Ab170904

The protein expression levels were measured by western blot. In brief, total protein was obtained using the specific protein extraction kit (BestBio Institute of Biotechnology, Wuhan, China). The amounts of total protein were quantified by the BCA assay (Keygen Institute of Biotechnology, Nanjing, China). Protein was resolved by 6–15% SDS/PAGE and transferred onto poly(vinylidene difluoride) membranes (EMD Millipore, Billerica, MA, USA), then blocked with 5% nonfat milk. Primary antibodies, including P‐gp (1 : 1000; ab170904; Abcam, Cambridge, UK), STAT3 (1 : 1000; ab76315; Abcam), IL‐8 (1 : 1000; ab18672; Abcam), IL‐23 (1 : 1000; ab45420; Abcam), VEGF (1 : 1000; ab46154; Abcam), p‐STAT3 (1 : 1500; 9145; Cell Signaling Technologies, Danvers, MA, USA), MRP1 (1 : 1000; 72202; Cell Signaling Technologies), IL‐1β (1 : 1000; 12703; Cell Signaling Technologies) and p‐NF‐κB (1 : 800; sc‐136548; Santa Cruz Technology, Santa Cruz, CA, USA), were added and incubated overnight at 4 °C. The anti‐IgG secondary antibodies (ab205718, ab190475; Abcam) were subsequently applied to the membranes and incubated for 2 h. Immunoreactive signals were revealed by the enhanced chemiluminescence detection system (GE Healthcare, Muenchen, Germany). imagej software (National Institute of Health, Bethesda, MD, USA) was applied to analyze protein expressions.

The total protein of tissues or cells was extracted with the radioimmunoprecipitation assay (RIPA) lysis buffer (Solarbio Science Technology) in accordance with the instructions. Protein concentration was determined using the bicinchoninic acid (BCA) kit. The proteins were transferred to the polyvinylidene fluoride (PVDF) membrane and then blocked with non‐fat milk for 1 hour at room temperature. The member was incubated with the primary antibodies (anti‐FOXO3, Abcam, ab47285, anti‐ABCB1, Abcam, ab170904, 1:1000). The ImageJ software was used for protein quantification analysis according to the ratio of the grey value of control GAPDH band.

Whole ovaries, livers, and kidneys and mitochondrial isolates were run on a western blot and incubated with mouse monoclonal anti-COX IV (abcam ab14744, 1:1000), mouse monoclonal anti-Actin (abcam ab8226, 1:1000), and rabbit monoclonal anti-P-gp (abcam ab170904, 1:1000) primary antibodies in 2% BSA overnight at 4 °C. Blots were labeled with corresponding rabbit anti-mouse or goat anti-rabbit HRP-conjugated secondaries, (1:3000) for 1 hour. HRP-labeled proteins were developed in ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA).

The transfected cells were washed and collected and a protein extraction kit (Beyotime) was used on ice to lyse the cells, followed by centrifugation to collect the supernatant. The protein samples were treated at high temperature, and then 10% SDS-PAGE was used for protein isolation. The protein was then used for transferring to the PVDF membrane (Millipore, Billerica, MA, USA). The PVDF membrane was then sealed with 5% BSA, and was incubated with primary antibodies and second antibody successively. Finally, chemiluminescence reagent (ECL, Beyotime) was applied to develop the PVDF membrane. Primary and secondary antibodies were bought from Abcam (Cambridge, UK), including multidrug resistance mutation (MDR1, 1:1,000, ab170904), multidrug resistance-associated protein 1 (MRP1, 1:1,000, ab233383), GAPDH (1:2,000, ab8245), anti-vascular endothelial growth factor A (VEGFA, 1:1,000, ab52917), anti-HMGA1 (1:1,000, ab129153), anti-β-actin (1:2,000, ab179467), and horse radish peroxidase-conjugated secondary antibody (1:5,000, ab288151).

Total protein was extracted, separated and transferred to membranes, which were then blocked with 5% skim milk. Next, the protein‐loaded membrane was probed with antibodies against APE1 (ab92744, 1:500, Abcam), MDR1 (ab170904, 1:500), MRP (ab261871, 1:500), LRP (ab273093, 1:500), ABCG2 (ab207732, 1:500), STAT3 (ab68153, 1:500, Abcam), p‐STAT3 (ab267373, 1:1000, Abcam), Bcl‐2 (ab196495, 1:1000, Abcam), Bax (ab53154, 1:500, Abcam), cleaved caspase‐3 (ab2302, 1:1000, Abcam) and β‐actin (ab8227, 1:500, Abcam) and then with HRP‐labelled anti‐rabbit IgG secondary antibodies (No. 7074, 1:5000; CST, Danvers, MA) at 37°C for 1 h. The blots were visualized via an ECL reagent (32106, Thermo Fisher Scientific), normalized to β‐actin.

HT29 cells were lysed and centrifuged. The protein content was measured using the BCA kit (Beyotime Institute of Biotechnology, Jiangsu, China), and the concentration was calculated. SDS-PAGE electrophoresis was performed and the proteins were transferred to the nitrocellulose membrane. The membrane was then stained with a 2% Ponceau dye solution. After washing, the membrane was blocked. Then, primary antibodies TRIM29 (ab108627, 1 : 5000, Abcam), MDR1 (ab170904, 1 : 2000, Abcam), P53 (60283-2-Ig, 1 : 5000, Proteintech), HA (ab1424, 1 : 7000, Abcam), Flag (ab205606, 1 : 10,000, Abcam), and β-actin (66009-1-Ig, 1 : 5000, Proteintech) were added, with an incubation time of 2–4 hours at room temperature. After washing, a horseradish peroxidase-labeled secondary antibody HRP-goat anti-mouse IgG (SA00001-1, 1 : 5000, Proteintech) or HRP-goat anti-rabbit IgG (SA00001-2, 1 : 6000, Proteintech) was added, with an incubation time of 1 hour at room temperature. ECL chemiluminescence was utilized for visualization.