Fig. 6. µCH1 binds to ERGIC-53 in a Ca²⁺-dependent manner. (A) Ca²⁺-dependent co-localization of ERGIC-53 and µCH1. After 5 days of culture without tetracycline, H[µCH1] cells were treated for 2 hours with or without 50 µM CPA (top right and left panels, respectively), a drug that reversibly depletes intracellular Ca²⁺ stores. CPA was then washed out and cells were cultured in Ca²⁺-containing medium for 15 and 45 minutes (bottom left and right panels, respectively) before fixation and co-staining with anti-µ (red) and anti-ERGIC-53 (green); bar 5 µm. (B) ERGIC-53 is recruited to the insoluble fraction in cells expressing µCH1. Detergent-soluble (s) and insoluble (i) fractions were obtained from cells treated with or without tetracycline as indicated. In order to preserve Ca²⁺-dependent interactions, EDTA was omitted from lysis buffer, and 1 mM CaCl₂ added (lanes 3-6). An aliquot of cells was lysed in the presence of EDTA (lanes 1-2). Aliquots were resolved under reducing conditions, and blotted. The filter was sequentially decorated with anti-ERGIC-53 and anti-µ. (C) Ca²⁺-dependent co-immunoprecipitation of ERGIC-53 and µCH1. H[µCH1] cells were cultured without tetracycline for 2 days and then transfected with ERGIC-53 wt, the KKAA or the lectin-deficient mutant N156A. After a further 40 hours in culture without tetracycline, cells were treated with 50 µM CPA for 2 hours to deplete intracellular Ca²⁺ stores (lanes 3-4), and cross-linked with DSP. Equal aliquots of the lysates were immunoprecipitated with anti-µ or anti-ERGIC-53, as indicated. Immunoprecipitates were resolved by SDS-PAGE, transferred to nitrocellulose and probed with anti-Myc, in order to detect co-precipitated exogenous ERGIC-53. (D) The amount of ERGIC-53 co-immunoprecipitated with µCH1 was quantified and expressed as the percentage relative to total immunoprecipitable material (IP with anti-ERGIC-53). Average of two independent experiments.