Figure 4.

The expression of Orai1 and SOCE amplitudes in IP₃Rs-TKO cells could be regulated by modulating activities of NEDD4L. (A). Representative Western blotting results of GIPK or WT GCaMP cells after incubation of MG132 (10 μM), a proteasome inhibitor. Densitometric analysis of all repeats showed that, after 12-h treatments, Orai1 levels in GIPK cells were significantly increased. (Normalized against GAPDH, * P < 0.05, Paired t-test, n = 3). (B–C). Effects of NL.1 or NL.3 overexpression in GIPK cells. (B), Typical traces; (C), Statistics. Left, statistics of mScarlet fluorescence, indicating the relative expression of mScarlet-P2A-NL.1/NL.3; Right, statistics of mean SOCE responses shown in (B). Compared to blank controls, inhibition of NEDD4L with NL.3 significantly increased SOCE, while NL.1 significantly reduced SOCE in GIPK cells (* P < 0.03; *** P < 0.002, Student’s t-test, n = 3). (D) Typical Western blotting results. Densitometric analysis of all repeats showed that both NL.1 and NL.3 significantly altered Orai1 protein levels (NL.1 vs blank, P < 0.02; NL.3 vs blank, P < 0.04; Student’s t-test, n = 3). (E) mRNA levels of NEDD4L in WT or GIPK cells with or without overexpression IP₃R3. (** P < 0.002; * P < 0.02, Paired t-test, n = 3).