P53 antibody

The following drugs and reagents were used in the study: DMEM/F12 media without phenol red (Gibco), fetal bovine serum (FBS, Sciencell), RNA isolation kit, Color SYBR Green qPCR Master Mix and 4× reverse transcription Mix (EZBioscience, A0012), enzyme linked immunosorbent assay (ELISA) kits (E2, Labor Diagnostika Nord, FR E-2000; FSH, Immunoway, KE1425; LH, Immunoway, KE1475; AMH, Jingmei, JM11692M1; insulin, Alpco, 80-INSMSU-E01, E10), Normal rabbit IgG (CST, 2729), Normal mouse IgG (CST, 7076), p53 antibody (Abcam ab131442), p16 antibody (Cell Signaling Technology Cat, sc1661), p21 antibody (Cell Signaling Technology, sc-6246), AGER antibody (Abcam, ab3611), Senescenceβ-Galactosidase Staining Kit (Beyotime, C0602), Cell Cycle Staining Kit (Multi Sciences, CCS021), insulin (Absin, abs42019847), metformin (Topscience, T8526), high-fat diet (Jiangsu Xietong Pharmaceutical Bio-engineering Co., Ltd., D12451).

LNCaP and DU145 cells were lysed with ice-cold RIPA lysis buffer (Millipore) and total protein was collected. Subsequently, protein concentration was detected by a BCA Protein Assay Kit (HyClone-Pierce). Equal amount proteins (20 µg) were separated by 10% SDS-PAGE (Invitrogen) and transferred to PVDF membrane (BIO-RAD). The membrane was blocked in TBST solution containing 5% non-fat milk for 1 h and incubated with primary antibodies (CACYBP antibody, 1:3000, Cat. #ab171972, Abcam; p53 antibody, 1:2000, Cat. #10442-1-AP, Proteintech; p-p53 antibody, 1:2000, Cat. #28961-1-AP, Proteintech; Bax antibody, 1:2000, Cat. #ab182733, Abcam; Bcl-2 antibody, 1:2000, Cat. #ab182858, Abcam; GAPDH antibody, 1:3000, Cat. #60004-1-lg, Proteintech) at room temperature for 2 h. Then, the membrane was continuingly incubated with the secondary antibody Goat Anti-Rabbit (1:3000, Cat. #A0208, Beyotime) at room temperature for 1 h. Protein bands were visualized by ECL plus TM Western blotting system kit (Millipore).

Protein was extracted from cardiac tissues or cell samples using RIPA lysis buffer. The cell lysate was fractionated by 10% SDS‐PAGE and subsequently transferred to PVDF membranes (Millipore) and incubated with different primary antibodies overnight. After incubated with the primary antibodies, membranes were washed with TBS‐T and incubated with the corresponding secondary antibodies for 1h at room temperature. Finally, the blots were scanned by a Bio‐Rad ChemiDoc XRS+system (Bio‐Rad). The following antibodies were used: Runx1 antibody (Cell Signaling Technology; #4334; dilution 1:1000), p53 antibody (Abcam; #ab26; dilution 1:1000), p‐p53 antibody (Abcam; #ab223868; dilution 1:2000), Noxa antibody (Cell Signaling Technology; #14766; dilution 1:1000), Bax antibody (Cell Signaling Technology; #5023; dilution 1:1000), Puma antibody (Cell Signaling Technology; #24633; dilution 1:1000), Anp antibody (Abcam; #ab181242; dilution 1:10 000) and Gapdh antibody (Abcam; #ab8245; dilution 1:50 000).

Primary keratinocytes were seeded in a 12-well plate at 100,000 cells/well in LabTek Chambered -Microscopic slides (Thermofisher, Australia) and exposed to UV and/or heat stress. For immunocytochemistry, cells were fixed with 4% paraformaldehyde, washed twice with TBS. Skin tissues were processed and stained according to previous protocols [14 (link)]. Primary NHEK and skin sections were incubated with antibodies to either thymine dimer (CPD) (mouse monoclonal, 1:500 dilution; Kamiya Biomedical, USA), p53 antibody (rabbit monoclonal 1:50 dilution; Abcam, USA), p53 acetyl K382 (rabbit monoclonal 1:200, Abcam USA), SIRT1, SIRT1-p, Casp-3 (cleaved), Survivin or anti-pan Cytokeratin and ki67 (rabbit polyclonal, 1:50 dilution; Abcam, USA). The mouse monoclonal antibody to pan-cytokeratin (1:50; Abcam, USA) was used to label keratinocytes. The secondary antibodies anti-mouse Alexa Fluor 488 (for keratin and CPD) and anti-rabbit Alexa Fluor 550 (for caspase 3, p53 and ki67) were used for detection. Three sections were analysed per exposure replicate and five images of randomly selected fields-of-view were captured from each section. To determine percentages of keratinocytes positive for each individual antibody, positive cells were quantified within 5 images randomly chosen per section.

Total protein extract from rat HCC JM1 cells was immunoprecipitated with p53 antibodies using a Dynabeads^® Protein G Immunoprecipitation kit (ThermoFisher Scientific). Briefly, p53 antibody (Abcam) was covalently coupled to the beads using BS3 (Thermo Scientific) as a cross-linker, according to the manufacturer's instructions. Total HCC JM1 cell extract was incubated with Dynabeads-Ab complexes for 10 min at room temperature to allow antigen binding. After elution of the bound p53-protein complexes by heating at 70°C, the samples were analyzed by Western blotting using anti-CRM1 and anti-p53 antibodies.