4.1. Cell Culture and Protein Extraction

The wild-type strain of Synechocystis was grown in BG-11 liquid medium bubbling with filtrated air at 30 °C under continuous illumination at 40 µmol photons m⁻² s⁻¹ [66]. Cells were grown under several conditions and harvested at different growth stages. For stress treatments, the cultures were grown to the exponential phase (OD₇₃₀ = 0.7~0.8) and immediately resuspended in medium deficient of nitrogen [67], iron [68], potassium [69], or A5 (trace metal elements). Briefly, cells in the exponential phase of growth were harvested by centrifugation, washed twice with nitrogen deficiency medium BG11 (lacking the specified nutrient), and immediately resuspended in medium BG11 without the specified nutrient: in Fe-free medium BG11 that added an Fe-binding chelator to the growth medium; in potassium-free medium BG11 that contained Na₂HPO₄ instead of K₂HPO₄; in nitrogen-free medium without nitrate (NaNO₃); or in A5-free medium BG11 (without the trace elements H₃BO₃, MnCl₂•4H2O, ZnSO₄•7H₂O, Na₂MoO₄•2H₂O, CuSO₄•5H₂O, and Co(NO₃)₂•6H₂O). For HL treatment, cells in the exponential growth phase (OD₇₃₀ ≈ 0.7~0.8) were harvested and adjusted to an OD₇₃₀ of ~0.3 with fresh medium in tubes (φ40 × 200 mm) containing 100-mL cultures that were exposed to HL (400 µmol photons m⁻² s⁻¹) for designated times. For high salt stress and heterotrophic conditions, cells grown to the exponential phase were transferred to fresh medium supplemented with 0.685 M NaCl or 5 mM glucose. Cells with different treatments were harvested by centrifugation at 6000 g for 5 min at 4 °C, washed twice with ice cold PBS buffer, and resuspended in ice-cold lysis buffer (20 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 50mM Nicotinamide, pH 7.5 and 1 × protease inhibitor cocktail). The mixture was then disrupted by sonication (2s on, 2s off) for 30 min on ice with an output of 135 W by a JY92-IIN sonicator (Ningbo Scientz Biotechnology Co., Ltd., Ningbo, China), and centrifuged at 5000× g for 30 min at 4 °C. The supernatants were stored in aliquots at −80 °C until further use. Protein concentrations were measured using a BCA Protein Assay Kit (Beyotime, Jiangsu, China).