Chaps

After protein labelling, samples were prepared for the isoelectric focusing (IEF) step. Strip gels of 24 cm and a linear pH range of 4–7 (GE Healthcare) were used. Strips were initially rehydrated with labelled protein samples (7 M urea, 2 M thiourea, 2% CHAPS (w/v), 0.5% IPG buffer (v/v) (GE Healthcare), 2% DTT). Strips were then processed using an Ettan IPGPhor IEF system (GE Healthcare) in a total of 35,000 Volts.h^-1 and, subsequently, reduced and alkylated for 30 min under slow agitation in Tris-HCl solution (75 mM), pH 8.8, containing 2% SDS (w/v), 29.3% glycerol (v/v), 6 M urea, 1% DTT (w/v) and 2.5% iodocetamide (w/v). The 2D- DIGE conditions were performed as described by Weiss and Görg [32 ]. Gels were immediately scanned with a FLA-9000 modular image scanner (Fujifilm Lifescience, Dusseldorf, Germany). To ensure maximum pixel intensity between 60,000 and 90,000 pixels for the three dyes, all gels were scanned at a 100 μm resolution and the photomultiplier tube (PMT) voltage was set between 500 and 700 V. As described by Agapito-Tenfen et al. [2 (link)], we performed preparative gels for each plant variety in order to extract relevant spots. These were performed with a 450 μg load of total protein pools in 24 cm gels from each variety, separately, and stained with colloidal Coomassie Brilliant Blue G-250 (MS/MS compatible).

Human MP was kept in tissue culture medium for 24 h with and without 5 μg/ml bacterial lipopolysachharide (Sigma). After this stimulation, the plexus pieces were collected, frozen in liquid nitrogen and then homogenized (FastPrep24, MP Biomedicals) in lysis buffer containing 7 M urea, 2 M thiourea, 4% CHAPS (Invitrogen, Germany), protease-inhibitor cocktail (Roche, Germany) and a nuclease-mix (GE-Healthcare, Germany). The proteins were purified using the 2-D clean-up kit (GE-Healthcare, Germany). Afterward, the protein pellets were re-equilibrated in buffer with 7 M urea, 2 M thiourea and 4% CHAPS and the amount of purified protein was analyzed (2-D quant-kit, GE-Healthcare, Germany).
Equal amounts of the two samples were labelled with different fluorescent dyes: Cy3 and Cy5. A mixture of both proteins was used as an internal standard and labelled with Cy2.
All three samples were then mixed, and a total amount of 55 μg of protein was applied to an IPG-Strip (Immobilized pH-Gradient) with an 18 cm, nonlinear gradient from pH 3–11 (GE) and focused for 8 h on the IPGphor3 (GE). The strips were transferred to a 12.5% polyacrylamide-gel and run on an EttanDalt12-separation unit overnight. After separation, the gels were scanned on a TyphoonTrio laser scanner (GE) and the spots were analyzed using the DeCyder (GE) software.

GD201008-001 total cell protein precipitations were performed as described previously [24 (link)]. Briefly, 30 mL exponential growth cultures were centrifuged at 10, 000 × g for 15 min at 4°C and washed twice in PBS. Pellets were resuspended in Mutanolysin working buffer (30 mM Tris–HCl, 3 mM MgCl₂, 25% sucrose) containing 125U/mL Mutanolysin (Sigma) and incubated for 90 minutes at 37°C. The components of solution B (7 M urea, 2 M thiourea, 4% CHAPS, and 65 mM DTT; GE Healthcare) were added directly into the mixed solution. The turbid solution gradually became transparent and was then sonicated in an ice bath for 50 cycles of 5 s on/10 s off at 100 W. After 30 min incubation at 25°C, unbroken cells was removed by centrifugation at 10,000 × g for 15 min at 4°C. Proteins in the supernatant were precipitated in 10% pre-chilled trichloroacetic acid (TCA) and incubated in ice-water for 30 min. After centrifugation at 10,000 × g for 10 min at 4°C, the pellet was resuspended in 10 mL of pre-chilled acetone and washed twice. The final pellet was dried in air.

Protein extraction was performed according to Xu et al.⁶¹ with minor modifications to six samples of each group. Briefly, frozen samples were minced and homogenized individually on ice in buffer containing 8 M urea, 65 mM DTT, 4% CHAPS and protease inhibitor (50 µl/g of sample, GE Healthcare, Little Chalfont, United Kingdom) and incubated on ice three hours under stirring. Then the samples were centrifuged at 4 °C for 30 min at 10,000 × g, the supernatant was carefully collected, and the proteins were precipitated in ice-cold acetone. The pellet was solubilized in buffer containing 8 M urea in 100 mM ammonium bicarbonate and stored at −80 °C until use.

After Morris water maze test, mice were anesthetized with 4% (w/w) chloral hydrate intraperitoneal injection (i.p.), and then were decapitated. Cortex and hippocampus were immediately dissected via surgery on ice and frozen in liquid nitrogen. All the samples were stored at −80°C until analysis.
Samples for 2D‐DIGE of each mouse were sonicated (5 × 10 s with an interval of 15 s) on ice in lysis buffer containing 30 mM Tris, 2 M thiourea, 7 M urea, 4% (w/v) CHAPS, 0.5% (v/v) IPG buffer (pH 3‐10NL; GE Healthcare, USA), and 1% protease inhibitor. The suspension was then centrifuged at 15,000 rpm for 30 min at 4 °C. The supernatant was collected and stored at −80°C until further use. Protein quantification was accurately conducted with a 2‐D Quant Kit (GE Healthcare).
Samples for western blot were sonicated (5 × 10 s with an interval of 15 s) on ice in lysis buffer containing 10 mM Tris, 1 mM EDTA, 1% (v/v) Triton, 0.1% (w/v) SDS, 0.2% (w/v) sodium deoxycholate, 101 mM Na₄O₇P₂, 1 mM Na₃VO₄, and 1% protease inhibitor. The suspension was then centrifuged at 15,000 rpm for 30 min at 4°C. Protein quantification was performed with a BCA Quantification Kit (Applygen, China).

50 mg of frozen mouse tumor tissues were extracted using TissueLyser LT (QIAGEN, Hilden, Germany) with urea/thiourea lysis buffer (1:10 w/v). For cell lines, the cell pellets were extracted with urea/thiourea lysis buffer [7 M urea, 2 M thiourea, 4 % (w/v) CHAPS, 30 mM Tris/HCl and protease inhibitor, pH 9.0, (GE healthcare)]. The supernatants were processed with 2-D Clean Up kit and re-suspended in the urea/thiourea lysis buffer for 2D-DIGE or in Dissolution buffer containing 5 % SDS provided in iTRAQ Reagent 4-Plex kit (AB SCIEX) for iTRAQ experiment as previously described [26 (link), 27 (link)]. MS/MS data was processed using Bruker Compass Data Analysis software, and the generated peaklists were submitted to MASCOT search engine against SwissProt 51.6 database. Detail methodologies were described in supplementary information.

Peptides from the N-terminus of Dop (ISTSTPQKNDEHQEQC and MSRQEGAASRPADGAC) were synthesised and used to immunise rabbits and to affinity purify antibodies (anti-Dop 1303) (Eurogentec). Protein extracts from staged embryos were prepared in lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.1% β-mercaptoethanol) and run on 3-8% Tris acetate SDS-PAGE (Invitrogen) and transferred to PVDF membrane (Whatman, GE Healthcare) for immunoblotting. For 2D PAGE, protein extracts from visually staged embryos were acetone precipitated and redissolved in rehydration buffer [7 M urea, 2 M thiourea, 1.2% CHAPS, 0.25% ampholytes (GE Healthcare), 0.4% ASB-14, 43 mM DTT]. Protein concentration was established using 2-D Quant (GE Healthcare). Equal amounts of protein were separated on pH 4-7 Immobiline DryStrip gels (Amersham) with the IPGphor isoelectric focusing system (Amersham). The second dimension was performed on 4-12% bis-Tris Zoom gels (Invitrogen). Signal intensity on immunoblots was measured using ImageJ.
For dephosphorylation assays, 100 μg protein lysate was incubated with 5 µl λ-protein phosphatase (New England BioLabs) at 30°C for 30 min. As control, phosphatase was heat inactivated for 1 hour at 65°C in the presence of 50 mM EDTA.