Ca²⁺-dependent CSQ2 oligomerization and CSQ2–triadin interaction. (A) The Ca²⁺ sensitivity of light (350 nm) scattering of CSQ2-WT (filled circles), CSQ2-R33Q (triangles), and CSQ2-L167H (inverted triangles) proteins in presence of 100 mM CsCl. Samples were stirred for 2 min before measurement. The curve fit to the CSQ2-WT data has an EC₅₀ of 18.1 ± 5.23 mM and a 2.1 Hill coefficient. The curve fit to the CSQ2-R33Q data has an EC₅₀ of 16.4 ± 1.18 mM and a 3.0 Hill coefficient. Both curves were fit with V_MAX arbitrarily fixed at 0.6. (B) At left, top panel (i) depicts the Coomassie blue–stained SDS-PAGE of purified, recombinant CSQ2-WT (arrow, MW of ∼52,000). Bottom panel (i) depicts the Western blot with anti-triadin antibodies, revealing two bands having MW of ∼45,000 (glycosylated form) and 40,000 (unglycosylated form), respectively. At right (ii), the Ca²⁺ sensitivity of the interaction of glycosylated and unglycosylated triadin with CSQ2-WT, CSQ2-R33Q, and CSQ2-L167H was measured with either very low Ca²⁺ (1 mM EGTA) or 1 mM free Ca²⁺ present, and data are shown as means ± SEM (n = 5). Filled bars represent glycosylated triadin, open bars represent unglycosylated triadin. Asterisk indicates P < 0.05 using an unpaired Student's t test.