Anti p21

Cell lysates were collected in 1X sample buffer (2% SDS, 10% glycerol, 0.01% bromophenol blue, 62.5mM Tris, pH=6.8, 0.1M DTT) and boiled (10 min, 95° C). Protein concentration was determined using Bradford assay (Bio-Rad, cat#5000006). Proteins were resolved using SDS-PAGE gels and transferred to nitrocellulose membranes (GE Healthcare Life Sciences, cat#10600001) as previously described [8 (link)]. Antibodies used include: anti-BRAF (Santa Cruz Biotechnology, cat#sc-5284, 1:1000), anti-RAS (BD Sciences, cat#610001, 1:1000), anti-p16 (Abcam, cat#ab108349, 1:1000), anti-p21 (Abcam cat#ab109199, 1:1000), anti-cyclin A2 (Abcam cat#ab181591, 1:2000), anti-vinculin (Sigma-Aldrich cat#V9131, 1:1000), β-Actin (Sigma-Aldrich, cat#A1978, 1:10000), anti-mouse HRP (Cell Signaling Technology, cat#cst7076, 1:10,000), and anti-rabbit HRP (Cell Signaling Technology, cat#cst7074, 1:5000).

Immunoprecipitation assays were performed as previously described [31 (link)]. Cells were washed twice with PBS, collected and homogenized with RIPA buffer. After cell debris was removed by centrifugation, extracts were aliquoted and either used immediately or stored at -80°C. Whole-cell lysates in lysis buffer were cleared with 1.0 μg nonimmune rabbit IgG (Santa Cruz) together with 30 μl of protein A-Sepharose beads (Pierce). After centrifugation, the lysates were immunoprecipitated for 1 h at 4°C with 1 μg of the anti-Stat6 antibody or nonimmune rabbit IgG and then incubated overnight at 4°C with protein A-Sepharose. The immunoprecipitates were washed three times with lysis buffer and once with PBS and then resuspended in electrophoresis sample buffer. Samples of immunoprecipitated or total proteins (30 μg) were analyzed by Western blotting using the anti-ppRB-Ser807/811 antibody (Cell Signaling Technology) against a pRB peptide phosphorylated on the Ser807/811 residue, which is phosphorylated by both CDK2 and CDK4/6 kinases [32 ], or the anti-pRB against underphosphorylated pRB (BD Biosciences-Pharmingen), the anti-PR antibody (abcam), anti-p21(abcam), anti-p27(abcam), and anti-GADPH (as control antibody). The blots represent typical results from at least three independent experiments.

Renal cortex was homogenized and extracted in a cold buffer containing 0.1 mol/l Tris (hydroxymethyl) aminomethane HCl. The tissue extracts were then partially purified by ethanol extraction. Proteins from GENs or mesangial cells were isolated from using RIPA buffer (Thermo Scientific, USA). 50 μg of protein samples were isolated in 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride (PVDF) membranes (Invitrogen, USA) by electroblotting. The membranes were blocked in 5% non-fat dried milk for 60 min at room temperature. For the detection of RhoA, the membrane and cytoplasm proteins were extracted from mesangial cells using Membrane and Cytosol Protein Extraction Kit (Beyotime Biotechnology), then membrane and cytosolic proteins were separated on SDS-PAGE. The membranes were probed with first primary antibody anti-ETBR (Abcam, USA), anti-ETAR (Abcam, USA), anti-p-p65 (Invitrogen, USA), anti-p65 (Invitrogen, USA), anti-CTGF (Abcam, USA), anti-RhoA (Abcam, USA), anti-collagen IV (Abcam, USA), anti-Fibronectin (Abcam, USA), anti-p21 (Abcam, USA) and anti-β-actin (Invitrogen, USA) and incubated at 4°C overnight. After washing with PBST, membrane was cultivated with secondary antibody for 60 min at room temperature. β-actin was used as internal control.

Protein lysates of ESCC cells were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to 0.22-mm NC membranes (Sigma), and incubated with specific antibodies: anti-CDK6, anti-CDK4, anti-CDK2, anti-cyclinD1, anti-cyclinD3, anti-p27, anti-p21, anti-CDKN2C (Abcam, Shanghai, China), anti-EZH2, anti-EED, anti-SUZ12, anti-EZH1, and anti-β-actin (Cell Signaling Technology). The dilution ratio of the primary antibodies was 1:1,000, although anti-β-actin was diluted to 1:8,000 for Western blotting. Protein bands were visualized with Super Signal Chemiluminescence Substrate (Thermo Scientific) and β-actin was used as a control.

Protein from the treated and untreated HCT116 and LOVO cells was extracted by RIPA (Beyotime, Nantong, China). The protein lysates were separated by SDS-PAGE and then transferred onto PVDF membranes. ECL chromogenic substrate was used for densitometry quantification after incubating specific antibodies at 4 °C for 12 h. Protein detection was performed using rabbit monoclonal anti-CCND1 (Abcam, Cambridge, UK, ab134175), anti-CDK4 (Abcam, ab108357), anti-P21 (Abcam, ab109520), anti-casepase3 (CST, #14220), anti-PARP (Proteintech, Rosemont, IL, USA, 13371-1-AP), anti-PDCD4 (Abcam, ab80590), anti-Vimentin (CST, #5741), anti-N-cadherin (Abcam, ab76011), anti-E-cadherin (Abcam, 76011), anti-METTL3 (Proteintech, 15073-1-AP), anti-IGF2BP2 (Proteintech, 11601-1-AP),anti-FLAG (Proteintech, 20543-1-AP), anti-pAKT (CST, #4060), anti-AKT (CST, #9272), anti-PI3K (CST, #4249), anti-pPI3K (CST, #4228). GAPDH (Affinity, West Bridgeford, UK, AF7021) was applied as the internal control.

Western blot was used for the expression of senescence markers P16 and P21 in NPCs in each group. According to the instructions, the total protein of each group of NPCs was extracted in RIPA lysis buffer (Millipore, Billerica, MA, USA), Equal amounts of protein were subsequently added to each well and separated by sodium dialkylsulfonate-polyacrylamide gel electrophoresis (SDS–PAGE) and then, transferred to polyvinylidene fluoride membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Next, the membranes were incubated with 5% bovine serum albumin for 1 h, followed by overnight incubation at 4 °C with various primary antibodies: anti-p16 (Abcam, USA), anti-P21 (Abcam, USA) and GAPDH (Ambion, Austin, TX), all primary antibodies were diluted 1:1000. Next, the membrane was washed and incubated with secondary antibody (1:8000, Bioworld technology, China) for 2 h at room temperature. Protein signals on the bands were visualized by enhanced chemiluminescence (Thermo Fisher Scientific, Waltham, USA). Protein expression levels were quantified by densitometry using ImageJ 64 software, and relative protein expression was compared to GAPDH.