Anti vinculin

Cell lysates’ preparation and immunoblot analyses were performed as previously reported [35 (link)]. The filters were probed with the indicated primary antibodies: anti-EGFR, anti-PDGFRβ, anti-vinculin and anti-α-tubulin (Cell Signaling Technology Inc.). The blots shown are representative of at least three independent experiments.

BMDCs (4 × 10⁶) isolated, differentiated and cultured as described above were aliquoted into 250 μl PRF HBSS in 1.5 ml screw-cap polypropylene tubes. Cells were starved for 2 h at 37 °C prior to stimulation by the addition of 250 μl 2×-concentrated stimuli at the appropriate time points. Stimulation was stopped with the addition of 1 ml ice-cold HBSS and transfer to wet ice. Cells were immediately centrifuged at 8000g for 1 min at 4 °C, snap-frozen on dry ice or liquid N₂, and stored at −80 °C until analysis.
Cells were lysed using RIPA buffer (50 mM Tris–HCl pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA). Lysates were clarified by centrifugation at 10,000g for 10 min and the supernatants transferred to new 1.5 ml tubes. Protein contents of the lysates were quantified using the BCA 96-well plate assay (ThermoFisher) according to the manufacturer’s instructions. Samples were fractionated on 4–12% gradient NuPAGE Bis–Tris precast polyacrylamide gels, followed by transfer to polyvinylidene difluoride (PVDF) membranes according to the manufacturer’s instructions (Invitrogen; ThermoFisher). Blots were probed with anti-phospho-NFκB p65 (Ser536), anti-vinculin, anti-β-actin (Cell Signaling), and anti-NFκB p65 (Santa Cruz) antibodies. Uncropped immunoblot scans are presented in Supplementary Fig. 9.

Cell and tumor lysates preparation and immunoblot analyses were performed as previously reported [34 (link), 35 (link)]. Filters were probed with the indicated primary antibodies: anti-EGFR, anti-phospho-44/42 MAPK (extracellular signal-regulated kinase 1/2, ERK1/2, indicated as pERK1/2), anti-caspase 3 and anti-vinculin (Cell Signaling Technology Inc., Danvers, MA), anti-ERK1 (C-16) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-histone H2A.X (Ser139, indicated as γH2AX), clone JBW301 (Upstate Biotechnology, Inc., Lake Placid, NY).
Densitometric analysis was performed on at least two different expositions to assure the linearity of each acquisition using ImageJ (v1.46r). Blots shown are representative of at least three independent experiments.