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2 protocols using pi3k c2β

1

Characterization of PI3K-C2α and PI3K-C2β Signaling

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Albumin-dinitrophenyl (DNP-BSA), anti-dinitrophenyl IgE, 4-nitrophenyl N-acetyl-β-D-glucosamide and A23187 were purchased from Sigma-Aldrich Japan (Tokyo, Japan). Wortmannin and PMA were obtained from Kyowa Medex (Tokyo, Japan) and Funakoshi (Tokyo, Japan), respectively. The monoclonal antibodies against PI3K-C2α, PI3K-C2β, CD63 (AD1) and PtdIns(3,4)P2 were obtained from BD Biosciences (San Jose, CA, USA), AbD Serotec (Kidlington, Oxford, UK) and Echelon Biosciences (Salt Lake City, UT, USA), respectively. The polyclonal antibody against PI3K-C2α was obtained from GeneTex (Irvine, CA, USA). Alexa Fluor 488-, 555- and 647-conjugated F (ab)2 fragments of goat anti-mouse and anti-rabbit IgG were purchased from Cell Signaling Technology (Danvers, MA, USA).
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2

Molecular Markers of Synaptic Signaling

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PI3K-C2β (BD Biosciences, #611342, 1:1000), phospho-p70 S6 kinase (Thr389; Cell Signaling, #9205, 1:500), p70 S6 kinase (Cell Signaling, #9202, 1:1000), S6 ribosomal protein (Cell Signaling, #2217, 1:1000), phospho-S6 ribosomal protein (Ser235/236; Cell Signaling, #2211, 1/1000), phospho-Akt (Ser473) antibody (Cell Signaling, #9271, 1:1000), Akt (pan; Cell Signaling, #4691, 1:1000), GAPDH (Cell Signaling, #2118, 1/2500), PSD95 (Synaptic Systems, #124 011, 1:1000), vGAT (Synaptic Systems, #131011, 1:1000), vGLUT (Synaptic Systems, #135302, 1:1000), synaptotagmin 1 (1Synaptic Systems, #105 011, 1:5000), mTOR (Cell Signaling, #7C10, 1:1000), GluA1 (Millipore, # MAB2263, 1:1000), Homer1 (Synaptic Systems, #160003, 1:1000), Rab7 (Cell Signaling, #9367, 1:1000), p62 (Cell Signaling, #5114, 1:1000), LC3 (Abcam, #ab48394, 1:1000), GFP (Abcam, #ab290, 1:1000), Vinculin (Abcam, #ab129002, 1:5000).
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