Measurement of PI4P Levels in Intact Chloroplasts Isolated from Arabidopsis thaliana

Materials and Reagents

1. Isolation of intact chloroplasts
   
   1. Miracloth (Merck Millipore Corporation, Calbiochem^®, catalog number: 475855 )
      
   2. Gauze (Hakujuji Co., model: FC-gauze )
      
   3. Seedlings of Arabidopsis thaliana
      Wild-type (Columbia-0) plants, phosphatidylinositol 4-kinase (PI4K) α1 knockdown plants, pi4kβ2-1 mutants and PI4Kα1 knockdown plants of pi4kβ2-1 mutants were grown for 4 days on MS agar plates and then transferred onto agar plates with or without 5 μM dexamethazone (DEX) and grown for 1 week. The down-regulation of PI4Kα1 expression was induced by DEX treatments in PI4Kα1 knockdown plants and PI4Kα1 knockdown plants of pi4kβ2-1 mutants. For treatments with inhibitors, wild-type 4-d-old seedlings were transferred onto agar plates with PI4K inhibitors, 200 μM wortmannin (WM) or 25 μM phenylarsine oxide (PAO), or a phosphatidylinositol 3-kinase (PI3K) inhibitor, 50 μM LY294002 (LY), or without inhibitors (DMSO) and grown for 3 days.
      
   4. Percoll (GE Healthcare, Dharmacon, catalog number: 7-0891-01 )
      
   5. Bradford assay kit (Bio-Rad Laboratories, catalog number: 500-0006JA )
      
   6. 1x protease inhibitor cocktail (Nakarai, catalog number: 03969-21)
      Note: Currently, it is “Nacalai tesque, catalog number: 03969-21 ”.
      
   7. Sorbitol
      
   8. HEPES-KOH (pH 7.5)
      
   9. EDTA
      
   10. Grinding buffer (see Recipes)
       
   11. 80% Percoll or 40% Percoll (see Recipes)
   
   
2. Extraction of PI4P
   
   1. 0.75 M Trichloroacetic acid (TCA) (Wako Pure Chemical Industries, Siyaku, catalog number: 203-04952 )
      
   2. 5% (w/v) TCA with 1 mM EDTA
      
   3. Methanol (Wako Pure Chemical Industries, Siyaku, catalog number: 137-01823 ): Chloroform (Wako Pure Chemical Industries, Siyaku, catalog number: 038-02601 ) (2:1, v/v)
      
   4. Methanol: chloroform: 12 N HCl (Wako Pure Chemical Industries, Siyaku, catalog number: 080-01066 ) (80:40:1, v/v/v)
   
   
3. Measurement of PI4P levels
   
   1. PI(4)P Mass Strip Kit (Echelon Biosciences, catalog number: K-4000E )
      
   2. Albumin from bovine serum (BSA), fatty acid free (Wako Pure Chemical Industries, Siyaku, catalog number: 017-15146 )
      
   3. HRP substrate solution (Thermo Fisher Scientific, Pierce, catalog number: NCI32132 )
      
   4. Phosphate (pH 7.4)
      
   5. NaCl
      
   6. 0.1% (w/v) Tween-20
      
   7. Phosphate buffered saline (PBS) (see Recipes)
      
   8. PBST (see Recipes)

Equipment

1. Homogenizer (Microtec Co., model: NS-51 )
   
2. Centrifuge (TOMY SEIKO CO., models: MX-200 and GX-250 )
   
3. Swing rotor (TS-7C)
   
4. Paintbrush
   
5. Pasteur pipette (Sansyo, Iwaki, catalog number: IK-PAS-9P )
   
6. Vacuum dryer (Centrifuge evaporator) (Shimadzu Corporation, model: SPE-200 )
   
7. Sonicator (SHARP CORPORATION, model: UT-106 )
   

Procedure