Figure 3.

Effects of Sig-1R ligands on the dissociation of ankyrin B (ANK220) from IP₃R-3 in NG-108 cells. NG-108 cells were incubated with respective drugs and then lysed for coimmunoprecipitation (IP) studies. (A) Time- and concentration-dependent dissociation of ankyrin B (ANK220) from IP₃R-3 by the (+)PTZ treatment. The concentration of (+)PTZ in the top row was 100 nM. For the concentration-dependent curve, immunoblots were digitally scanned and densitometrically analyzed by the National Institutes of Health image program. Data were normalized to total immunoprecipitated IP₃R-3 proteins and expressed as percentage relative to the level of coimmunoprecipitated ANK220 obtained in the absence of (+)PTZ. Note: Ten-minute treatment time for all concentrations. Data represent mean ± SEM from two to four separate determinations. (B) Effects of selective Sig-1R ligands on ANK220 coupling to IP₃R-3. Sig-1R agonists (100 nM, 10 min) decreased the amount of ANK220 coimmunoprecipitated with IP₃R-3 without altering the level of IP₃R-3. NE-100 (100 nM), a Sig-1R antagonist, did not by itself affect the ANK220 dissociation from IP₃R-3. (C) Inhibition of (+)PTZ-induced ANK220 dissociation from IP₃R-3 by NE-100. NE-100 (100 nM) was applied 5 min before (+)PTZ (100 nM, 10 min). Data were analyzed as described in A and represent mean ± SEM from six to seven separate determinations. *, P < 0.05 compared with “control”; ##, P < 0.01 compared with “NE-100 + (+)PTZ”.