Ab196575

Western blotting was performed using the primary antibodies, anti‐SH2B1 (1:1000, ab196575, Abcam, Cambridge, MA), anti‐E‐cadherin (1:3000, ab40772, Abcam), anti‐N‐cadherin (1:1000, ab76011, Abcam), anti‐Vimentin (1:2000, ab92547,Abcam), anti‐β‐catenin (1:5000, ab32572, Abcam), anti‐TCF‐4 (1:25000, ab76151,Abcam), anti‐CyclinD1 (1:10000, ab134175, Abcam), anti‐C‐Myc (1:1000, ab32072, Abcam), anti‐IRS1 (1:1000, ab40777), anti‐DVL‐2 (1:500, ab137528), antibodies (Abcam), and anti‐GAPDH antibody (1:3000. D110016, Sangon Biotech, Shanghai, China). Following the Western blotting assay, the membranes were stripped, and the band intensities were relative to GAPDH. The quantification of protein bands was performed using ImageJ software.

IHC analysis was performed on the 159 paraffin‐embedded LADC tissue sections, using the following primary antibodies, anti‐SH2B1 (1:100, ab196575, Abcam), anti‐E‐cadherin (1:500, ab40772, Abcam), anti‐N‐cadherin (1:200, ab76011, Abcam), anti‐Vimentin (1:200, ab92547, Abcam), anti‐β‐catenin (1:500, ab32572, Abcam) antibodies (Abcam, Cambridge, MA, USA). The IHC staining scores were determined by combining the intensity of staining and the proportions of positively stained cells. The standard of staining intensity was graded: 1, no staining; 2, weak staining (light yellow); 3, moderate staining (yellow‐brown); and 4, strong staining (brown). The positive cell proportions were scored according to the following standard: 0, <5% positive cell; 1, 6‐25% positive cells; 2, 26‐50% positive cells; 3, 51‐75% positive cells; 4, 76‐100% positive cells. We evaluated protein expression using staining index (SI),41 which was calculated in the proportion of positive cells and the staining intensity score as possible total scores of 0, 2, 3, 4, 6, 8, 9, 12, and 16. Samples with SI < 8 were defined as low (negative) expression and SI ≥ 8 as high (positive) expression. The degree of SI was reviewed and scored separately by two independent pathologists.