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. Author manuscript; available in PMC: 2011 Oct 1.
Published in final edited form as: Cell. 2010 Oct 1;143(1):84–98. doi: 10.1016/j.cell.2010.08.040

Figure 4. SPCA2 interacts with Orai1 to mediate Ca2+ entry.

Figure 4

(A) Confocal micrographs of immunofluorescence staining of endogenous SPCA2 in MCF-7 cells showing partial plasma membrane localization. (B) Cell surface biotinylation of endogenous SPCA2 and Orai1 in MCF-7 cells. T and B represent total lysate and biotinylated fraction, respectively. (C) Co-immunoprecipitation of endogenous SPCA2 and Orai1 in MCF-7 cells. (D) Co-immunoprecipitation of HA-SPCA with Orai1-Myc following expression in HEK293. (E) Cell surface biotinylation of HA-tagged SPCA1, SPCA2 or D379N SPCA2 expressed in HEK293. (F) Basal Ca2+ in MCF-7 cells after knock down of endogenous SPCA2 or Orai1. ControlKD, n = 47; SPCA2KD, n = 54; Orai1KD, n = 52. ** P < 0.01 (Student’s t-test). (G) Normalized proliferation of MCF-7 cells with SPCA2 or Orai1 knockdown. n = 3. (H) Immunoblot of ERK 1/2 phosphorylation and Cyclin D1 expression in MCF-7 cells with SPCA2 or Orai1 knockdown. (I) Normalized growth of MCF-7 cells with SPCA2 or Orai1 knockdown in soft agar. n = 3. (J) Tumor incidence in nude mice injected with MCF-7 cells; ControlKD, n = 10; SPCA2KD, n = 8, P = 0.007 (log-rand test); Orai1KD, n = 9, P = 0.045 (log-rank test). Immunoblot (K) and normalized growth in soft agar (L) of MCF-10A cells with knockdown of Orai1 in control and cells overexpressing SPCA2. n = 3 in (L). Error bars represent standard error (F) or standard deviation (G, I and L).