Tyr397

Total cell protein extracts were prepared using RIPA buffer as described previously [12 (link)]. Membranes were blocked for 1 h with 10% non-fat milk or 5% BSA in TBS containing 0.1% Tween-20, and then incubated overnight at 4°C with the primary antibody at the dilutions recommended by the manufacturer.
The primary antibodies against FAK and phospho-FAK (Tyr397) were purchased from Cell Signaling (Danvers, MA, USA), and the anti-β-actin from Sigma-Aldrich (Steinheim, Germany). HRP-conjugated anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as the secondary antibody. Blots were developed using Lumi-Light Plus Reagent (Roche, Barcelona, Spain), and the autoradiograms were scanned using a GS-800 calibrated densitometer and analyzed using Quantity One software (Biorad, Berkeley, CA,USA).

FAK activation was assessed by performing western blotting with phospho-FAK (mouse Tyr925; catalog no. 3284 and human Tyr397; catalog no. 8556) (1:500) and FAK (1:1000) antibodies (catalog no. 13009) (Cell Signaling, Massachusetts, USA). Western blotting with IκB (catalog no. 4812) (1:1000) and phospho-IκB (catalog no. 2859) (1:1000) antibodies (Cell Signaling) was performed to examine NFκB activation status. As indicated, α5 integrin (catalog no. ab150361) (1:500) and αv integrin (catalog no. ab179475) (1:1000) antibodies (Abcam, Cambridge, UK) were also used for western blot analyses. The actin antibody (catalog no. A300-485A) (1:5000) was purchased from Bethyl Laboratories (Texas, USA). In some experiments, protein bands from the western blots were quantified by using ChemiDoc™ XRS + software Image Lab 5.1 (BioRad). Uncropped western blots are shown in Supplementary Fig. 10.

Gemcitabine (GEM) was purchased from Eli Lilly (Indianapolis, IN, USA). AMD3100, MTT, Hoechst 33342, PD98059 (an ERK inhibitor), PF573228 (a FAK inhibitor), SB20938 (a P38 inhibitor) were all supplied by Sigma-Aldrich (St. Louis, MO, USA). Recombinant human SDF-1α (rhSDF-1α), the anti-IL-6 mouse monoclonal antibody (mAb) and the IL-6 ELISA kit were purchased from R&D Systems (Minneapolis, MN, USA). The anti-αSMA mouse mAb was from Dako. The anti-vimentin and anti-SDF-1α mouse mAbs, the anti-CXCR4 rabbit polyclonal antibody and the SDF-1α neutralizing antibody (SDF-1α Ab) were from Abcam. Rabbit mAbs against pAKT (Ser473), pP38 (Thr180/Tyr182), ERK1/2, pERK1/2 (Thr202/Tyr204) and pFAK (Tyr397) were from Cell Signaling Technologies (Beverly, MA, USA). The mouse mAb against FAK, rabbit mAbs against AKT and and β-actin, the rabbit polyclonal antibody against P38, and the secondary horseradish peroxidase-conjugated anti-rabbit and anti-mouse antibodies were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).