Hybond c membrane

DMEM (Dulbecco’s modified Eagle’s medium)/F-12 medium, FBS (fetal bovine serum), and TRIzol were from Invitrogen (Carlsbad, CA, USA). The Hybond C membrane and ECL (enhanced chemiluminescence) Western blot detection system were from GE Healthcare Biosciences (Buckinghamshire, UK). The phospho-c-Src (Tyr416) (Cat. #6943), phospho-EGFR (Tyr845) (Cat. #2231), phospho-Akt (Ser473) (Cat. #4060), phospho-ERK1/2 (Thr202/Tyr204) (Cat. #4370), phospho-p38 (Thr180/Tyr182) (Cat. #4511), phospho-JNK1/2 (Thr183/Tyr185)(Cat. #4668), and phospho-p65 NF-κB (Ser536) (Cat. #3033) antibodies were from Cell Signaling (Danver, MA, USA). Anti-ZO-1 (Cat. #sc-33725) antibody was from Santa Cruz (Dallas, TX, USA). Anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) antibody was from GeneTex (Irvine, CA, USA). MMP2/9 inhibitor (2/9i), apocynin, PP1, AG1478, SH-5, U0126, SB202190, SP600125, Bay11-7082, and were from Enzo (Farmingdale, NY, USA). BCA (Bicinchoninic acid) protein assay reagent was from Pierce (Rockford, IL, USA). IL-1β (interleuline-1β) was from R&D Systems (Minneapolis, MN, USA). NAC (N-acetyl-cysteine), enzymes, and other chemicals were from Sigma (St. Louis, MO, USA).

DMEM/F-12 medium, fetal bovine serum (FBS), 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA), DAPI, and Trizol were purchased from Invitrogen (Carlsbad, CA, USA). Antibodies against cPLA2α, COX-2, phospho-c-Jun, GAPDH, and OB-R were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phospho-Plus p47^phox was acquired from Assay Biotechnology (Sunnyvale, CA, USA). N-acetylcysteine (NAC), apocynin (APO), and tanshinone IIA were purchased from Biomol (Plymouth Meeting, PA, USA). Leptin was acquired from BioVision (Milpitas, CA, USA). Hybond C membrane and Hyperfilms were acquired from GE Healthcare Biosciences (Buckinghamshire, UK). An enhanced chemiluminescence (ECL) Western blotting detection system was purchased from Visual Protein Biotechnology Co (Taipei, Taiwan). Enzymes and other chemicals were purchased from Sigma (St. Louis, MO, USA).

The mycelial proteome of R. solani was resolved in a 2-DE gel and electrophoretically transferred onto a Hybond-C membrane (GE Healthcare) with a blotting buffer (39 mM glycine, 48 mM Tris base, 20 % methanol, and 0.037 % SDS) using a semidry blotting apparatus (TE77; Amersham Pharmacia Biotech). The electrotransfer was run for 60 min at a current of 56 mA, 25 V. The membrane was temporarily stained with Ponceau S (Sigma-Aldrich, USA) to ensure the protein transfer from gel to Hybond-C membrane. The membrane was incubated for 15 min in Ponceau S staining solution with gentle agitation. Finally the membrane was rinsed in distilled water for two washes of 5 min each until the background is clean. Then the membrane was blocked overnight in 10 ml blocking buffer [5 % nonfat milk (Merck, Germany) in 1 × TBST]. Next day, the membrane was washed with three changes of TBST for 2 min each time and further incubated with mASAL (20 μg) for 2 h at 37 °C. Finally, the blot was incubated using a primary anti-mASAL polyclonal antibody (1:8000) and an anti-rabbit IgG HRP-conjugated secondary antibody (1:20,000, Sigma-Aldrich, USA). Membranes incubated without mASAL served as negative controls (data not shown).

Cells were harvested and washed once with phosphate buffered saline (PBS) then resuspended in DISC lysis buffer [37 (link)] supplemented with 1X complete protease inhibitor cocktail (Roche), 10 mM sodium fluoride, 2 mM sodium pyrophosphate, 1 mM sodium molybdate and 5 mM ß-glycerophosphate and incubated for 30 min on ice and centrifuged at 16 000g for 10 min at 4°C. Clarified lysates (50 μg) were mixed with sample buffer, boiled and separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) on Tris-glycine gels then transferred to Hybond C membrane (GE, Castle Hill, NSW). Membranes were blocked in 5% skim milk powder in Tris-buffered saline containing 0.1% Tween-20 (TBST) and incubated with primary antibody. Membranes were washed with TBST, incubated with horseradish peroxidase-conjugated secondary antibody (GE) and washed with TBST before detection with enhanced chemiluminescence (Millipore).

The assay was performed as described previously [33 (link)]. Briefly, 500 pmol of each lipid [Ptdins, PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(3,4)P₂, PtdIns(4,5)P₂, Ptdins(3,5)P₂ and PtdIns(3,4,5)P₃] were spotted on to Hybond-C membrane (GE Healthcare) and let dry for 1 h. The membrane was blocked in blocking buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.1% Tween-20 and 2 mg/ml fatty acid-free BSA) for 1 h. The membrane was incubated overnight at 4°C in a 10 nM solution of each recombinant protein. After washing in TBST buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20), the signal was detected by using HRP-conjugated anti-FLAG or HRP-conjugated anti-GST antibodies (Roche) at a 1:3000 dilution. All lipids were purchased from Echelon and dissolved in 1:1 solution of methanol and chloroform.

The tuber proteins were extracted by homogenization in extraction buffer [50 mM Tris-HCl (pH 8.2), 2 mM EDTA, 20% glycerol, 5 mM DTT and 2 mM PMSF]. The homogenates were centrifuged at 10,000 × g for 10 min at 4 °C, and the protein concentration in the supernatant was determined using Bradford protein assay kit (Bio-Rad). An aliquot of 50 μg protein from each sample was fractionated on 12.5% (w/v) SDS-PAGE and electrotransferred onto Hybond-C membrane (GE Biosciences). The AmA1 was detected with anti-AmA1 antibody29 (link) in combination with alkaline phosphatase conjugated goat anti-rabbit IgG using NBT-BCIP and HRP reaction. The protein quantification was performed with a Fluor-S MultiImager (Bio-Rad) and the band intensity corresponding to AmA1 was quantified using Quantity One software (Bio-Rad).

Foetal bovine serum (FBS), DMEM medium, TRIZOL and 5‐(and‐6)‐chloromethyl‐2′,7′‐dichlorodihydrofluorescein diacetate, acetyl ester (CM‐H2DCFDA) were purchased from Invitrogen (Carlsbad, CA, USA). Antibodies against COX‐2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). PhosphoPlus p47^phox were from Assay Biotechnology. PhosphoPlus p42/p44 MAPK antibody and GAPDH were obtained from New England Biolabs (Beverly, MA, USA). N‐acetylcysteine (NAC), diphenylene iodonium chloride (DPI), apocynin (APO), U0126, NS398, SC51089, AH6809, L798, 106 and GW627368X were obtained from Biomol. Hybond C membrane, Hyperfilms and enhanced chemiluminescence (ECL) Western blotting detection system were obtained from GE Healthcare Biosciences. XTT assay kit was purchased from Biological Industries. SDS‐PAGE supplies were from MDBio Inc. LPS, Oil red O, enzymes and other chemicals were obtained from Sigma.