Ab108349

Immunohistochemical analysis for p16 was performed on 4-μm thick tissue sections cut from the FFPE tissue. A monoclonal antibody to p16 (ab108349, Abcam, UK; dilution 1:100) was used with a BenchMark Autostainer (Ventana Medical Systems, Tucson, USA) following the manufacturer’s recommendations. Expression of p16 was considered positive if strong and diffuse nuclear and cytoplasmic staining was detected in ≥70% of the tumor [11 (link)] Two head and neck pathologists reviewed p16 expression and were blinded to the clinical and follow-up data. Discrepancies that occurred between observers were resolved by consensus review.

After treatment with rapamycin, hGFs were harvested and lysed with RIPA Lysis and Extraction buffer (Pierce Biotechnology, Rockford, IL, USA) and Halt Protease and Phosphatase Inhibitor (Pierce Biotechnology). Protein concentration was quantified using a BCA protein assay kit (Pierce) and fractionated by sodium dodecyl sulphate-polyacryalamide gel electrophoresis (SDS-PAGE), then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore Co., Bedford, MA, USA). The membrane was blocked with 5% nonfat milk and 2% BSA in TBST for 1 h at room temperature and incubated with certain primary antibodies, rabbit anti-human p21 antibody (ab109520; Abcam, Cambridge, UK), rabbit anti-human p16 antibody (ab108349; Abcam), rabbit anti-human phospho-p70 S6 kinase antibody (Thr421/Ser424) (Cell Signaling Technology), rabbit anti-human p70 S6 kinase antibody (Cell Signaling Technology), and rabbit anti-human β-tubulin antibody (ab151318; Abcam) at 4°C overnight. The membrane was washed 4 times and incubated with a 1 : 3000 dilution of horseradish peroxidase-conjugated donkey anti-rabbit or anti-mouse IgG antibody (R&D Systems) for 1 h at room temperature. After being washed 3 times with TBST, the immunodetection was developed with the ECL-chemiluminescent kit (Thermo Scientific).

Total protein extracts from cultured cells were obtained using Ripa Buffer (50 mM TrisHCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40, and 0.1% SDS) containing protease and phosphatase inhibitor cocktails (Sigma). Equal amounts of proteins (20 μg) were separated by SDS-PAGE and transferred to nitrocellulose membranes by western blotting. Membranes were then blocked and incubated with the following primary antibodies: anti-P16 (ab108349, Abcam), anti-P21 (ab109199), anti-SOD2 (sc-30080, Santa Cruz Biotechnology), anti-phospho-rpS6^S240/244 (number 5364, Cell Signaling Technology), and B-actin (A2228, Sigma) for loading control. Detection was performed using horseradish peroxidase-coupled anti-mouse or anti-rabbit secondary antibodies (Cell Signaling), ECL substrate (GE Healthcare), and conventional developing using X-ray films. The intensity of the bands was determined by densitometry using Totalab TL120 software (Nonlinear Dynamics, Newcastle upon Tyne, United Kingdom).

Total cell proteins were prepared from the DM1 model cells, as previously described (Nakamori et al., 2017 (link)). Then, the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with the following primary antibodies: mouse anti-IGFBP3 (1:500; MAB305, R&D Systems), rabbit anti-PAI-1 (1:2000; NBP1-19773, Novus), rabbit anti-AKT (1:500; GTX121937, GeneTex), rabbit anti-phospho-Akt (Ser473) (1:500; 4,058, Cell Signaling Technology), rabbit anti-p53 (1:100; 2,527, Cell Signaling Technology), rabbit anti-p21 (1:1,000; 2,947, Cell Signaling Technology), rabbit anti-p16 (1:1,000; ab108349, Abcam), and rabbit anti-GAPDH (1:1,000; G9545, Sigma-Aldrich). After incubation, the immunoblots were washed, incubated with horseradish peroxidase-conjugated anti-mouse immunoglobulin (Ig) G or anti-rabbit IgG (GE Healthcare), and detected by ECL Prime Western Blotting Detection Reagent (GE Healthcare) using a ChemiDoc Touch Imaging System (Bio-Rad).

Male Bl6 mice (Sankyo Laboratories, Inc., Tokyo, Japan), at 9 weeks old (young) or 80 weeks old (old), were intravenously treated with the vehicle alone or with anti-ApoD antibody and PBD-conjugated IgG with a cleavable linker (Moradec LLC, San Diego, CA, USA), each at a concentration of 0.3 mg/kg and 3 mg/kg in a single dose. Each group contained five mice. The mice were kept with free access to food and water; after 1 month, the mice were euthanized, and tissue samples were collected. The frozen specimens were sliced into 7 µm thick sections, mounted on glass slides, and fixed in acetone for 10 min at room temperature. To block nonspecific binding sites, the slides were incubated with 3% goat serum in PBS for 30 min at room temperature. The slides were then incubated overnight at 4 °C with the primary antibody p16 (ab108349, abcam, 1:200). After washing three times with PBS, the slides were incubated with a Alexa Fluor 488-conjugated goat anti-rabbit antibody (ThermoFisher Scientific) diluted at a ratio of 1:1000 in PBS for 1 h at room temperature. Nuclear contrast staining was performed using ProLongGold with the DAPI anti-fading sealant (ThermoFisher Scientific).

Cell Lysis Buffer (CST) was used to extract protein lysates and the Subcellular Protein Fractionation Kit for Cultured Cells (ThermoFisher Scientific) was used for fractionation according to manufacturers’ instructions. The following primary antibodies were used: EZH2 (5246; CST), GAPDH (MAB374; Merck Millipore), Histone H3 (ab1791; Abcam), H3K27me3 (ab6002; Abcam), MYOG (556,358; BD Biosciences), p16 (ab108349; Abcam), p21 (ab80633; Abcam), PARP (9542; CST), RARα (E6Z6K; CST), α-Skeletal Muscle Actin (ab28052; Abcam), α-Tubulin (SC-8035; Santa Cruz Technology). Immunostained bands were detected by chemiluminescence (GE Healthcare). Full length blots in Additional File 1.