Western Blot Analysis of Activated Signaling Pathways in C. reinhardtii

C. reinhardtii cells were harvested 5th day after the treatment for Western blot analysis. Approximately 1.5 × 10⁶ cells of C. reinhardtii were harvested by centrifugation at 4000 rpm for 10 minutes and washed with PBS. The cell number was quantified with a hematocytometer (INCYTO, Korea) under a microscope (Nikon TS100, Tokyo, Japan). The cells were resuspended in a 100 μL mixture of Laemmli lysis buffer, beta mercaptoethanol, protease inhibitor and phosphatase inhibitor (19:1:1:1, v/v/v/v). The mixture was then heated at 100 °C for 30 min. for protein extraction. The supernatant was recovered by centrifuging the cells at 13000 RPM for 5 minutes. The protein lysate was run on a SDS page gel, which was subsequently electroblotted to a PVDF (polyvinylidene fluoride) membrane with the trans-blot turbo semi-dry transfer system (Bio-Rad). Immunodetection for the activation of ERK1/2 and MEK1/2 was done with phospho-p44/42 MAPK (Thr202/Tyr204) (Cell Signaling Technology, Beverly, MA, USA)^{49 (link)} and phospho-MEK1/2 (Ser217/221)^{50 (link)} antibodies. JNK was detected with the phospho-SAPK/JNK antibody (Cell Signaling Technology, Beverly, MA, USA)^{51 (link)}. β-subunit of ATP synthase (ATPβ) antibody was used as an experimental loading control^{52 (link)}. Signals were detected by using exposure in ChemiDoc system (Bio-Rad) with enhanced chemiluminescence substrate (Bio-Rad, USA).