Figure 4. PPI of CaM and Myc family members.

Whole cell extracts of distinct cell lines and tissues were used in CaM-agarose (CaM-ag) pull-down experiments. A. For comparison, authentic CaM binding partners were analyzed first. Endogenous CaM-dependent protein kinase from whole mouse brain extracts was detected using monoclonal antibodies directed against α-CAMKII. Ras proteins were analyzed in extracts from the human colon adenocarcinoma cell line SW480 using monoclonal anti-pan-Ras antibodies. B. To analyze the binding of distinct Myc-family members to CaM, HA-tagged v-Myc, N-Myc, and L-Myc proteins were overexpressed in QT6 cells. Whole cell extracts were prepared and subjected to CaM-ag binding assays. Proteins eluted from the affinity matrix (upper panel) and aliquots of whole cell extracts (lower panel) were analyzed by immunoblotting using HA-specific antibodies. C. Endogenous c-Myc proteins were analyzed in CaM-ag binding assays using extracts from SW480 cells and monoclonal anti-c-Myc antibodies. For direct comparison, Ras proteins were analyzed from the same extracts using monoclonal anti-pan-Ras antibodies. GAPDH was used as a negative control, using monoclonal anti-GAPDH antibodies. Input: 0.25% (Ca²⁺), or 0.1% (EDTA). D. Analyses as in (C) of endogenous c-Myc proteins from the human embryonic kidney cell line HEK293. All binding assays were done in the presence of 2 mM EDTA, or 0.5 mM CaCl₂ (Ca²⁺). E. Left panel: CaM-ag binding analysis of endogenous c-Myc proteins from HEK293 cell extracts containing 2 mM EDTA and supplemented either with no metal ions (no Me²⁺), or an excess (8 mM) of Ca²⁺ or Mg²⁺, respectively. Right panel: Analysis as before with variation of the excess Ca²⁺ concentration.