, 2004). Briefly, anti-HA polyclonal sera (Molecular Probes) was diluted (1:1,000) in worm injection buffer and injected into the pseudocoelomic space. To test the pH sensitivity of superecliptic phluorin (SEP), transgenic worms that expressed SEP::GLR-1 in AVA neurons were dissected to expose the ventral nerve cord (VNC). Dissected worms were bathed in extracellular

fluid (ECF) (pH 7.4) ( Mellem et al., 2002), and the VNC was imaged both before and after solution exchange to ECF (pH 6.5). Dissected worms were then bathed in ECF (pH 7.4) containing 50 mM NH4Cl to change intracellular Romidepsin pH. Images were acquired using a Roper Cascade 512B CCD camera and a Zeiss 100× 1.0 NA water immersion lens. Confocal images were acquired using Nikon Ti-eclipse equipped with a Yokogawa CSU10 spinning disc head and captured by a Cascade 1224B EMCCD camera. Electrophysiological recordings from Xenopus oocytes were performed using standard two-electrode voltage-clamp techniques ( Walker et al., 2006a). Plasmids for cRNA are described in Supplemental Experimental Procedures. Recordings VX-809 manufacturer from AVA interneurons and muscle cells from dissected transgenic worms were performed as previously described ( Mellem et al., 2002). Rapid perfusion experiments were performed on outside-out patches obtained from AVA interneurons in dissected C. elegans preparations. Control ECF and 3 mM glutamate

ECF solutions were delivered via theta tubing mounted on a piezoelectric manipulator (MXP ZT-300, very Siskiyou). The rate of solution exchange was measured as the 10%–90% change in open-tip potential. Statistical significance was determined using Student’s

t test. Nose touch response and osmotic avoidance assays were performed as described in (Mellem et al., 2002). Reversal frequency was recorded manually and quantified using a computer program written in python. A reversal was defined as a switch from forward to backward movement. Statistical significance was determined by using the standard Student’s t test. Error bars throughout represent the SEM. HEK293 cells were cultured in DMEM medium with 10% bovine fetal serum and transiently transfected using Lipofectamine 2000 (Life Technologies) in the presence of Opti-MEM. Plasmids for transfection are described in Supplemental Experimental Procedures. Forty-eight hours posttransfection, cells were lysed in ice-cold immunoprecipitation (IP) buffer (25 mM Tris [pH 7.5], 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EDTA, and Complete Protease Inhibitor Cocktail [Roche Diagnostics]). Cell lysates were spun at 16,100 × g for 20 min and 79,000 × g for 1 hr. The supernatants were incubated with rabbit polyclonal antibodies (Santa Cruz Biotechnology, Inc.) on ice for 1 hr followed by the addition of Protein A-Agarose (Santa Cruz Biotechnology, Inc.) and gentle mixing at 4°C for 2 hr in ice-cold IP buffer.