Figure 6: Menin interacts directly with calcium and regulates the levels of active histone marks and SKP2 transcription.

A-B) HT-29 cells were treated for 30 hours, then ChIP assay was performed to look for menin (A), RNA polymerase II, H3K4me3, and total H3 (B) at amplicon 1 of the SKP2 promoter. 10 μM gefitinib, 1 μM MI-2–2. *p < 0.05 compared to Control. C-D) HT-29 cells were treated for 30 hours, then ChIP assay was performed to look for menin (C) and RNA polymerase II (D) at amplicon 1 of the SKP2 promoter. 10 μM gefitinib, 1 μM MI-2–2, 2 μM BAPTA-AM. *p < 0.05 compared to Control and BAPTA-AM. E) Chelex resin eluent after incubation with HT-29 cell lysate. Uncharged resin or resin prepared with NaOH or CaCl₂. Equal volumes of eluent were utilized to analyze protein by western blot. Ponceau S demonstrated no significant non-specific protein binding to resin. F) Chelex resin eluent after incubation with purified recombinant menin protein. Uncharged resin or resin prepared with NaOH or CaCl₂. Equal volumes of eluent were utilized to analyze protein by western blot. G) After incubation with recombinant menin protein, uncharged and CaCl₂ Chelex resin was washed with different calcium concentrations with resulting protein eluent examined by western blot. H-I) Differential scanning fluorimetry was performed on purified recombinant menin protein with different concentrations of CaCl₂, with example fitted curves (H) and average melting temperatures (I).