Journal: The Journal of Biological Chemistry
Article Title: The Transient Receptor Potential (TRP) Channel TRPC3 TRP Domain and AMP-activated Protein Kinase Binding Site Are Required for TRPC3 Activation by Erythropoietin *
Figure Lengend Snippet: Subcellular localization of TRPC3, TRPC6, TRPC3/TRPC6 chimeras, PLCγ, and Epo-R. A , proteins from HEK 293T cells transfected with FLAG-TRPC3, FLAG-TRPC3-C6TRP, FLAG-TRPC6, FLAG-TRPC6-C3TRP-C3C2, and Epo-R were fractionated, purified, and analyzed by Western blotting ( WB ). Transfected FLAG-tagged constructs were detected by probing with anti-FLAG antibody. Transfected Epo-R was detected with anti-Epo-R antibody, and endogenous PLCγ was detected with anti-PLCγ antibody. Results from four fractionation experiments using FLAG-tagged constructs and two experiments using V5-tagged constructs were similar, and representative results with FLAG constructs are shown. B , representative results showing quality of fractionation by probing Western blots with anti-GAPDH, anti-Na + K + -ATPase, anti-lamin, and anti-vimentin (markers for cytosol, membrane, nuclear, and cytoskeletal fractions, respectively).
Article Snippet: Blots were incubated with anti-TRPC3 (1:400; Alomone Laboratories, Jerusalem, Israel), anti-TRPC3-C targeted to the human C-terminal sequence RRRRLQDIEMGMGNSKSRLN (1:500 to 1:1000, Bethyl Laboratories, Inc., Montgomery, TX) , anti-TRPC3-N targeted to the murine N-terminal sequence LNGDLESAEPLERHGHKASL (1:1000; Bethyl Laboratories, Inc.) , anti-TRPC6 (1:250; Alomone Laboratories), anti-V5-HRP (1:10,000; Invitrogen), anti-FLAG (1:1000; Sigma), anti-PLCγ (SC-81, 1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-Epo-R (SC-697; 1:1000; Santa Cruz Biotechnology, Inc.), anti-actin (1:10,000; Sigma), anti-tubulin (1:10,000; Sigma), anti-GAPDH (1:5000; Cell Signaling Technology, Inc., Boston, MA), anti-Na+ K+ -ATPase (1:1000; Cell Signaling Technology, Inc.), anti-lamin (1:1000; Cell Signaling Technology, Inc.), anti-vimentin (1:1000; Cell Signaling Technology), or streptavidin-HRP (Pierce) antibodies.
Techniques: Transfection, Purification, Western Blot, Construct, Fractionation