Anti p50

The Magna ChIP^TM HiSens Chromatin Immunoprecipitation Kit (Cat. No. 0025; Millipore) was used to perform the ChIP assay according to the manufacturer's recommendations. Briefly, ≈2 × 10⁶ primary CFs cultured in a 10 cm petri dish were used per immunoprecipitation sample. CFs were fixed for 10 min at room temperature (23–27 °C) and sonicated on a Bioruptor UCD‐200TM‐EX for 16 cycles of 30 s ON and 30 s OFF at high power. DNA bound to NKRF, p65, and p50 was precipitated using anti‐NKRF (Cat. No. 14693‐1‐AP; Proteintech), anti‐p65 (Cat. No. ET1603‐12; HUABIO, Hangzhou, China), and anti‐p50 (Cat. No. 14220‐1‐AP; Proteintech). Rabbit IgG (Cat. No. 2729; Cell Signaling Technology) was used as a control. The precipitated DNA was subjected to PCR following agarose gel electrophoresis and RT‐PCR to calculate the fold enrichment of ChIP DNA using specific HuR promoter primers. These primer sequences were listed in Table S2 (Supporting Information).

For the Co‐IP assay, post‐treatment CFs were lysed in IP buffer (150 mM saline, 50 mM Tris‐HCl, 1% NP‐40, pH 7.8) containing mammalian cell‐specific protease inhibitor cocktail (Merck, Darmstadt, Germany). The concentration of the extracted protein was measured using a BCA Protein Assay Kit (Thermo Fisher Scientific) and adjusted to similar concentrations (across extracts) using the extraction reagent. Briefly, 1 mg protein was incubated with magnetic protein A/G beads (Cat. No. HY‐K0202; MedChemExpress, Shanghai, China) precoated with 5 µg of rabbit IgG (Cat. No. 2729; Cell Signaling Technology), anti‐NKRF (Cat. No. 14693‐1‐AP; Proteintech), anti‐p65 (Cat. No. ET1603‐12; HUABIO), or anti‐p50 (Cat. No. 14220‐1‐AP; Proteintech) antibody at 4 °C overnight with constant rotation. Then, the beads were washed five times with PBST (136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na₂HPO₄, 1.76 mM KH₂PO₄, and 0.5% Tween 20). The precipitated proteins were eluted from the magnetic beads using 2x loading buffer (Cat. No. P0015F; Beyotime) and boiled for 5 min. Immunoprecipitated proteins were subjected to western blot analysis.