Ecl western blot detection system

Total protein was extracted from the first systemically infected leaf tissue using an extraction buffer consisting of 220 mM Tris-HCl, 250 mM sucrose, 1 mM MgCl₂, 50 mM KCl, and 10 mM β-mercaptoethanol. After incubation for 10 min, the extract was centrifuged twice for 10 min at 10,000 × g at 4℃ to pellet the protein. The proteins detected were visualized using the ECL Western Blot Detection System (Bio-Rad, Shanghai, China).

Cells in experiments (adenoviruses and inhibitors) were collected and lysed for total protein, according to the methods of Zhang et al [19 (link)]. Western blots were conducted according to the methods of Xu et al. and Gou et al. [22 (link),23 (link)]. In brief, 30 ug of total protein samples from three biological replicates was separated by electrophoresis on 7.5–10% sodium dodecyl sulfatepolyacrylamide gels and then transferred onto nitrocellulose paper and incubated with antibodies against β-actin (CW0096, CW Biotech; 1:1000), EGFR (AF1330, Beyotime; 1:1000), pY1068-EGFR (ab40815, Abcam; 1:1000), pS473-Akt (4060, CST; 1:1000), pT202/Y204-ERK1/2 (4370, CST; 1:1000) and pS1248-PLC-γ1 (4510, CST; 1:1000). Eventually primary antibodies were detected with HRP-conjugated secondary antibodies, followed by light detection with chemiluminescent ECL Western blot detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The densities of bands were analyzed by ImageJ (http://imagej.nih.gov/ij/), and relative protein abundance was normalized to β-actin.

Cells were lysed in RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific) containing 1% protease and phosphatase inhibitors (Cell Signaling Technologies). Protein concentrations were assessed through BCA assays (Thermo Fisher) according to the manufacturer’s instructions. Cell lysates were electrophoresed on SDS-PAGE gels and then electroblotted on polyvinylidene difluoride (PVDF) membranes using the Trans-Blot Turbo Transfer System (Biorad). Nonspecific binding was blocked with 5% non-fat milk or 5% Bovine Serum Albumin (BSA) in TBS-Tween-20 (TBST; Sigma) for 1 hour. Primary antibodies for VE-Cadherin (sc-9989), PTP1B (sc-133259), Claudin-5 (sc-374221), PP1α (sc-271762), Ets-1(sc-55581), TGFβ RII (sc-17792), Rac-2 (sc-517424) and α-actinin-4 (sc-393495) (all Santa Cruz Biotechnology) were diluted in TBST with 5% non-fat milk, Phospho-VE-Cadherin antibody (Tyr658, Cat. No.44-1144G; Thermo Fisher) was diluted in TBST with 5% BSA. β-Actin (#4970; Cell Signaling Technologies) served as the loading control. Immunoreactive bands were visualized by using peroxidase-conjugated secondary antibodies (Anti-rabbit IgG, #7074; Anti-mouse IgG, #7076; Cell Signaling Technologies) and the ECL western blot detection system (BioRad).