Minilys

Total genomic DNA was extracted from herbarium samples according to Krinitsina et al. (2015) (link). Following the homogenisation of plant fragments (MiniLys, Bertin Technologies, France), total DNA was extracted using the CTAB-method (Doyle and Doyle 1987 ) and further purified using AMPure Beads (Beckman Coulter, USA).
PCRs for two chloroplast markers (atpB-rbcL and trnL-trnF) and nrDNA (ITS region) were carried out in a Thermal Cycler T100 (Bio-Rad, USA) using primers and cycler programmes listed in Table 2. A 10 ng aliquot of DNA was used to make a 25 μl total volume reaction, containing 1 μM of each primer, 200 μM of each dNTP and 0.5 U Encyclo polymerases (Evrogen, Russia). PCR products were checked on 1.2% agarose gels and purified using AMPure Beads (Beckman Coulter, USA) according to the owner’s manual. AMPure Beads suspension was mixed with a solution containing PCR-product ratio 1 vol. PCR-mix: 1.2 vol. AMPure Beads for atpB-rbcL and ITS primer pairs and 1 vol. PCR-mix: 1.4 vol. AMPure Beads for rbcL, Tab C/Tab D and Tab E/Tab F primer pairs.

Following the electrophysiological recordings, slices were harvested, snap frozen and stored at -80° until needed. Each slice was lysed in 100 µl Ripa Buffer (50 mM Tris–HCL pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP40, 0.1% SDS) supplemented with protease inhibitors (Calbiochem set III) and phosphatase inhibitors (1 mM Na₂MO₄,1 mM NaF, 20 mM β-glycerophosphate, 1 mM Na₃V0₄, 500 nM Cantharidin) using a tissue homogenizer (Minilys, Bertin instruments). Homogenates were centrifuged at 20,000 G and supernatant was collected for further protein quantification analysis using BCA Thermo Scientific Pierce Protein Assay. 30 µg of protein was loaded on an SDS-PAGE gel and western blot analysis was performed as previously described^{49 (link)}. Anti-Drebrin (M2F6, Enzo) was used at a concentration of 1:1000 and anti-α-Tubulin (Sigma) was used in 1:8000. Quantification of band densities was performed using FIJI. The area of the band and the mean grey value were measured to obtain a relative density. For relative quantifications, measurements were normalized to tubulin loading control.

Hair was cut as close as possible to the scalp from the posterior vertex of the head from mothers and their children. The samples were kept at room temperature until they were analyzed. From each hair sample, 3 cm was used for the analysis. Hair was placed in tubes (Precellys Lysing Kits, Bertin Technologies, Paris, France), grinded and pulverized at 5000 rounds per minute via a homogenizer (Minilys, Bertin Technologies, Paris, France). Following this, 1 mL of methanol was added to each tube at room temperature and stirred for 16 h. The samples were centrifuged (Biofuge 13, Heraeus Instruments, Athens, Greece) and the extracts were transferred to a glass tube and left at room temperature in order to evaporate until they dried. Afterwards, they were reconstituted with phosphate buffered saline (100 μL) (pH 8.0) and placed in a vortex for 1.5 min. Before analysis, the samples were placed in a vortex once more. Finally, to estimate cortisol levels, the automatic electrochemiluminescence immunoassay “Cortisol II” was used in the automatic analyzer Cobas e411-Roche Diagnostics (GmbH, Mannheim, Germany). The detection threshold, as indicated from the manufacturer’s instructions, was set at 0.054 μg/dL. Hair cortisol was then calculated and expressed in pg/mg of hair.