P c src

Cell lysates were separated by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE). Primary antibodies used were: PRLR (D01PID5618, Abnova, Taiwan); Moesin (SC6410, Santa Cruz, Texas, USA); p-Moesin (SC12895, Santa Cruz, Texas, USA); FAK (SC271195, Santa Cruz, Texas, USA); p-FAK (SC11765-R, Santa Cruz, Texas, USA); c-Src (SC5266, Santa Cruz, Texas, USA); p-c-Src (SC166860, Santa Cruz, Texas, USA); GAPDH (SC59540, Santa Cruz, Texas, USA). The secondary antibodies Anti-rabbit (SC2357, Santa Cruz, Texas, USA) (1:3000) for PRLR; Anti-goat (SC2768, Santa Cruz, Texas, USA) (1:3000) for Moesin, p-Moesin and Actin, and Anti-Mouse (SC2005, Santa Cruz, Texas, USA (1:3500) for FAK, p-FAK, c-Src, p-c-Src, and GAPDH were incubated for 2 h in room temperature. Primary and secondary antibodies were incubated with the membranes, followed by three 5-min washings with TRIS-buffered saline-Tween 20. Immunodetection was carried out using enhanced chemiluminescence and was recorded with a quantitative digital imaging system (Quantity One, BioRad, Hercules, CA, USA), enabling us to assess saturation. Band densitometric analysis was quantified, as previously described (17 (link)), with the ImageJ image program (National Institute of Health – NIH, USA) using conditions ensuring analysis in the linear range of detection.

Fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT, USA). The following antibodies were purchased: Rac1 antibody (BD Biosciences, Franklin Lakes, NJ, USA); c-Jun N-terminal kinase (JNK), p-JNK, p-p38, p38, p-ERK, ERK, p-PKC, PKC, p-c-Src, c-Src, p-NF-κBp65, NF-κBp65, p-c-Jun, c-Jun, Bax, p-Bcl-2, Bcl-2, caveolin-1, cleaved caspase-3, caspase-9, and β-actin antibodies (Santa Cruz Biotechnology, Paso Robles, CA, USA); Bax (6A7) monoclonal antibody (Thermo Fisher Scientific, Rockford, IL, USA); LC-3, NCF-1 and Beclin-1 antibodies (Novus Biologicals, Littleton, CO, USA). VvhA-specific antibody was acquired from Professor Sang Ho Choi (Seoul National University, Seoul, Korea). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse IgG antibodies (Jackson Immunoresearch, West Grove, PA, USA). SP600125 was purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of the highest purity commercially available and were used as received.

Anti-mouse and anti-rabbit IgG-conjugated horseradish peroxidase, rabbit polyclonal antibodies specific for p-FAK, FAK, p-c-Src, c-Src, p-EGFR, EGFR, p-ERK, ERK, HIF1-α, β-actin, CD31, and WISP-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); VEGF-A antibody was purchased from Abcam (Cambridge, MA, USA). Recombinant human VEGF-A was purchased from R&D Systems (Minneapolis, MN, USA). Dulbecco's modified Eagle's medium (DMEM), F-12 medium, fetal bovine serum (FBS) and all other cell culture reagents were from Gibco-BRL Life Technologies (Grand Island, NY, USA). ON-TARGETplus siRNAs were purchased from Dharmacon Research (Lafayette, CO, USA). All other chemicals were from Sigma-Aldrich (St Louis, MO, USA). The efficacy of all inhibitors were provided in supplementary information (Figure S6). The methods of Transwell migration assay, Western blot analysis, Quantitative real-time PCR, Enzyme-linked immunosorbent assay, Tube formation, Establishment of the WISP-1 knockdown SCC4 cell line and Chick chorioallantoic membrane assay are provided in Supplementary Information.