α1 4

Immunohistochemistry on 5-μm paraffin sections using antibodies against Ki-67 (Novocastra Laboratories), cleaved caspase-3 (Cell Signaling), CD31 (Abcam), α1(IV) (Abgent) or DDR1 (Santa Cruz) was performed as described [40 (link)]. For α5(IV) immuno-staining, 8-μm frozen tissue sections were fixed in cold acetone for 10 min. Samples were incubated with α5(IV) antibody (rat monoclonal antibody clone b14) (1:50–1:100) for 16 hours at 4°C, followed by incubation with Alexa Fluor 555/488 conjugated anti-rat IgG antibody. Immunohistochemistry or immunofluorescence sections were viewed under microscope (IX71; OLYMPUS, Inc.) with a UPlan-FLN 4×objective/0.13 PhL, a UPlan-FLN 10×objective/0.30 PhL, or a LUCPlan-FLN 20×objective/0.45 PhL. Images were captured with a digital camera (IX-SPT; OLYMPUS, Inc.) and Digital Acquire software (DPController; OLYMPUS, Inc.). Perfused blood vessels in Matrigel plugs were viewed by UV-illumination under microscope (SZX16; OLYMPUS, Inc.) with a SDF-PLAPO 1×PF. Images were captured with a digital camera (U-LH100HGAPO; OLYMPUS, Inc.) and Digital Acquire software (DPController; OLYMPUS, Inc.).

Total cell lysates were harvested in hot SDS sample buffer. For immunoprecipitation, cells were lysed in RIPA buffer. DDR1 was immunoprecipitated with anti-DDR1 (Santa Cruz) antibody. Immunoprecipitated proteins were eluted with SDS sample buffer. Proteins were separated by SDS-PAGE. After electrophoresis, the proteins were transferred to nitrocellulose membrane. The membrane was incubated overnight at 4°C with primary antibodies, washed with TBS-T (TBS with 0.1% Tween-20), and incubated with HRP-conjugated secondary antibodies at room temperature for 1 hour. Immuno-reactive protein was detected using SuperSignal West Pico Chem KIT (Thermo Scientific, USA). Primary antibodies used were against ERK, ERK pT202/pY204, Akt, Akt pS473, Src, Src pY416 (Cell Signaling), FAK (BD Transduction Laboratories), FAK pY397 (Millipore), α1(IV) (Abgent), α2(IV) (Abcam), DDR1, phosho-tyrosine (pY99), ubiquitin (Santa Cruz), α5(IV) (Proteintech), MEK1 (Abmart) and Actin (Sigma-Aldrich). Western blots were scanned and analyzed with Image J.

The antibodies used are ERK, ERK pT202/pY204, Akt, Akt pS473, Src, Src pY416, cleaved caspase-3 (Cell Signaling), FAK (BD Transduction Laboratories), FAK pY397 (Millipore), Ki-67 (Novocastra Laboratories), α1(IV) (Abgent), α2(IV) and CD31 (Abcam), α5(IV) (rabbit ployclonal antibody from Proteintech (western blot) and rat monoclonal antibody clone b14 (immunostaining) provided by Dr. Yoshikazu Sado, Shigei Medical Research Institute [36 (link)]), DDR1, phosho-tyrosine (pY99), ubiquitin (Santa Cruz), MEK1 (Abmart), Actin (Sigma-Aldrich), biotinylated goat anti-rabbit secondary antibody (Zymed), Alexa Fluor 555/488 conjugated anti-mouse, rat or rabbit IgG secondary antibodies (Invitrogen). Expression level of integrins on A549 cell surface was determined by immunofluorescence flow cytometry with anti-β1 (Thermo Scientific Pierce), α1, α2 and α11 (Santa Cruz) integrin antibodies as described [37 (link)]. Cycloheximide, MG132 and NH₄Cl were purchased from Sigma-Aldrich.