Chemidoc xrs software image lab 5

Total RNA was extracted using TRIzol reagent (Life Technologies) following supplier’s instructions. Isolated RNA was treated with RNase-free DNase I (Thermoscientific) and cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). RT-PCR was performed using 2X Taq Red master mix (Apex) in a final reaction of 25 μl. The amplification cycle was as follows: an initial denaturing step (95°C for 3 min) was followed by 34 cycles of denaturing (95°C for 30 s), annealing (55.7°C for 30 s–), and extending (72°C for 1 min), followed by 5 min at 72°C for elongation. Following amplification, the PCR products were analyzed on 2% agarose gels and bands were visualized by ChemiDoc XRS (BioRad). The PCR product bands were quantified using ChemiDoc™ XRS + software Image Lab 5.1 (BioRad). Housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. The primers used to detect the indicated genes are listed below:
Human GAPDH forward, (5′ GATCATCAGCAATGCCTCCT-3′) and human GAPDH reverse, (5′ TGTGGTCATGAGTCCTTCCA-3′).
Human HBD3 forward, (5′ TCCAGGTCATGGAGGAATCAT-3′) and human HBD3 reverse, (5′ CGAGCACTTGCCGATCTGT-3′).

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.

A549 cells were lysed using 1%-Triton X-100 (pH 7.4), EDTA-free protease inhibitor cocktail (Roche Diagnostics) in PBS. Cell lysates were subjected to SDS-PAGE and separated proteins were transferred onto 0.2 μm nitrocellulose membrane (GE Health care) and blotted with specific antibodies. β-catenin and FLAG antibodies were purchased from Sigma-Aldrich. LRP5 antibody was obtained from Cell signaling. β-actin antibody was purchased from Bethyl Laboratories. HBD3-8A antibody was deposited to the DSHB by Starner, T. (DSHB Hybridoma Product hBD-3-8A). An anti-RFP antibody was purchased from Invitrogen. Western blots were quantified using ChemiDoc™ XRS + software Image Lab 5.1 (BioRad).