Skim milk powder

Two hundred micrograms of total protein extracted from Jatropha buds and 5x Laemmli buffer (50 mM Tris, 1% SDS, 0.05% bromophenol blue and 10% glycerol [pH 6.8]) were mixed together and incubated at 100 °C for 5 min. The samples were then resolved on 5% spacer and 15% separation discontinuous polyacrylamide SDS gels. Electrophoresis was subsequently conducted at 60 V for 30 min and, followed by 120 V for 60 min. The proteins were transferred onto NC membranes (No. F619511, BBI, Shanghai, China) with a wet electroblotting device (Bio-Rad, Hercules, CA, USA). The transfer conditions included a constant 55 V for 2 h. After transfer of the proteins, the membrane was saturated with 5% skim milk powder (Merck, Darmstadt, Germany) in Tris-buffered saline containing Tween-20 (TBST; 10 mM Tris, 150 mM NaCl, 0.2% Tween-20 [pH 7.5]). Incubation with the primary antibody (polyclonal rabbit anti-FT/Actin antibody, PytoAB, San Jose, CA 95131, USA) was performed overnight at 4 °C; after washing three times in TBST, incubation with the secondary antibody (goat anti-rabbit IgG H&L [HRP]; Dianova, Hamburg, Germany) in TBST was performed at room temperature for 60 min. Bound antibodies were detected with an Immobilon Western Chemiluminescent HRP Detection Kit (Millipore Corporation, Billerica, MA, USA) in conjunction with a Gel Doc™ XR+ system (Bio-Rad).

All chemical materials used in this study were analytical grade unless stated otherwise. 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPC), cholesterol, tween-20, dimethyldioctadecylammonium bromide (DDAB), complete freund’s adjuvant (CFA), incomplete freund’s adjuvant (IFA), and o-phenylenediamine dihydrochloride (OPD) tablets were purchased from Sigma-Aldrich. p-methylbenzhydrylamine (MBHA)^. HCl resin was purchased from Novabiochem. N, N’-dimethylformamide (DMF), trifluoroacetic (TFA) acid, acetonitrile, protected (tert-butyloxycarbonyl) Boc-amino acids and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate (HATU) were purchased from Mimotopes. Chloroform, methanol, skim milk powder, and other solvents were purchased obtained from Merck. Goat antimouse IgG (H+L)-HRP conjugate secondary antibody was purchased from Bio-Rad, Australia).
Analytical reverse-phase high performance liquid chromatography (RP-HPLC) and preparative RP-HPLC were performed on a Shimadzu instrument using Vydac analytical (C4 and C18) and preparative (C4 and C18) columns. HPLC solvent A: 0.1% TFA in water and solvent B: 0.1% TFA in acetonitrile: water (90:10); flow rate: 1 mL/min for analytical RP-HPLC and 20 mL/min for preparative RP-HPLC; detection: UV absorbance at 214 nm.

20-mer, N-acetylated peptides on cellulose membranes were ordered from JPT (PepSpots). The membranes were activated with 5 ml methanol for 5 min at room temperature and washed with 10 ml TBST (50 mM Tris, 137 mM NaCl, 2.7 mM KCl, pH adjusted to 8.0 with HCl, 0.05% Tween-20) three times for 3 min at room temperature. The membranes were then incubated with 10 ml blocking buffer (5% skim milk powder (Merck Millipore, 115363) in TBST) for 2 h at room temperature while rotated. The blocked membranes were incubated with concentrated GST-HA-tagged proteins of interest in blocking buffer overnight, at 4 °C, while rotated. After three quick rinses with ice-cold TBST, the membranes were incubated with HRP-conjugated anti-GST antibody (Cytiva, RPN1236; 1:3000 dilution) in blocking buffer for 1 h at 4 °C, while rotated. Following three quick washes with ice-cold TBST, the chemiluminescent readout was carried out using ECL reagent (Clarity Max Western ECL substrate, 1705062, Bio-Rad) and ChemiDoc Imaging system (Bio-Rad). The acquired raw tiff images were analyzed using Image Studio Lite Ver. 5.2., and all values were normalized to the wild-type results and the mean ± SD were calculated.

Cortical neuron cultures from WT and Doc2a, b DKO mice were plated to 200 K cells per well, infected at DIV 1 with lentiviral Doc2b constructs (LIP #1984, #1985, #1986 as above) and harvested at DIV 17. Cells were washed 2 times with PBS, lysed in Laemmli sample buffer, loaded with 50% or the totality of each well for WT and DKO cells respectively, separated by SDS-PAGE and blotted on PVDF membrane (Biorad). Membranes were blocked in 2% skim milk powder (Merck) and 0.5% FCS (Gibco) in PBS with 0.01% Tween-20 (Sigma-Aldrich). Doc2B polyclonal antibody 13.2 was used as primary antibody (1:500) for incubation overnight at 4 °C. Goat anti rabbit alkaline phosphatase (Jackson lab) was used as secondary and Attophos (Promega) as substrate for 30 min incubation at RT. Reprobing was made for actin immunostaining with monoclonal anti-actin antibody C4 (1:3000; Chemicon) and Goat anti mouse alkaline phosphatase as secondary Ab (1:10000).

RPMI-1640 medium was purchased from Biosera, England, fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), penicillin – streptomycin, trypsin, trypsin – EDTA were purchased from PAA Laboratories, Austria. 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT), Bovine serum albumin (BSA), trypan blue dye, ammonium sulfate, Dimethyl sulfoxide (DMSO) were purchased from Sigma Aldrich, USA. β- Mercaptoethanol, sodium dodecyl sulfate, tris (hydroxymethyl) aminomethane, glycine, hydrochloric acid, sodium carbonate, Skim milk powder, Muller Hinton broth, peptone, agar, agarose, and acrylamide were purchased from Merck, Germany.

Tris-acetate-ethylene diamine tetra acetic acid (Tris-acetate-EDTA) was purchased from Thermo Fisher Scientific, USA. Pepsin, pancreatin, [sodium taurocholate, sodium cholate and sodium deoxycholate (sodium salt TDCA)], ethidium bromide, ox-bile salt and antibiotics (ampicillin, cefoxitin, doxycycline, streptomycin, neomycin, cephalexin, gentamycin, norfloxacin, ciprofloxacin and lincomycin) were obtained from Oxiod Ltd. (Altrincham, Cheshire, England WA14 2DT). MRS medium was obtained from BIOKAR Diagnostics, France. Agarose, agar, glycerol, di-potassium phosphate, sodium bicarbonate, hydrochloride acid, sodium hydroxide, hydrogen peroxide, potassium phosphate, toluene, sodium chloride, calcium chloride and potassium chloride were obtained from El Nasr Pharmaceutical Chemicals Company, Cairo, Egypt. Skim milk powder (Merck, Germany) was obtained from a local market. Defibrinated sheep blood was obtained from Thermo Scientific™ Oxoid.

A commercial product, freeze-dried LGG from CHR Hansen (Hørsholm, Denmark), was procured. Maltodextrin (GLUCIDEX^® IT 19) was provided by Roquette (Lestrem, France) and Dextrose Equivalent value of 19 was used. Instantgum BB^TM (arabic gum) purified from instant acacia gum was purchased from Nexira (Rouen, France). Skim milk powder was provided by Merck (St. Quentin Fallavier, France). Trypton salt broth, MRS (de Man, Rogosa and Sharpe) broth, and agar were purchased from Biokar (Beauvais, France).