Anti p16

Liver lysates and serum samples were prepared, and Western blotting was performed as previously described (Liu et al., 2014). Proteins were resolved with sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by transferred to polyvinylidene difluoride membranes. The membranes were incubated overnight with primary antibody at 4°C. Polyclonal rabbit anti‐LC3B (1:1000, Abcam, ab48394), anti‐p62 (1:1000, Sigma‐Aldrich, P0067), anti‐Atg7 (1:1000, Cell Signaling Technology, 8558), anti‐p16 (1:1000, Proteintech Group, 10883‐1‐AP), anti‐p21 (1:1000, Proteintech Group, 10355‐1‐AP), anti‐GRP78 (1:1000, Proteintech Group, 11587‐1‐AP), anti‐GRP94 (1:1000, Cell Signaling Technology, 2104), and anti‐glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) antibody (1:20,000; Sigma‐Aldrich, G9545) were used for immunoblotting. Western blotting was visualized on the Kodak Image Station (Carestream Health Inc., Rochester, NY) and analyzed with the ImageJ software.

We conducted Western blot analysis following the method described previously [16 (link)]. The relevant antibodies were anti-HMGB1 (1:10,000; Abcam, USA), anti-Akt(AKT3 + AKT2 + AKT1,1:5000; Abcam) anti-Akt (phosphor S473, 1:5,000; Abcam), anti-PI3K p110(1:1000; Abcam), anti-Ki67 (1:1,000; Abcam), anti-ATM (1:2,000; Abcam), anti-ATM (phosphor Ser1981, 1:1,000; Novus), anti-P16(1:1,000; Proteintech, Chicago, USA), anti-CDK4 (1:2,000; Proteintech), anti-cyclin D1(1:5000; Proteintech) and anti-β-actin (1:10,000; Bioworld Technology Inc. USA), phosphatase inhibitor cocktail III(1:100; MCE, MedChemExpress, USA). The blotted protein bands were revealed by an Odyssey system (LI-COR Biosciences, USA). Finally, we measured the intensity of protein bands with ImageJ and calculated the ratio of the protein to the corresponding β-actin for the purpose of reflecting the changes in expression levels.

After being ground in liquid nitrogen, the samples were lysed with RIPA lysis buffer containing 1% PMSF (Beyotime, China) for 30 minutes at 4°C. The lysates were centrifuged (12,000 rpm, 8 minutes) at 4°C. After determining protein concentration, the qualified protein samples spread in SDS-polyacrylamide gel electrophoresis and transferred to a PVDF membrane band by electroblotting. The PVDF membrane bands were then incubated with the primary antibodies (anti-nitrotyrosine (NT), anti-MIF, anti-LC3, anti-P62, anti-PINK1 (1 : 1000; Abcam, UK), anti-P53, anti-P21, anti-P16, anti-aggrecan, anti-collagen II, anti-β-actin (1 : 500; Proteintech, China), and anti-Parkin (1 : 1000; Cell Signaling, USA)) overnight at 4°C. After the bands were washed with Tris-Buffered Saline with Tween (TBST) thrice, they were then incubated with the fluorescent secondary antibody for 80 minutes. Being washed with TBST thrice, the intensity of the proteins was determined and analyzed by Image Lab software (Bio-Rad, USA).

The cells were prepared in RIPA buffer (Solarbio, #R0020). A BCA™ Protein Assay kit (Applygen, #P1511) was used to determine the protein concentration. The proteins (40 μg/sample) were separated by different polyacrylamide gel electrophoresis, transferred to PVDF membranes (Millipore, #IPVH00010), and incubated with the corresponding primary antibody at 4°C overnight. Then, the membranes were incubated with the corresponding secondary antibodies at room temperature for 1 h and measured with a chemiluminescence reagent ECL kit (Advansia, #K-12045-D50).
The primary antibodies used in the experiment used were: anti-PRPS1 (Proteintech, #15549-1-AP), anti-cyclin E1 (Proteintech, 11554-1-AP), anti-CDK2 (Proteintech, 10122-1-AP), anti-P16 (Proteintech, #10883-1-AP), anti-Bax (Proteintech, 50599-2-Ig), anti-Bcl2 (Proteintech, 12789-1-AP), anti-Cleaved-caspeas3 (CST, #9664), anti-MMP2 (Abcam, ab37150), anti-MMP9 (Abcam, ab76003), anti-MMP13 (Proteintech, 18165-1-AP), anti-E-Cadherin (Proteintech, 20874-1-AP), anti-N-Cadherin (Proteintech, 22018-1-AP), anti-Vimentin (Proteintech, 10366-1-AP), anti-NRF2 (Abcam, ab89443), anti-β-actin (Bioss, bs-0061R), and Tubulin (Abcam, #ab7291). The secondary antibodies used in the experiment were anti-rabbit IgG (Abcam, #ab6721) and anti-mouse IgG (Jackson ImmunoResearch Laboratories, 115-035-003).

The specimens were collected after sacrificing the mice. Then they were fixed in 10% formalin and embedded in paraffin. Paraffin tissue sections were stained with Masson’s trichrome or Sirius Red for histomorphometric assessment. The liver samples of cirrhotic patients in the form of tissue array (Alenabio, China) were collected from Tongxu County People’s Hospital, China. The study was approved by the Ethics Committee of Tongxu County People’s Hospital. The length of the biopsy specimen is around 2 mm. 5 cirrhosis samples and 5 healthy liver samples used as control were included in the present study. The patients’ information is summarized in Supplementary Table S1. Sections were deparaffinized and dehydrated. For antigen retrieval, they were heating in a microwave in EDTA repairing buffer (pH 9.0) for 15 min followed by blocking with 5% BSA. The sections were incubated with anti-p16 (1:200, Proteintech, catalog# 10883-1) and anti-Desmin (1:200, Thermo Fisher Scientific, United States, catalog# MA5-13259) overnight at 4°C. After briefly washing, the sections were incubated with goat anti-rabbit IgG and goat anti-mouse IgG secondary antibodies and DAPI.

As our previous studies (Yu et al., 2015 (link); Wang et al., 2017 (link)), virus-infected and mock-infected cells were collected at indicated times. The following antibodies were used: anti-cyclinE1 (Proteintech), anti-CDK2 (Cell Signal), anti-cyclinD (Cell Signal), anti-CDK6 (Cell Signal), anti-CDK4 (Cell Signal), anti-P53 (Cell Signal), anti-P21 (Proteintech), anti-P16 (Proteintech), anti-cyclinB1 (Santa Cruz), anti-CDK1 (Boster), anti-VP1 (Genetex) and anti-histone (GenScript). Secondary antibodies from mouse or rabbit were obtained from Jackson Immuno Research.

Cells were washed with phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4) and lysed in radioimmunoprecipitation assay (RIPA) buffer (20 mM Tris-HCl, 1% NP-40, 0.25% deoxycholic acid, 150 mM NaCl, 1 mM EDTA) containing 1 mM PMSF and 1x protease inhibitor (539134, Calbiochem). Total cell lysates were then collected by sonication of the cells followed by centrifugation at 16,000g for 5 min at 4 °C. Total cell lysates (30–50 μg) were separated by SDS-PAGE and then transferred onto nitrocellulose membranes. The membranes were hybridized with anti-p53 (Proteintech Group), anti-p53-S15 (Cell Signaling Technology), p53-T18 (GeneTex, Inc), p53-T55 (Abcam), p53-T81 (Cell Signaling Technology), p53-T155 (Santa Cruz Biotechnology, Inc), anti-Cdc25B (Cell Signaling Technology), anti-Cdk1 (Santa Cruz Biotechnology, Inc), anti-pCdk1-Y15 (Abcam), anti-p27 (GeneTex, Inc), anti-p21 (Cell Signaling Technology), anti-p16 (Proteintech Group), anti-pRB (S780, Cell Signaling Technology), or anti-RB (Proteintech Group). Horseradish-peroxidase-conjugated donkey anti-rabbit (GE Healthcare Life Sciences) or anti-mouse antibodies (Proteintech Group) were used as the secondary antibodies. The T-Pro LumiLong Plus Chemiluminescent Substrate Kit (T-Pro Biotechnology) was used to visualize the antibody-bound proteins.