Isolation of inositol 1,4,5-trisphosphate receptor-associating proteins and selective knockdown using RNA interference.
Inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) are IP(3)-gated Ca(2+) release channels localized on intracellular Ca(2+) stores and play a role in the generation of complex patterns of intracellular Ca(2+) signals. We show herein experimental protocols for the identification of associating proteins of IP(3)R isoforms from various cells and tissues using affinity column chromatography and for the specific knockdown of the expression of IP(3)R isoforms and their associating proteins using RNA interference. These methods will provide clues to understand the exact nature of how the signaling complex contributes to the generation of spatio-temporal patterns of intracellular Ca(2+) signals.
Mizutani
Akihiro
A
Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, Saitama, Japan.
Kawaai
Katsuhiro
K
Hisatsune
Chihiro
C
Ando
Hideaki
H
Michikawa
Takayuki
T
Mikoshiba
Katsuhiko
K
D000818:Animals; D002460:Cell Line; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011506:Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D034622:RNA Interference
D053496:Inositol 1,4,5-Trisphosphate Receptors; D020033:Protein Isoforms; D011506:Proteins