Protocol detail

Cloning and Characterization of hTPC2

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The cDNA for hTPC2 was cloned from HEK293 cells by RACE-PCR. Northern hybridization was performed using a multi-tissue human mRNA blot (BD Biosciences). For stable expression, the N-terminal HA-tagged hTPC2 was placed in pIRESneo (BD Biosciences), transfected in HEK293 cells, and stable clones selected and maintained using G418. [³²P]NAADP synthesis, membrane purification and radioligand binding studies were carried out as previously described 23 (link),25 (link). Caged-NAADP was synthesized as described 30 . TPC2 knockout mice were developed from an ES cell line (YHD437) containing a gene trap mutation in the Tpcn2 gene. Details for flash photolysis of caged-NAADP, intracellular NAADP dialysis, Ca²⁺ imaging, and measurement of Ca²⁺-activated cation currents are described in additional methods.

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Calcraft P.J., Arredouani A., Ruas M., Pan Z., Cheng X., Hao X., Tang J., Rietdorf K., Teboul L., Chuang K.T., Lin P., Xiao R., Wang C., Zhu Y., Lin Y., Wyatt C.N., Parrington J., Ma J., Evans A.M., Galione A, & Zhu M.X. (2009). NAADP mobilizes calcium from acidic organelles through two-pore channels. Nature, 459(7246), 596-600.

Publication 2009

A gene Cdna Cell line Clones Dialysis Es cell G418 Hek293 cells Human Hybridization Intracellular Knockout mice Membrane Mrna Mutation Naadp Photolysis Synthesis Tissue Tpcn2

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Protocol cited in 19 other protocols

Variable analysis

independent variables

Stable expression of N-terminal HA-tagged hTPC2 in HEK293 cells
Generation of TPC2 knockout mice from an ES cell line (YHD437) containing a gene trap mutation in the Tpcn2 gene

dependent variables

[32P]NAADP synthesis
Membrane purification
Radioligand binding studies
Flash photolysis of caged-NAADP
Intracellular NAADP dialysis
Ca2+ imaging
Measurement of Ca2+-activated cation currents

control variables

Maintenance of stable clones using G418

positive controls

Radioligand binding studies carried out as previously described 23 (link), 25 (link)

negative controls

Not explicitly mentioned

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