Anti myc

The following monoclonal mouse antibodies were used: anti-Rich2 (Cat# sc-390609, Santa Cruz Biotechnology, CA, USA), anti-β-tubulin (Sigma-Aldrich, Cat# SAB4200715, RRID: AB_2827403), and anti-Myc (Cat# M047-3, RRID: AB_591112; Medical & Biological Laboratories, Nagoya, Japan).

RIP assay was performed using Magna RIP Kit (17–701, Millipore) according to the instructions by the manufacturer. Briefly, 5 μg of anti-Myc (MEDICAL & BIOLOGICAL LABORATORIES CO., LTD.) or anti-Mouse IgG (Millipore) was incubated with 50 μL of magnetic beads before cell lysates were added (approximately 2 × 10⁷ cells per sample). Subsequently, RNAs were extracted after the removal of proteins and purified for qPCR analysis. The relative enrichment was normalized to the input as follows: %Input = 2^^{-(Ct [IP]–(Ct [input]-LOG2[10])}. The RIP-seq process and analysis were performed by SEQHEALTH Co., Ltd. (Wuhan, China). The raw data could be found in GEO (GEO accession number GSE241111).

Cells were harvested and extracted using RIPA buffer. Lung tissues from mice were homogenized in an appropriate amount of RIPA buffer. Total protein was quantified using a BCA (Bicinchoninic acid) protein assay reagent kit (Tiangen, China). Equal amounts of proteins were separated on a 10% SDS‐PAGE gel and transferred onto a PVDF membrane (PALL, USA). Membranes were incubated with anti‐Smad2/3, anti‐pSmad2/3 (Cell Signaling Technology, USA), anti‐Myc (Medical & Biological Laboratories Co, Japan), anti‐β‐actin (Santa Cruz Biotechnology, USA), and anti‐GAPDH (Santa Cruz Biotechnology, USA) overnight at 4°C. After three washes in 1 × TBST buffer, the blots were incubated with either horseradish peroxidase‐conjugated goat anti‐rabbit antibody or horseradish peroxidase‐conjugated goat anti‐mouse antibody (1:2000; Jackson Immuno Research, USA) for 1 h at room temperature, rewashed, and then developed using an ECL chemiluminescence kit (Thermo Fisher Scientific, USA).