Cleaved caspase 3 asp175 9661s

Antibodies to α-tubulin (Millipore, 05-829); Erk (9102), phospho-Erk (9101), Ras (3965), Raf-1 (12552S), p-Raf-1/Ser259 (9421S), FGFR2 (23328S), phospho-FGFR (3471S), H3 (4499), p-H3^Ser10 (9701), cyclins D1, D3, A, B, E (Cyclin Antibody Sampler Kit, 9869T); CDKs 4 and 6 (CDK Antibody Sampler Kit, 9868T); cleaved caspase 3/Asp175 (9661S) (Cell Signaling); GPC1 (GeneTex, GTX104557); GAPDH (SAB2100894), laminin α1 (SAB4501255), FGF2 (SAB2100814), FGF1 (SAB1405808), β-catenin (C2206) (Sigma); α1(XI) (Oncomatrix, IS002); α3(V)-NTD (see below) and α1(V)46 (link) were all diluted 1:1,000. All were rabbit polyclonal except for CDK4 and 6, and α-tubulin antibodies, which were mouse monoclonal. Uncropped, full scan versions of immunoblots can be found in Supplementary Figs 9–14.

The immunohistochemistry (IHC) and IHC scoring were carried out according to protocols that have been described previously [14 (link)]. Protein lysates were prepared by homogenization in 1% NP40 containing 1 mM PMSF and 20 μg protein was separated by SDS-PAGE. The immunoreactive bands were visualized using an Immobilon™ Western Kit (Millipore, Billerica, MA) using the SYNGENE G: BOX imaging system (Frederick, USA). Antibodies specific to CLDN7 (ab27487), BCL-2 (ab32124), PARP1 (ab32064), Caspase-3 (ab13847), E-cadherin (CDH1, ab76055), N-cadherin (CDH2, ab18203), Vimentin (ab92547) and TGFB1 (ab25121) were purchased from Abcam (Hong Kong, China). GAPDH (TA309157) and Ki-67 (TA500265) were purchased from ZSGB-BIO, Beijing, China. Cleaved-Caspase3 (Asp175) (9661S) was purchased from Cell Signaling Technology, MA, USA. The antibodies for the IHC and Western blot assays were diluted with phosphate buffered solution (PBS) or PBS plus Tween-20 (PBST), according to the manufacturer’s instructions.