Control mouse igg1

For AMPAR Co-IP 2 mg total protein was incubated with 4 μg of either mouse anti-GluA1 (Clone RH95; Millipore), mouse anti-GluA2 (Clone 6C4; Millipore), or control mouse IgG1 (Clone G3A1; Cell Signaling Technology) antibodies overnight at 4°C, with gentle rocking. Immune complexes were precipitated for 2 h at 4°C using 20 μl protein A/G agarose beads (Santa Cruz Biotechnology). For NSG2-mC Co-IP, 1.5 mg total protein was incubated with either 50 μl anti-RFP mAb-agarose (MBL International Corporation) or RFP-Trap_M (ChromoTek Inc.) according to manufacturer’s instructions. Agarose beads from the above reactions were washed and denatured in 50 μl 1% SDS, and 20 μl was loaded on an SDS-PAGE gel for Western blotting or 2 μl of the supernatant was subject to capillary electrophoresis.

Stromal CAFs were fixed in 1% formaldehyde for 10 min, followed by incubation in 1.25 M glycine for 5 min. Cells were lysed in IP lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% NP40, pH 7.4) supplemented with protease inhibitor cocktail for 20 min on ice. Following optimised sonification, the cell lysates were clarified and precleared with Protein A/G agarose beads and salmon sperm DNA (Thermo Fisher, #15632011) for 1 h at 4 °C. The precleared cell lysates were incubated with the anti-p53 antibody (Cell Signaling, #2524, 1:200) or control mouse IgG1 (Cell Signaling, #5415, 1:200) at 4 °C overnight on a rotator, followed by incubation with precleared Protein A/G agarose beads and salmon sperm DNA (Thermo Fisher, #15632011) for 1 h at 4 °C. The immunocomplexes were sequentially washed with low-salt wash buffer once, high-salt wash buffer once and TE buffer twice and eluted with elution buffer (1% SDS, 0.1 M NaHCO₃) twice. The eluted DNA–protein complexes were incubated with 0.2 M NaCl overnight at 65 °C, RNase A for 30 min at 37 °C and proteinase K (Thermo Fisher, #AM2548) for 1.5 h at 45 °C. The bound DNA was purified using a kit (Sigma-Aldrich, #NA1020) according to the manufacturer’s instructions, and then subjected to PCR or qPCR analysis (primer sequences are shown in Supplementary Table 1).