Anti ilk

Preparation of cell lysates from SCC4 cells in 6-well plates and protein concentration was determined for each cell lysate using the BCA Protein Assay Kit (Thermo Fisher Scientific; Waltham, MA, USA). An amount of 30–50 μg of protein was loaded into each well containing 10% SDS-PAGE gel then transferred to polyvinylidene difluoride (PVDF) membranes (EMD Millipore; Temecula, CA, USA) for Western blot analysis, according to our previous research [32 (link),37 (link)]. Membranes were probed with their respective antibodies; anti-ILK (Santa Cruz; Dallas, TX, USA), anti-GSK3β(Santa Cruz; Dallas, TX, USA) anti-FAK (Cell Signaling; Danvers, MA, USA), anti-Akt (Cell Signaling; Danvers, MA, USA), anti-Snail (Santa Cruz; Dallas, TX, USA), anti-Twist (Santa Cruz; Dallas, TX, USA), anti-E-cadherin (Abcam; Cambridge, MA, USA), and anti-α-tubulin (Santa Cruz; Dallas, TX, USA). Immunoblot images were acquired using the ImageQuant™ LAS 4000 biomolecular imager (GE Healthcare Life Sciences; Pittsburgh, PA, USA).

The following agents were obtained from commercial sources: vascular endothelial growth factor‐A 165 (Merck Millipore, Billerica, MA, USA); anti‐phospho‐VEGFR‐2 (Y951) (Abcam, Cambridge, UK); anti‐phospho‐p70^S6K (T421/S424), anti‐phospho‐Akt (S473), anti‐phospho‐ERK (T202/Y204), anti‐phospho‐p38^MAPK (T180/Y182), anti‐phospho‐pRb (S780) and anti‐phospho‐pRb (S807/S811) (Cell Signaling, Beverly, MA, USA); anti‐phospho‐tyrosine (BD Biosciences, Bedford, MA, USA); anti‐VEGFR‐2, anti‐vascular endothelial (VE)‐cadherin, anti‐integrin β1, anti‐ILK, anti‐p70^S6K, anti‐Akt, anti‐ERK, anti‐p38^MAPK, anti‐Cdk2, anti‐Cdk4, anti‐cyclin D, anti‐cyclin E, anti‐actin antibodies and mouse and rabbit IgG‐horseradish peroxidase conjugates (Santa Cruz Biotechnology, Santa Cruz, CA, USA); Alexa Fluor 488–conjugated goat anti‐mouse IgG (Life Technologies, Grand Island, NY, USA).

After electrophoresis of total cell proteins, samples were immunoblotted as previously reported^{82 (link),83 (link)}. Membranes were then incubated overnight at 4 °C with the following polyclonal antibodies [dilution, -fold]: anti-VCAM1 antibody (Abcam,) [5000], anti-E-Cadherin antibody (BS Transduction Laboratories) [1000], anti-tenascin-C antibody (Santa Cruz Biotechnology, Santa Cruz CA) [500], anti- NPHS2 antibody (Abcam) [5000], anti-nephrin antibody (Abcam) [5000] and anti-tubulin (Sigma–Aldrich, Saint Louis, MO [5000]. Anti-vimentin (Santa Cruz Biotechnology) [1000], anti-cofilin-1 (Santa Cruz Biotechnology) [1000], anti-vinculin (Santa Cruz Biotechnology) [1000], anti-ILK (Santa Cruz Biotechnology) [1000], anti-β-catenin (Santa Cruz Biotechnology) [2000].
Coomassie staining (Sigma) of the membrane was used as an internal loading control. Blots were analyzed by densitometric scanning with Image J. Western blot studies in cultured cells were performed in at least three independent experiments, and a representative figure is shown.