Figure 4

In vitro CSQ1 binding to trisk95 and junctin. CSQ1 IP. Western blot showing binding of junctin and trisk95 to CSQ1 coupled to anti-CSQ1/protein A/G sepharose. (A) and (C). Binding of trisk95 (A) and junctin (C) at 1 mM Ca²⁺ to the following CSQ constructs, from left to right; WT CSQ1; CSQ1_DA1; CSQ1_DA2; CSQ1_DA3; CSQ1_ΔC7, and CSQ1_ΔC14. (B) Binding of WT CSQ1, CSQ1_ΔC7 and CSQ1_ΔC14 to trisk95 at 100 μM Ca²⁺. WT CSQ1 binding to trisk95 at 1 mM Ca²⁺ is shown in in the first lane for comparison. Each blot in (A) to (C) was immunoprobed with antibodies against CSQ1, trisk95, and junctin. Binding was repeated three to four times. (D) Quantitation of trisk95 and junctin association with CSQ1 C-terminal mutants at 1 mM Ca²⁺. The relative percentage binding of trisk95 (gray bin) and junctin (black bin) is presented as mean ± SE. Data is presented relative to the association of trisk95 and junctin with WT CSQ1. No band was detected for trisk95 binding to CSQ1_ΔC14. Asterisks (*) indicate average data significantly different (P ≤ 0.05) from binding of trisk95 or junctin to WT CSQ1. Molecular weight markers are to the left of the blots in (A) and (C).