Almond β glycosidase

Oleuropein was purchased from Extrasynthese and deglycosilated by treatment with almond β-glycosidase (EC 3.2.1.21, Fluka, Sigma-Aldrich, St. Louis, MI, USA), as previously described [45 (link)]. Briefly, a 10 mM solution of Oleuropein in 310 μL of 0.1 M sodium phosphate buffer, pH 7.0, was incubated with 8.9 I.U. of β-glycosidase overnight at room temperature. The reaction mixture was centrifuged at 18,000 rpm for 10 min to precipitate OleA, which was resuspended in dimethylsulfoxide (DMSO) in stocks at 50 mM concentration. The complete Oleuropein deglycosylation was confirmed by assaying the glucose released in the supernatant with a Glucose (HK) Assay kit (Sigma-Aldrich, St. Louis, MI, USA). Stocks of OleA were kept frozen protected from light and were used within the same day once opened.

N. benthamiana leaves were collected 6 days after infiltration
and immediately frozen in liquid nitrogen. Samples were lyophilized
(CHRIST Alpha 1-4 LD Plus, Martin Christ, Osterode am Harz, Germany)
and powdered with a ball mill grinder (MM301, RETSCH, Haan, Germany)
for 2 × 1 min at 29 Hz. Lyophilized samples were weighed with
a precision scale, and 100 mg of each was suspended in 5 mL of 80%
(v/v) aqueous methanol and sonicated for 20 min, followed by centrifugation
for 15 min at 3000 rpm. Two methanol extractions were performed for
each sample, and supernatants were combined into 50 mL Falcon tubes
and evaporated to dryness (SpeedVac SPD 300DDA, Thermo Scientific,
Bellefonte, USA).
Crude plant extract samples were mixed with
500 μL of 10 mM (MES)-KOH (2-(N-morpholino)
ethanesulfonic acid) and 150 μL of 5 mg/mL almond β-glycosidase
(Sigma-Aldrich, St. Louis, USA) and incubated at 37 °C for 15
h with gentle shaking. Samples were then transferred into glass KIMAX
tubes, and raspberry ketone was extracted with 4 mL of methyl tert-butyl ether (MTBE), transferred into 2 mL Agilent vials,
and evaporated to dryness under nitrogen flow.

Oleuropein was from Extrasynthese (Lyon, France). DMEM (high glucose + GlutaMAX), HAM F-12, fetal bovine serum (FBS), and horse serum were purchased from Gibco, Life Technologies. Medium 199, Earle's salts, pancreatin, gelatin solution, phenol red (solution), Percoll, chloroquine (CQ), and almond β-glycosidase were from Sigma-Aldrich. Collagenase A (Clostridium hystolyticum) was from Roche. Ad-MAO-A adenovirus was made as previously described [4 (link)]. RFP-GFP-LC3 plasmid was from Addgene (Cambridge, USA).