G box imaging system

Immunohistochemistry (IHC) experiments and IHC scoring were carried out as previously described according to previous protocols [25 (link)]. Protein lysates were prepared by homogenization in 1% NP-40 containing 1 mM PMSF, and samples containing 20 μg of protein were separated by SDS-PAGE. The immunoreactive bands were visualized using an ImmobilonTM Western kit (Millipore, Billerica, MA) using the Syngene G:BOX imaging system (Frederick, USA). According to the manufacturer’s instructions, western blot assays were carried out with antibodies diluted in PBS plus Tween-20 (PBST). Information on all primary antibodies used is listed in Supplementary Table 2.

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.