A23910

Total proteins were extracted using RIPA (Beyotime, Shanghai, China) with 1 mM PMSF (Beyotime, Shanghai, China). Nuclear and cytoplasmic proteins were extracted using Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China). Western blotting was conducted following previous procedures [21 (link)]. Antibodies used for western blotting and their dilutions were as follows: anti-DHX15 (1:1000, sc-271686, Santa Cruz Biotechnology, USA), anti-GFP (1:1000, sc-9996, Santa Cruz Biotechnology, USA), anti-NF-κB p65 (sc-8008, Santa Cruz Biotechnology, USA), anti-p-NF-κB p65 Ser 536 (1:1000, sc-136548, Santa Cruz Biotechnology, USA), anti-β-Actin (1:1000, sc-47778, Santa Cruz Biotechnology, USA), anti-cyclin D1 (1:1000, 2978s, CST, USA), anti-Lamin A/C (1:1000, 10298-1-AP, proteintech, USA), Dylight 800-goat anti-rabbit IgG (1:2000, A23920, Abbkine, USA), and Dylight 800-goat anti-mouse IgG (1:2000, A23910, Abbkine, USA).

RIPA buffer (cat. no. R0010; Solarbio) was used to lyse cells, and protein concentrations were determined with a BCA protein assay kit (Beijing Leagene Biotech Co., Ltd.). Protein samples (25 µg/lane) were separated by SDS-PAGE on 10% gels and transferred onto PVDF membranes. The membranes were blocked by protein-free rapid block buffer (Epizyme Pharmaceutical Biotechnology Co, LTD) for 15 min at room temperature and were incubated overnight at 4˚C with primary antibodies against AGPS (1:500, sc-374201, Santacruze), MDM2 (1:500,sc-965, Santacruze), PMP70(1: 1000, ab3421, Abcam), Flag (1: 1000, 201,126-3A6, ZENBIO), c-Myc (1: 1000, sc-40, Santacruze), TrkA (1:1000,2510,CST), p-TrkA (1:1000,PA5-104,674, ThermoFisher), and β-tubulin (1:1000, ABL1030,Abbkine). Following primary antibody incubation, the membranes were further incubated with Dylight 800-Goat Anti-Rabbit IgG (1:100, A23910 Abbkine) or Dylight 800-Goat Anti-Mouse IgG secondary antibodies (1:100, A23920 Abbkine) at 25˚C for 1 h. The membranes were then scanned by an imaging system (ODYSSEY ® CLx, Gene Company limited), and the optical density was measured using Image Studio Lite (LI-COR Biosciences).