Ab10484

Cell or tissue lysates were prepared in RIPA buffer (50 mM Tris-HCl [pH 8 (link)], 150 mM NaCl, 0.1% SDS, and 0.5% Na deoxycholate, 1% NP40 and fresh proteinase inhibitor cocktail). Protein extracts were analyzed by Western blotting according to standard protocols using primary antibodies specific for p53 (CM5, Leica Microsystems), P-S15p53 (9284, Cell Signaling Technologies), p21 (SX118, Santa Cruz), PUMA (p4743, Sigma), ATM (ab2631, Abcam), phosphor-ATM (ab81292, Abcam), Kap1 (ab10484, Abcam), phosphor-Kap1 (ab70369, Abcam), Slc7a11 (ab37185, Abcam) and β-actin (A3853, Sigma). HRP-conjugated anti-rabbit,-mouse and -rat secondary antibodies (GE Healthcare) were used and signal was detected using an ECL Western blotting detection system (GE Healthcare).

Whole-cell lysates were prepared by lysing the cells with radioimmune precipitation assay (RIPA) buffer (Sigma-Aldrich, St. Louis, MO, USA) completed with Complete Protease Inhibitor Mixture (Roche) according to the manufacturer’s protocol. Protein lysates were stored in −80 °C for further analyses. Protein concentration was measured with PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), and then samples were subjected to SDS-PAGE (using Mini-PROTEAN^® Tetra Cell System, Bio-Rad) followed by immunoblotting with antibodies for TRIM28 (ab10483 or ab10484, Abcam, Cambridge, UK) and GAPDH (ab9485, Abcam). The blots were visualized using an enhanced chemiluminescence detection kit (ECL-Plus, Amersham Biosciences, Little Chalfont, UK) and a G:BOX F3 Gel Documentation System (Syngene, Bangalore, India).

HEK293T cells (4 × 10⁵) were lysed in 100 µL Passive Lysis Buffer (Promega). Cell lysates (10 µL) were boiled with 2.5 µL 4× gel loading buffer for 5 min at 98 °C and run on a NuPAGE 4–12% Bis-Tris polyacrylamide gel for 45 min at 200 V. Gels were blotted by using an iBlot Gel Transfer Device and Transfer Stacks (ThermoFisher). Blots were blocked with 5% milk in PBS solution for 1 h at room temperature and incubated with primary antibody overnight at 4 °C. Rabbit anti-KAP1 antibody (ab10484; Abcam) was diluted 1:10,000, whereas rabbit anti-actin antibody (ab219733; Abcam) was diluted 1:2,000. Blots were incubated with fluorescent secondary antibodies for 30 min at room temperature. Secondary anti-rabbit antibodies were diluted 1:10,000. Blots were imaged by using an Odyssey CLx gel scanner (LI-COR Biosciences).

MEFs were lysed in RIPA buffer with protease and phosphatase inhibitors and lysates were processed using standard methods. We used the following primary antibodies: VHL (sc-5575, 1:250; Santa Cruz), PBRM1 (A301-590A, 1:1000; Bethyl Laboratories), alpha-Tubulin (T5168, 1:2000; Sigma), RAD51 (sc-8349, 1:100; Santa Cruz), RPA32 (A300-244A, 1:500; Bethyl Laboratories), pATM (ab36810, 1:1000; Abcam), ATM (ab85213, 1:1000; Abcam), pDNA-PKc (ab18192, 1:1000; Abcam), DNA-PKc (ab70250, 1:1000; Abcam), pCHK1 (#2348 S, 1:500; Cell Signalling), CHK1 (#2360, 1:1000; Cell Signalling), H3K9me3 (ab8898, 1:1000; Abcam), H3K27me3 (ab6002, 1:1000; Abcam), H3K9me2 (ab8898, 1:1000; Abcam), H4K20me3 (ab9053, 1:1000; Abcam), HP1a (ab77256, 1:1000, Abcam), KAP1 (ab10484, 1:1000; Abcam) and H3 (4499, 1:2000; Cell Signalling). Secondary antibodies were conjugated to IRDye 680 or 800 (Li-Cor). Fluorescent signals were imaged using the Odyssey Infrared Imaging System (Li-Cor). Western blot band quantifications were performed with Fiji^{58 (link)}. Uncropped scans of presented blots are shown in Supplementary Fig. 7.

Cells for immunofluorescence observation were fixed in 4% PFA (Affymetrix, 19943 1 LT) at room temperature for 15 min and further treated with 0.2% triton for 10 min. They were then blocked by 3% BSA (SIGMA, A-7030) at room temperature for 1 h. Primary antibody for RAD51 (ab63801, Abcam, 1:200)), RAD52 (sc-365341, Santa Cruz, CA, USA, 1:500), TRIM28 (ab10484, Abcam, 1:500) and γH2AX ser139 (JBW301, 05–636, EMD Millipore, 1:200) were incubated with cells at 4°C overnight. After washing with 0.05% PBS-Tween (PBST), the cells were incubated with secondary antibody for 1 h at room temperature.
For m⁵C staining using the heat method, the cells or tissues were fixed and permeabilized in a 35 mm glass-bottom dish using a standard protocol, incubated in buffer (10 mM Tris-HCl, 2 mM EDTA, pH 9), and steamed on a 95°C heating block for 20 min to expose the antigen. The dish was cooled, washed three times with PBS and blocked using 5% BSA in 0.1% PBST for 0.5 h at room temperature. Primary antibody for m⁵C (33D3, ab10805, Abcam) and secondary antibody were diluted in the same buffer (5% BSA in 0.1% PBST) and followed the standard IF protocol. This protocol is modified from the classic heat-induced antigen retrieval method for paraformaldehyde-fixed tissues using Tris-EDTA buffer.

Whole cell lysates were prepared by lysing the cells with radioimmune precipitation assay (RIPA) buffer (Sigma) plus Complete Protease Inhibitor Mixture (Roche Applied Science, Indianapolis, IN, USA) and were subjected to SDS-PAGE followed by immunoblotting with antibodies for TRIM28 (ab10483 or ab10484, Abcam, Cambridge, MA, USA) and β-actin (ab75168, Abcam). The blots were visualized using an enhanced chemiluminescence detection kit (ECL-Plus, Amersham Biosciences, Piscataway, NJ, USA) and a G:BOX F3 Gel Documentation System (Syngene). The results of the western blot analyses shown in this report are representative of four independent experiments.

Cell or tissue lysates were prepared in RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% SDS and 0.5% sodium deoxycholate, 1% NP40 and fresh proteinase inhibitor cocktail). Protein extracts were analysed by western blotting according to the standard protocols using primary antibodies specific for PAXX (ab126353, Abcam, 1:1,000 dilution), XLF (A300-730A-1, Bethyl Laboratories, 1:1,000 dilution), Flag (M2, Sigma-Aldrich, 1:10,000 dilution), KAP1 (ab10484, Abcam, 1:1,000 dilution), phospho-KAP1 (ab70369, Abcam, 1:1,000 dilution). HRP-conjugated anti-rabbit and mouse secondary antibodies (GE Healthcare) were used and signal was detected using an ECL western blotting detection system (GE Healthcare). Uncropped images of all blots are available in Supplementary Fig. 6.