Anti actin

The following antibodies are used in immunohistochemistry studies: anti-Lin28A (ab124765, Abcam, USA), anti-RAN (ab53775, Abcam, USA), anti- HSBP1 (ab83247, Abcam, USA), anti-Ki67 (D154094, BBI Life Sciences, China), anti-E-cadherin (20874-1- AP, Proteintech, China). The antibodies used for western blot analysis are as follows: anti RAN (10469- 1-AP, Proteintech, China), anti-HSBP1 (DF8954, Affinit, China), anti-Actin (AC004, ABclonal, China), anti-E-cadherin (20874-1-AP, Proteintech, China), anti-N-cadherin (22018-1-AP, Proteintech, China), anti-Vimentin (10366-1-AP, Proteintech, China), Apoptosis Antibody Sampler Kit (#9915T, CST, USA), Epithelial-Mesenchymal Transition (EMT) Antibody Sampler Kit (#9782, CST, USA).

Total cellular proteins were extracted using SDS lysis buffer (250 nM Tris–HCL, pH 7.4, 2.5% SDS) with 100 mM phenylmethanesulfonyl fluoride (PMSF) (Beyotime, Biotechnology, Haimen, China). After denaturing at 100°C for 10 min, aliquots of protein (20 μg/sample) were electrophoresed on 10 or 12% polyacrylamide gel (according to the molecular weight of goal proteins) using an electrophoresis system (Bio-Rad Laboratories Inc., CA, United States). After electrophoresis, proteins were transferred to a PVDF membrane, blocked with 5% skim milk in Tris-buffered saline/Tween 0.05% (TBST) for 2 h and then incubated overnight at 4°C with a primary antibody of anti-ANXA6 antibody (1:2000, Abclonal), anti-P62 (1:1000, Cell signaling Technology), anti-LC3 (1:1000, Cell signaling Technology) or anti-Actin (1:20000, Abclonal). Then the membrane was triply washed with TBST at room temperature for 10 min and labeled with a peroxidase-conjugated secondary antibody (1:5000, Beyotime Biotechnology) for 2 h. Proteins in the membrane were detected by the enhanced chemiluminescence system (ECL kit, Millipore, St. Louis, MO, United States), and band images were analyzed with the Bio-Rad ChemiDoc XRS system.

Total protein was extracted using an extraction buffer (Thermo Fisher Scientific, Inc.). The proteins were separated by SDS-PAGE (12% acrylamide gel) and transferred onto a PVDF membrane. The membranes were subsequently blocked with 5% skim milk for 2 h at room temperature to inhibit nonspecific protein binding. This incubation was followed by an incubation with the primary antibodies anti-GAPDH (1:10,000; cat. no. AC002; ABclonal), anti-actin (1:1,000; cat. no. ab8226; Abcam), anti-HMGB1 (1:1,000; cat. no. Ab184532; Abcam), anti-TNF-α (1:1,000; cat. no. ab1793; Abcam), anti-IL-1β (1:1,000; cat. no. A1112; ABclonal), anti-pIKK-β (1:1,000; cat. no. Ab194519; Abcam) and anti-IKK-β (1:1,000; cat. no. A2087; ABclonal) at 4 °C overnight. The secondary antibodies were a horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody (1:10,000; cat, no. ab6721; Abcam) and a horseradish peroxidase-conjugated anti-mouse IgG antibody (1:20,000; cat. no. AS003; ABclonal), which were incubated with the membranes for 1 h. The results were imaged and quantitated using Bio-Tanon Imagine (Tanon Science & Technology Co., Ltd., Shanghai, China). ImageJ software (version 1.4; National Institutes of Health, Bethesda, MD, USA) was used to analyse the specific protein band values of each group. All original pictures of western blot results are provided in supplementary file.