Anti p53 antibody

Western blotting was performed to determine the expression of inositol-requiring protein 1 (IRE1) p-IRE1/IRE1, eIF2, p-eIF2, PERK, p-PERK, transcription factor 4 (ATF-4), transcription factor 6 (ATF-6), C/EBP-homologous protein (CHOP), p53, cystine glutamate transporter (SLC7A11), and glutathione peroxidase (GPX4) in HK-2 cells. Protein concentrations were measured using a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Grand Island, NY, USA). Proteins of HK-2 cells were separated by SDS PAGE and then transferred electrophoretically onto polyvinylidenedifluoride (PVDF) membranes. The membranes were probed with anti-p-IRE1/IRE1 antibody, anti-ATF-4/6 antibody anti-CHOP antibody, anti-P53 antibody, anti-SLC7A11 antibody, anti-GPX4 antibody (Abcam, Cambridge, MA, USA), and polyclonal anti-actin antibodies (Santa Cruz Biotechnology, Dallas, USA). Protein bands were detected using an Odyssey System from LI-COR Biosciences, USA.

The levels of protein expression were measured using previously reported methods (12 (link)). Total proteins from the treated JEG-3 and BeWo cells were isolated using Radio-Immunoprecipitation assay (RIPA) buffer with 1% Phenylmethanesulfonyl fluoride (PMSF) (Abcam, Cambridge, UK). The protein concentrations were quantified using the Bicinchoninic acid (BCA) protein assay kit (Keygen, Beijing, China). The protein levels of the target genes were determined by Western blot analysis. The working concentration of primary anti-β-actin (ACTB) (Abcam, Cambridge, UK) was 1:2,000. The working concentrations of primary anti-cleaved Poly ADP-ribose polymerase (PARP) (Abcam, Cambridge, UK), anti-p21 (Abcam, Cambridge, UK), anti-MDM2 (Abcam, Cambridge, UK), anti-BCL2 (Abcam, Cambridge, UK), and anti-BAX (Abcam, Cambridge, UK) were 1:1,000. The working concentrations of primary anti-HIF1α (Abcam, Cambridge, UK) and anti-p53 antibody (Abcam, Cambridge, UK) were 5 μg/mL. The relative amount of each target protein was normalized to ACTB.

Cells were washed three times with pre-cooling PBS and then suspended in a cell lysis buffer (Cat No: P0013, Beyotime, Shanghai, China) with protease inhibitors. Protein concentrations were quantified using the Bradford Protein Assay Kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. For the assay, 40 μg of protein per sample was prepared for the following steps, including gel electrophoresis, transferring to polyvinylidene difluoride membrane, blocking for 1 h at 25°C with 5% nonfat milk, binding with antibodies, and detecting with an ECL detection system (Applygen Technologies, Beijing, China).
The following antibodies for the Western blot analysis were used: the anti-p53 antibody (Abcam, United States; ab1101, dilution: 1:5,000); antibody against TYMS (ABclonal, Wuhan, China, A10441, and dilution: 1:2000); anti-TCF21 antibody (ABclonal; A17451, dilution: 1:2000); anti-CD44 antibody (ABclonal; A1351, dilution: 1:2000); anti-cleaved PARP1 (ABclonal; A0942, dilution: 1:1,000); and anti-cleaved caspase 3 (Cell signaling technology, Danvers, MA, United States; #9664S, dilution: 1:1,000). The anti-RPS18 antibody was used for internal control protein (ABclonal; A11687, dilution: 1:1,000).

The protein was extracted from the cultured cells, and homogenized in RIPA lysis buffer (Beyotime, China) with Protease Inhibitor Cocktail (Sigma-Aldric,USA) on ice. Thirty micrograms of protein from each cell lysate were separated by SDS-PAGE using a 10% gel, and then electrophoretically transferred to a PVDF membrane (Millipore, MA, USA). Blots were probed with appropriate primary antibodies at 4°C overnight, and then incubated with an HRP-conjugated secondary antibody (Cell Signaling Technology, USA) for 1 h at room temperature. Protein bands were visualized using an Immobilon^TM Western HRP substrate (Millipore, USA). The antibodies used in western blot are list following: Anti-Dnmt1 antibody, Anti-p53 antibody, Anti-p21 antibody (Abcam, Cambridge, UK), Anti-p16 antibody, Anti-ZEB1 antibody Anti-β-actin antibody (SAB, USA). Image J software was used for quantitative analysis of scanning densitometric values of proteins as ratios to β-actin, which was used as a loading control.

To investigate the effects of fig latex on HPV oncoproteins (E6 & E7), tumor suppressor proteins (P53 & Rb) and HPV infection surrogate marker (P16), standard semi-dry western blotting technique was used. Cells were treated with concentration 0.125 $\mu$ g/ml of fig latex for 48 hrs then proteins were extracted using RIPA buffer (Life Technologies, UK) and equal amounts of protein were electrophoresed. Membranes were blocked with 5% (w/v) non-fat dry milk in TBS-T (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 1% (v/v) Tween20) at room temperature for 2 hours to reduce non-specific binding and incubated with the mAb Anti-HPV16 E6 + HPV18 E6 antibody (1:500; Abcam), mAb Anti-HPV16 E7 (1:500; Abcam) + HPV18 E7 (1:750; Abcam) antibody, Anti P53 antibody (1:500; Abcam), Anti Rb Antibody (1:1000; Abcam) Anti p16 antibody (1:750; Abcam) followed by Donkey anti-mouse (1:10000; Abcam) and actin antibody (1:10000). The membrane was then visualised by OdysseyCLx Imaging System (Li-COR).

Total protein was extracted from RA FLS. A BCA kit (Beyotime) was used for evaluating the total protein concentration. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Millipore). After blocking, the membranes were probed with primary antibodies and then incubated with specific horseradish peroxidase-conjugated secondary antibodies (Bio-Rad). Immunodetection was performed using an enhanced chemiluminescence kit (Thermo Fisher Scientific). GAPDH was used as a loading control for internal correction. The primary antibodies included an anti-proliferating cell nuclear antigen (PCNA) antibody (Abcam), an anti-cyclin D1 antibody (Abcam), an anti-IL-1 antibody (Abcam), an anti-IL-6 antibody (Abcam), an anti-MMP-3 antibody (Abcam), an anti-p53 antibody (Abcam), an anti-E2F1 antibody (Abcam), anti-ErbB1/2 antibodies (Abcam), and a GAPDH antibody (Sigma). The bands were exposed under x-ray using Gel Imager (Bio-Rad) (17 (link)).