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).