Anti sesn1

NRVMs were homogenized on ice in radioimmunoprecipitation assay lysis buffer (Santa Cruz Biotechnology, Dallas, TX) containing the protease inhibitors leupeptin (5 µg/mL), aprotinin (2 µg/mL), and phenylmethylsulphonyl fluoride (PMSF; 1 mM). Lysates were centrifuged at 12,000g for 20 min at 4 °C and the supernatants were collected. The protein concentrations were determined, and aliquots containing 50 mg of protein were separated by electrophoresis on 4–12% Blot Bis–Tris Gels (Invitrogen Japan) in a NuPAGE MOPS SDS Running Buffer system (Invitrogen Japan), and sequentially transferred onto nitrocellulose membranes, according to the manufacturer’s instructions. Proteins were blotted using iBind Western Systems (Thermo Fisher Scientific) and detected by chemiluminescence (#34096, Thermo Fisher Scientific; #NEL113001EA, PerkinElmer, Inc., Waltham, MA, USA; ImageQuant LAS500, GE Healthcare UK Ltd., Buckinghamshire, England). The following primary antibodies used in this study were: anti-Bax (#14796), anti-caspase3 (#14220), anti-Cleaved caspase3 (#9661; All of them; Cell Signaling Technology), anti-SESN1 (ab134091; Abcam, Cambridge, MA, USA), anti-p-mTOR (S2448), anti-mTOR (7C10), anti-LC3B (#2775), and anti-β-actin (All of them; Cell Signaling Technology).

Primary antibodies anti‐MYCN and anti‐SESN1 were purchased from Abcam (UK), and anti‐MyD88, anti‐GAPDH, and anti‐β‐Tubulin were purchased from Proteintech (Wuhan, Hubei, China). Cell Counting Kit‐8 (CCK‐8) was bought from Bimake (Shanghai, China). Matrigel was bought from Corning (NY, USA). HiSpeed Plasmid Maxi Kit was bought from Qiagen (Germany). jetPRIME was purchased from PolyPlus Transfection Co. (Illkirsch, France). TLR signaling pathway inhibitor Hydroxychloroquine‐d4 sulfate (HCQ) was purchased from MCE (USA).

The protein concentration in the supernatant was determined by BCA assay. Aliquots of 80 μg protein were separated in SDS-PAGE and transferred onto nitrocellulose membranes by an electric transfer (BIORAD Inc., USA). The membranes were blocked with 5% milk PBS-Tween20 (PBST) and incubated with primary antibody including: anti-Sesn1 (Abcam, USA), anti-Sesn2 (Santa Cruz Biotechnology, USA), anti-Sesn3 (Abcam, USA), anti-COL I (Abcam, USA), anti-COL III (Abcam, USA), anti-FN1 (Abcam, USA), and anti-Actin (Cell Signaling, USA) antibodies. They were then incubated with Alexa Fluor 700 antibodies (Molecular Probes, USA). Images were captured on an Odyssey Infrared Imaging System (LI-COR Biosciences, USA), and quantified using Odyssey v1.2.