Ab78393

Primary tumor samples were collected from 138 patients at The Second Affiliated Hospital of Xi'an Jiaotong University who underwent surgery for lung cancer between 2011 and 2014. This study protocol was approved by the Ethics Committee of The Second Affiliated Hospital of Xi'an Jiaotong University and written informed consent was provided prior to patient enrollment. Lung cancer and adjacent normal tissues were immediately flash frozen in liquid nitrogen following collection and used for quantitative real-time PCR analysis, otherwise formalin-fixed paraffin-embedded lung cancer specimens underwent immunohistochemistry (IHC) analysis using SMURF2 rabbit polyclonal antibody (Abcam; ab272897), TRIM27 rabbit polyclonal antibody (Abcam; ab78393), or SIX3 rabbit polyclonal antibody (Bioss Biotechnology Company, Beijing, China; bs-11970R). Immunoreactivity was scored by two investigators based on the percentage of positively stained cells and staining intensity, which ranged from 0 to 3. Our cohort of 138 patients with lung cancer was parsed into two subgroups by IHC staining comprised of a low-expression group with an IHC score less than 50% and a high-expression group with an IHC score over 50%.

Protein lysates were prepared using RIPA lysis buffer containing protease inhibitor cocktail (Sigma, St. Louis, MO, USA). After being separated by SDS‐PAGE, proteins were transferred onto nitrocellulose membranes (Millipore, Bedford, USA), blocked with 5% skim milk, and incubated with primary antibodies against METTL14 (Abcam; ab220030; 1:500), TRIM27 (Abcam; ab78393; 1:1000), STAT3 (Abcam; ab68153; 1:1000), p‐STAT3 (Abcam; ab267373; 1:1000), IGF2BP2 (Abcam; ab129071; 1:1000), and β‐actin (Cell Signalling Technology; 4970 s; 1:1000), followed by incubation with HRP‐conjugated secondary antibodies (Beyotime, Shanghai, China; A0208, A0216; 1:1000). Signals were detected using enhanced chemiluminescence system (Bio‐Rad, Richmond, CA, USA).