Specific detection and semi-quantitative analysis of TRPC4 protein expression by antibodies

被引:25
作者
Flockerzi, V [1 ]
Jung, C
Aberle, T
Meissner, M
Freichel, M
Philipp, SE
Nastainczyk, W
Maurer, P
Zimmermann, R
机构
[1] Univ Saarland, Fak Med, Expt & Klin Pharmakol & Toxikol, D-66421 Homburg, Germany
[2] Univ Saarland, Fak Med, D-66421 Homburg, Germany
来源
PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY | 2005年 / 451卷 / 01期
关键词
TRP channels; TRPC4; cell-free synthesis; anti-TRPC antibody;
D O I
10.1007/s00424-005-1443-1
中图分类号
Q4 [生理学];
学科分类号
071003 ;
摘要
In mouse tissues two variants of the transient receptor potential (canonical) (TRPC) 4 protein are expressed: the "full-length" TRPC4 protein and a slightly smaller variant, called TRPC4 Delta(761-864), which lacks 84 amino acid residues. Although the presence of mRNA encoding the TRPC4 protein in mammalian cells and the detection of the heterologously expressed TRPC4 protein by Western blot analysis have been reported, the unequivocal detection of endogenous TRPC4 proteins has proven difficult. In the present study we compared polyclonal antibodies for the detection of TRPC4 proteins in mouse tissues and monitored their specificity and reliability by analysing corresponding tissues from TRPC4-deficient mice. In addition we introduced a procedure that allows us to estimate the amount of TRPC4 protein expressed in a single cell. Using this technique it appears that the amount of TRPC4 protein expressed stably in HEK 293 cells is at least fourfold higher than the amount of TRPC4 protein expressed endogenously in the bovine adrenocortical cell line SBAC.
引用
收藏
页码:81 / 86
页数:6
相关论文
共 34 条
[1]   Removal of Ca2+ channel β3 Subunit enhances Ca2+ oscillation frequency and insulin exocytosis [J].
Berggren, PO ;
Yang, SN ;
Murakami, M ;
Efanov, AM ;
Uhles, S ;
Köhler, M ;
Moede, T ;
Fernström, A ;
Appelskog, LB ;
Aspinwall, CA ;
Zaitsev, SV ;
Larsson, O ;
de Vargas, LM ;
Fecher-Trost, C ;
Weissgerber, P ;
Ludwig, A ;
Leibiger, B ;
Juntti-Berggren, L ;
Barker, CJ ;
Gromada, J ;
Freichel, M ;
Leibiger, IB ;
Flockerzi, V .
CELL, 2004, 119 (02) :273-284
[2]   Rapid vesicular translocation and insertion of TRP channels [J].
Bezzerides, VJ ;
Ramsey, IS ;
Kotecha, S ;
Greka, A ;
Clapham, DE .
NATURE CELL BIOLOGY, 2004, 6 (08) :709-720
[3]  
Birnbaumer L, 2000, RECENT PROG HORM RES, V55, P127
[4]   STABLE AND FUNCTIONAL EXPRESSION OF THE CALCIUM-CHANNEL ALPHA-1 SUBUNIT FROM SMOOTH-MUSCLE IN SOMATIC-CELL LINES [J].
BOSSE, E ;
BOTTLENDER, R ;
KLEPPISCH, T ;
HESCHELER, J ;
WELLING, A ;
HOFMANN, F ;
FLOCKERZI, V .
EMBO JOURNAL, 1992, 11 (06) :2033-2038
[5]   TRPA1 is a candidate for the mechanosensitive transduction channel of vertebrate hair cells [J].
Corey D.P. ;
Garcia-Añoveros J. ;
Holt J.R. ;
Kwan K.Y. ;
Lin S.-Y. ;
Vollrath M.A. ;
Amalfitano A. ;
Cheung E.L.-M. ;
Derfler B.H. ;
Duggan A. ;
Géléoc G.S.G. ;
Gray P.A. ;
Hoffman M.P. ;
Rehm H.L. ;
Tamasauskas D. ;
Zhang D.-S. .
Nature, 2004, 432 (7018) :723-730
[6]   PURIFIED DIHYDROPYRIDINE-BINDING SITE FROM SKELETAL-MUSCLE T-TUBULES IS A FUNCTIONAL CALCIUM-CHANNEL [J].
FLOCKERZI, V ;
OEKEN, HJ ;
HOFMANN, F ;
PELZER, D ;
CAVALIE, A ;
TRAUTWEIN, W .
NATURE, 1986, 323 (6083) :66-68
[7]   Lack of an endothelial store-operated Ca2+ current impairs agonist-dependent vasorelaxation in TRP4-/- mice [J].
Freichel, M ;
Suh, SH ;
Pfeifer, A ;
Schweig, U ;
Trost, C ;
Weissgerber, P ;
Biel, M ;
Philipp, S ;
Freise, D ;
Droogmans, G ;
Hofmann, F ;
Flockerzi, V ;
Nilius, B .
NATURE CELL BIOLOGY, 2001, 3 (02) :121-127
[8]   Alternative splicing and tissue specific expression of the 5′ truncated bCCE 1 variant bCCE 1Δ514 [J].
Freichel, M ;
Wissenbach, U ;
Philipp, S ;
Flockerzi, V .
FEBS LETTERS, 1998, 422 (03) :354-358
[9]   Direct activation of human TRPC6 and TRPC3 channels by diacylglycerol [J].
Hofmann, T ;
Obukhov, AG ;
Schaefer, M ;
Harteneck, C ;
Gudermann, T ;
Schultz, G .
NATURE, 1999, 397 (6716) :259-263
[10]   Gating of store-operated channels by conformational coupling to ryanodine receptors [J].
Kiselyov, KI ;
Shin, DM ;
Wang, YM ;
Pessah, IN ;
Allen, PD ;
Muallem, S .
MOLECULAR CELL, 2000, 6 (02) :421-431