Figure 2.

Sub-organellar localization of STN8 and membrane association of the STN kinases. (A) Arabidopsis WT chloroplasts were fractionated into soluble and membrane components and subsequently separated by SDS-PAGE. After Western transfer, STN8, RbcL (stroma marker) and Lhcb2 (thylakoid marker) were detected using specific antibodies. (B) Extraction of membrane-associated proteins with alkaline buffers or chaotropic salt solutions. Thylakoid membranes from WT plants were resuspended at 0.5 mg chlorophyll/mL in HS buffer containing either 2 M NaCl, 0.1 M NaBr, 2 M NaSCN, 0.1 M Na₂CO₃, 0.1 M NaOH, or no additive. After incubation on ice, samples were fractionated into soluble (S) and membrane-associated proteins (P) and immunolabeled with antibodies specific for STN7, STN8, AtpB (a representative peripheral membrane protein), PetC (a membrane protein with a single hydrophobic domain predominantly anchored by electrostatic interactions), or Lhcb2 (an integral membrane protein with three transmembrane helices).