindly provided by Dr A. Cato, Institute of Genetics and Toxicology, Nuclear Research Center, Karlsruhe, Germany; LNCaP cells (ATCC number, CRL-1740).

Techniques: Labeling, Incubation, Transfection, Protein Binding

Journal: Journal of Investigative Dermatology

Article Title: Androgen Regulation of the Human Hair Follicle: The Type I Hair Keratin hHa7 Is a Direct Target Gene in Trichocytes

doi: 10.1111/j.0022-202x.2004.22336.x

Figure Lengend Snippet: Figure 8 Transient co-transfection experi- ments. (A) PtK2 cells were transiently co-transfected with expression vector pSG5-AR and pNass reporter con- structs of the hHa7 promoter frag- ments p0.2a7-N-p1.4a7-N indicated in Fig 4. Construct p3D1.4a7-N con- tains a 1.4 kb promoter version in which the three AREs were specifically deleted. Constructs p0.9a2-N and p0.9a5-N contain the proximal pro- moter regions of hair keratin genes hHa2 and hHa5, respectively (Rogers et al, 1998). MMTV represent control construct pMMTV--gal. Transfections were performed either in the absence (gray columns) or presence of R1881 (white columns). (B) The co-transfec- tion series described in A were re- peated with the GR expression vector pSG5-AR either in the absence (gray columns) or presence of dexametha- sone (white columns). Results are given as x fold -galactosidase activity relative to control (average of three experiments SD).

Article Snippet: Cell lines Rat kangaroo kidney epithelial cells PtK2 (ATCC number, CCL-56); PC3-ARwt cells (derived from the androgenindependent prostate cell line PC3 by stable transfection of the human AR), kindly provided by Dr A. Cato, Institute of Genetics and Toxicology, Nuclear Research Center, Karlsruhe, Germany; LNCaP cells (ATCC number, CRL-1740).

Techniques: Cotransfection, Transfection, Expressing, Plasmid Preparation, Construct, Control, Activity Assay