Ab134056

HK-2 cells were divided into two groups: control group transfected with negative control shRNA, and another group interfered with endogenous PARP1 using shRNA vectors. The cells were placed on a 35 mm cell culture dish, PBS washed three times. They were then fixed with 4% cold paraformaldehyde for 20 min, followed by PBS washing three times. Permeabilization was performed using 0.2% Triton X-100 for 10 min, followed by PBS washing three times. Serum blocking was carried out for 30 min, followed by PBS washing three times. Cells were incubated overnight at 4 °C in the primary antibodies PARP1 (1:500, ab191217, Abcam, Cambridge, UK) and XRCC1 (1:500, ab134056, Abcam, Cambridge, UK), followed by PBS washing three times. The cells were then incubated with secondary antibodies Alexa Fluor 555 and Alexa Fluor 488 (Cell Signaling Technology, Beverly, MA, USA) for 2 h at room temperature (protected from light), or at 37 °C for 1.5 h, followed by PBS washing three times. Finally, DAPI was used to stain the nuclei, and fluorescence images were captured directly. PBS was washed off with distilled water, and the samples were mounted with glycerol and immediately imaged using an Olympus FLUOVIEW FV1000 confocal laser scanning microscope (Olympus Corporation, Tokyo, Japan) [35 (link)].

RIPA lysate (P0013C, Beyotime, Shanghai, China) containing PMSF was used to extract the total protein from the tissues, which was incubated on ice for 30 min and centrifuged at 4 °C for 10 min at 8000 g, followed by extraction of the supernatant. The total protein concentration was measured by BCA kits (23227, Thermo Fisher Scientific). The protein was subjected to SDS-PAGE, and transferred to a PVDF membrane, followed by blockade with 5% skimmed milk powder at room temperature for 1 h. Then, the PVDF membrane was incubated at 4 °C overnight with diluted primary antibodies against PARP1 (1:1000, ab191217, Abcam. Cambridge, UK), XRCC1 (1:1000, ab134056, Abcam) and GAPDH (ab181602, 1:2500, Abcam) and with HRP-labeled secondary antibody goat anti-rabbit IgG H&L (HRP) (ab97051, 1:2000, Abcam) for 1 h.
The ECL fluorescence assay kit (abs920, Absin Bioscience Inc.) was used for development. The protein bands were photographed using the Bio-Rad Image Analysis System (Bio-Rad Laboratories, Hercules, CA). Grayscale quantification was performed on each group of bands in the Western blot images using Image J analysis software (Version: V1.8.0), in which GAPDH was the internal reference protein [34 (link)].

Proteins were extracted using RIPA lysis buffer containing 1% PMSF and 1% cocktail protease inhibitor. The protein concentration was determined using the BCA assay kit (PC0020, Solarbio, China). The protein samples were separated on a 10% SDS-PAGE gel and then transferred onto a PVDF membrane. The membrane was subsequently incubated with primary antibodies, including FTO (1:2000, ab126605, abcam, UK), POLQ (1:1000, SAB1402530, Merk, Germany), CDC6 (1:2000, ab109315, abcam, UK), CDT1 (1:1000, ab202067, abcam, UK), FEN1 (1:2000, ab109132, abcam, UK), NBN (1:2000, ab32074, abcam, UK), and XRCC1 (1:2000, ab134056, abcam, UK). Subsequently, appropriate secondary antibodies (1:10,000, abcam, UK) were applied for 1 h. The protein bands were visualized using a chemiluminescence imaging system (5200, Tanon, China) for image analysis.