Phospholipid standards

Unless otherwise stated, all reagents were of analytical grade and purchased from Sigma‐Aldrich (Buchs, Switzerland) or Merck (Darmstadt, Germany). Restriction enzymes were obtained from Fermentas (St. Leon‐Rot, Germany) and antibiotics from Sigma‐Aldrich, Invivogen (Nunningen, Switzerland) or Invitrogen (Basel, Switzerland). [³H]Serine (1 mCi ml⁻¹, 20 Ci mmol⁻¹) was purchased from American Radiolabeled Chemicals (St. Louis, USA) and [γ‐³²P]‐dCTP (3000 Ci mmol⁻¹) from PerkinElmer (Waltham, USA). Phospholipid standards were purchased from Sigma‐Aldrich or Avanti Polar Lipids Inc. (Alabaster, AL).

The qualitative and quantitative determination of individual phospholipid fractions—phosphatidylcholine (PC), phosphatidylethanolamine (PEA), phosphatidylserine (PS), phosphatidylinositol (PI), and lysophosphatidylcholine (LPC)—was performed by high-performance liquid chromatography (HPLC) and previously described in [11 (link)]. Phospholipid standards (Sigma-Aldrich, USA) were used for identifying and quantifying the phospholipid compounds in the sample. We identified six phospholipids (as total certain fractions without focusing on molecular species): PS, PE, PI, PC, LPC, and SM.

Palmitic acid (PA, sodium salt) and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA, sodium salt) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Palmitic acid was dissolved in 50% ethanol, while docosahexaenoic acid was dissolved in 100% ethanol to a concentration of 10 mg/mL; these stock solutions were stored at −80 °C under N₂ until use. The rabbit monoclonal anti-Calnexin (C5C9) and the rabbit monoclonal anti-Histone H3 (D1H2) antibodies were purchased from Cell Signaling Technology, Danvers, MA, USA. The mouse monoclonal anti-HSP60 (66041) antibody was purchased from Proteintech, Deansgate, Manchester, UK. Bound primary antibody was visualized by proper secondary horseradish peroxidase (HRP)-linked antibody, purchased from Cell Signaling Technology, Danvers, MA, USA. All solvents were purchased from Carlo Erba Reagents, Italy. Avanti Polar Lipids Inc. (Alabaster, AL, USA) supplied the fatty acid methyl ester (FAME) standards for GC analysis and neutral lipids (TG and CE) for LC analysis. Phospholipid standards were purchased from Sigma Aldrich, St. Louis, MO, USA.