Figure 3.

PGRMC1-S181-phosphorylation is essential for increased cell proliferation and PHB binding upon progestin treatment. (A) Western blot analysis of PGRMC1-S181-phosphorylation and PGRMC1 protein levels in whole cell lysates of MCF7/PGRMC1 cells after treatment with progestins (10⁻⁶ M) and DMSO. S181-phosphorylation occurs on both the endogenous PGRMC1 (lower band, ≈25 kDa) and exogenous HA-tagged PGRMC1 (upper band, ≈28 kDa). Densitometric analysis of Western blot results for S181-phosphorylation of (B) exogenous PGRMC1 and (C) endogenous PGRMC1 relatively to total PGRMC1 protein level. (D–F) Relative MTT signal as surrogate for cell number of (D) MCF7/PGRMC1-S57A, (E) MCF7/PGRMC1-S181A, (F) MCF7/PGRMC1-S57A/S181A cells treated with different progestins (all 10⁻⁶ M) or DMSO for 72 h. Values were normalized to DMSO treated cells. (G) Western blot analysis of immunopurified HA-tagged PGRMC1 and co-precipitated PHB1 from MCF7/PGRMC1 cells treated with different progestins (10⁻⁶ M) and DMSO (upper panel) and PHB1 protein level in whole cell lysates in the same cells (lower panel). (H) Densitometric analysis of co-precipitated PHB1 (I) Western blot analysis of immunopurified HA-tagged PGRMC1-variants and co-precipitated PHB1 after treatment with DYD (10⁻⁶ M) or DMSO. (J) Densitometric analysis of co-precipitated PHB1. (B,C,H,J) Signal intensity was normalized to corresponding DMSO-control and signal intensity of total PGRMC1 (B,C) or each precipitated PGRMC1-variant (H,J). Statistical analysis was performed by one-way ANOVA (A–H) or two-way ANOVA (J) and Bonferroni post-hoc tests. *: p < 0.05, **: p < 0.01, ***: p < 0.001.