**2.4. Enzymatic deglycosylation of patatin**

Patatin deglycosylation was performed with the enzyme protein-N-glycosidase F (PNGase F, New England Biolabs, Ipswich, MA, USA) according to the manufacturer specifications. A 75 μg sample of total protein extract from mature tuber was incubated with PNGase F (25 U/mL) and diluted in reaction buffer (New England Biolabs) until a final volume of 20 μL. The mixture was incubated for 12 h at 37°C. Patterns of deglycosylated patatin isoforms on 2-DE gels were obtained as described earlier.

#### **2.5. Pro-Q staining for phosphoproteins**

Pro-Q Diamond phosphoprotein stain (Pro-Q DPS, Molecular Probes, Leiden, The Netherlands) was used for the in-gel detection of phosphorylated patatin polypeptides, as described previously [31]. Briefly, gels were fixed with 50% methanol and 10% acetic acid for 60 min and washed twice for 15 min each with distilled water. The gels were subsequently incubated for 120 min with two-fold water-diluted Pro-Q DPS, destained four times (30 min per wash) with 50 mM sodium acetate and 20% ACN pH 4.0, and washed again twice with distilled water (5 min per wash). The PeppermintStick™ (Molecular Probes) phosphoprotein marker was added to tuber protein extracts before 2-DE. Phosphorylated (ovalbumin, 45.0 kDa; and β-casein, 23.6 kDa) and unphosphorylated (β-galactosidase, 116.25 kDa; bovine serum albumin, 66.2 kDa; avidin, 18.0 kDa; and lysozyme, 14.4) PeppermintStick proteins were used as positive and negative controls of phosphorylation, respectively.

#### **2.6. Chemical dephosphorylation of patatin**

The chemical dephosphorylation of patatin was performed with hydrogen fluoride-pyridine (HF-P) as previously described [28, 29], with some modification [10]. An amount of 1 mg of total protein extract from tuber of cv. Kennebec was dissolved in 250 μL of 70% HF-P and incubated on ice for 2 h. The mixture was neutralized by addition of 10 M sodium hydroxide solution. Proteins were desalinated using Amicon Ultra-4 centrifugal filter devices (Millipore, Billerica, MA, USA) and then eluted in 300 μL of lysis buffer. Prior to 2-DE, protein purification was performed using the Clean-up kit (GE Healthcare).
