FIGURE 7.

CRISPR/Cas9-mediated deletion of RNF170 and reconstitution with exogenous RNF170 constructs. A, levels of RNF170, IP₃R1, and other pertinent proteins in lysates from αT3 control and RNF170KO cells. B, GnRH (0.1 μm)-induced Ca²⁺ mobilization in αT3 control and RNF170KO cells. C, IP₃R1 ubiquitination in αT3 control and RNF170KO cells. Cells were incubated with 0.1 μm GnRH and anti-IP₃R1 IPs and input lysates were probed for the proteins indicated. D, IP₃R1 down-regulation in αT3 control and RNF170KO cells. Cells were incubated with 0.1 μm GnRH and lysates were probed for the proteins indicated. The histogram shows combined quantitated immunoreactivity (mean ± S.E., n = 4). E, reconstitution of IP₃R1 ubiquitination in RNF170KO cells. αT3 RNF170KO cells stably expressing ^WTRNF170 or ^R198CRNF170 were incubated without or with 0.1 μm GnRH for 20 min, and anti-IP₃R1 IPs were probed for the proteins indicated.