SDS-PAGE and Immunoblotting
Algal cells were harvested by centrifugation (5,000g, 5 min), resuspended in in 0.1 M Na2CO3containing 0.1 M DTT, and frozen at -20°C for 1 hr. Protein samples were solubilized with 5% (w/v) SDS and 30% (w/v) sucrose, and heated to 85°C for 5 min. The protein content was quantified using a protein quantification kit (Pierce BCA Protein Assay Kit, ThermoFisher Scientific) and loaded on equal protein basis. Electrophoresis and protein transfer was performed as described before (Szyszka-Mroz, Pittock, Ivanov, Lajoie & Hüner 2015). Membranes were probed with primary antibodies raised against C. reinhardtii and specific for HSP70A (1:3,000), HSP70B (1:10,000) (Agrisera, Vännäs, Sweden); CPN60A (1:5,000), HSP90A (1:1,500), HSP90C (1:5,000) (a kind gift from M. Schroda), predicted to bind specifically to their closest homologs in UWO241. HRP-conjugated secondary antibody was used (1:10,000; Sigma-Aldrich), and antibody-protein complexes were visualized using enhanced chemiluminescence detection reagents (GE Healthcare). Protein amounts were quantified by densitometry using ImageJ (National Institutes of Health, Bethesda, MA, USA).