Fig. 3.

Tmem178 is an ER-resident protein that targets RANKL/Ca²⁺ signaling. (A) Cytosolic Ca²⁺ measurements in WT preOCs. Ten representative cells are shown, representative of more than four independent experiments. (B) Cytosolic Ca²⁺ measurements in Tmem178^−/− preOC as in A. (C) Ratiometric imaging of HEK293T cells expressing empty vector pMX or Tmem178-HA cultured in Ca²⁺ free-HBSS and stimulated with 1 μM Tg, or Tg + 1.8 mM Ca²⁺. Traces are the average of more than 40 cells per condition, representative of three experiments. (D) Localization of Tmem178 in mature OCs by immunofluorescence. Phalloidin is used as plasma membrane marker, calnexin as an ER marker, and DAPI as a nuclear stain. (Scale bar: 100 μm.) Manually contoured white line denotes cell edge. (E) HEK293T cells transfected with Tmem178-HA (T), Stim1-Myc-His (S), and Orai1-FLAG (O) were untreated, stimulated for 10 min with 1 μM Tg in Ca²⁺-free media, or treated with Tg followed by the addition of extracellular Ca²⁺ as indicated. Lysates were immunoprecipitated and blotted as labeled. Representative of more than 10 experiments. (F) HEK293T cells transfected with Stim1-Myc-His and Orai1-FLAG with or without Tmem178-HA and treated as in E. Total cell lysates show ectopic protein expression. Representative of more than 10 experiments.