β actin

For the Western blotting, total cell lysates were prepared by extraction in RIPA buffer supplemented with 1% protease inhibitor (Bimake, B14002) and 1% phosphatase inhibitor (Bimake, B15002). The cell lysates were boiled at 100 °C for 10 min, separated by SDS‐polyacrylamide gel electrophoresis (PAGE) and transferred to PVDF membranes. Then, the membranes were blocked with 5% skim milk. Immunoblotting was performed with the corresponding primary antibodies. After incubation with HRP‐conjugated secondary antibodies, Western blot images were captured on a SmartChemi^TM 610 (Beijing SinSage Technology Co., Ltd., China). The following primary antibodies were used: TP53INP2 (1:1000; Abcam, ab273 012), ALDH3A1 (1:1000; Abcam, ab129 022), IDI1 (1:1000; Abcam, ab205617), DLD (1:1000; Abcam, ab133551), HMGCS1 (1:500; Proteintech, 17643‐1‐AP), CPOX (1:1000; Abcam, ab169766), ACACA (1:5000; Abcam, ab109368), USF1 (1:10 000; Abcam, ab125020), p‐ACACA (1:1000; Cell Signaling Technology, 11818T), β‐tubulin (1:2000, Beyotime Biotechnology, AF1216) and β‐actin (1:3000; TransGen Biotech, HC201‐01).

Cell lysates were extracted from the samples harvested at 18 hpi. The lysates were mixed with a sample buffer (Solarbio, Beijing, China) followed by heat treatment at 95 °C for 5 min. The samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Bands were detected with primary antibodies specific to SHP2 (3397T, Cell Signaling Technology, Danvers, MA, USA), phospho-SHP2 (Y542) (3751T, Cell Signaling Technology, Cambridge, MA, USA), PD-L1 (3751T, Cell Signaling Technology), ERK (4695T, Cell Signaling Technology, MA, USA), phospho-ERK (T202/Y204) (4370T, Cell Signaling Technology), β-actin (R019, Transgen biotech, China), and anti-H1N1-NP (generated in our laboratory) at 4 °C overnight prior to incubation with horseradish peroxidase-conjugated goat anti-mouse (125229, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) or anti-rabbit IgG (131879, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) incubation for 2 h. The blots were developed using the FluorChem M Imaging System (Protein Simple, San Jose, CA, USA). β-actin was used as a reference of internal standard.

All protein was extracted in radioimmunoprecipitation assay (RIPA) buffer (Solarbio Life Science, Beijing, China) mixed with protease inhibitor on ice-cold plates. Then, the protein concentration was determined with a bicinchoninic acid (BCA) protein assay kit purchased from Solarbio Life Science. Next, we loaded equal amounts of protein into a 10% SDS–PAGE gel for separation and transferred the protein onto nitrocellulose membranes (Millipore). The membranes were blocked with 5% BSA (Sigma-Aldrich) and incubated with primary antibodies at 4 °C overnight. On the second day, the membranes were washed with 1× TBS solution and incubated with secondary antibody conjugated with horseradish peroxidase (HRP) at normal temperature for 1 h. Finally, the results were obtained using a ChemiDoc^TM Imaging System (Bio-Rad, Hercules, CA, USA). Throughout the entire study, primary antibodies targeting the following proteins were used: RNF31 (ab46332, 1:1000, Abcam), p53 (ab1101, 1:1000 Abcam), PCNA (ab29, 1:1000, Abcam), Cyclin D1 (ab40754, 1:5000, Abcam), Bcl-xl (2764, 1:1000, CST), GAPDH (1:1000, TransGen Biotech, Beijing), Flag-tag (D6W5B, CST), HA-tag (C29F4, CST), and β-actin (1:1000, TransGen Biotech, Beijing). The secondary antibodies were purchased from TransGen Biotech (Beijing).

Total proteins were extracted by placing cell lines and HCC specimens in lysis buffer at 4 °C for 30 min. The protein samples were separated by using SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred onto PVDF membrane. The PVDF membranes were incubated with primary antibodies, anti-Pin1 (home made), anti-MMP9 (1:200; D261999; Sangon Biotech), anti-E-cadherin (1:500; #3195 S; Cell Signaling Technology), anti-vimentin (1:1,000; #3932 S; Cell Signaling Technology), anti-cyclinD1 (1:500; #2978 S; Cell Signaling Technology), anti-CDK2 (1:500; #2546 S; Cell Signaling Technology), anti-Akt (1:1,000; #9272 S; Cell Signaling Technology), anti-p-Akt(Ser 473) (1:500; #9271 S; Cell Signaling Technology), anti-ERK (1:500; 13-6200; Invitrogen), anti-pERK (1:1,000; #9101 S; Cell Signaling Technology), anti-NF-κB p65 (1:1,000; #8242 S; Cell Signaling technology) and then probed with a secondary antibody (1:5,000; Merck Millipore). β-actin (1:8,000; #HC201-02; TransGen Biotech) or GAPDH (1:8,000: #HC301-02; TransGen Biotech) was used as a protein loading control.

The samples were separated on 10% SDS‐PAGE gels. The membranes were incubated with 5% non‐fat milk in TBST at room temperature and then incubated with primary antibodies overnight at 4°C with gentle shaking: (a) CREB (1:1000, rabbit monoclonal; Cell signalling, CST9191S); (b) Ser133‐phosphorylated CREB (p‐CREB) (1:1000, rabbit polyclonal; Cell signalling, CST9197S); (c) BDNF (1:1000, rabbit polyclonal; Santa Cruz Biotech, SC 546); (d) β‐actin (1:2000, Mouse monoclonal; TransGen Biotech; #HC201). The membranes were then washed with TBST and incubated with goat anti‐rabbit lg G‐HRP conjugated secondary antibody (Proteintech, SA00001‐2; CREB (1:2000), p‐CREB (1:1500), BDNF (1:6000)) at room temperature for 30 minutes. The membranes were visualized by the ECL reagents after exposing the membranes to X‐ray film in a dark room. Western blots were scanned and quantified by densitometry, using Image J software.

Total protein was extracted from CRC cells using radioimmunoprecipitation assay (RIPA) buffer (Solarbio Life Science, Beijing, China), which was mixed with protease inhibitor on precooled plates. The details of the experimental conditions are described in the Methods section and in our previous paper (21 (link)). Throughout this study, primary antibodies targeting the proteins are listed as follows: GAPDH (1:1000, TransGen Biotech, Beijing,China), β-actin (1:1000, TransGen Biotech, Beijing,China), and USP20 (17491-1-AP, 1:1000, Proteintech, China).

Protein extraction and western blot assays were conducted according to the methods previously outlined [3 (link)]. The p65 (#8242), phospho-p65 (Ser536, #3033), PCNA (#2586), Bcl-2 (#15071), BAX (#41162), E-cadherin (#3195), N-cadherin (#13116), and vimentin (#5741) primary antibodies were acquired from Cell Signaling Technology (MA, USA), while β-actin (HC201–01) was obtained from TransGen Biotech (Beijing, China). All antibodies are diluted 1:1000.