Anti collagen 1

The detailed protocol for western blotting has been previously described (Zhang et al., 2020 (link)). Briefly, after transferring the kidney total protein to the poly-vinylidene difluoride membrane, the membrane was incubated overnight with primary antibodies. Followed by the corresponding secondary antibodies for 1 h at room temperature, signals were captured and visualized using enhanced chemiluminescence. The ratio of the protein of interest was subjected to α-tubulin and was analyzed by ImageJ version 1.46 (National Institutes of Health, United States). The following antibodies were used: anti-POLR2I (1:500; Santa Cruz Biotechnology, United States), anti-collagen I (1:500; Proteintech, United States), anti-α-SMA (1:1,000; Proteintech, United States), anti-α-tubulin (1:1,000; Proteintech, United States).

Protein isolation and Western blotting were carried out as described previously [35 ]. The following primary antibodies were used: anti-SIRT4 (1:1,000, Invitrogen, # PA5-114377), anti-α‐SMA, anti-collagen I and anti-‐actin rabbit monoclonal antibodies (mAbs, 1:1,000; Proteintech). The secondary antibodies, including HRP‐conjugated goat anti‐rabbit immunoglobulin G (IgG) or goat anti‐mouse IgG (Cell Signaling Technology, MA, United States), were diluted 1:2000.

Cell lysates and exosomal lysates were subjected to SDS‐PAGE. Western blot analyses were performed by using anti‐collagen I (1:1 000; 14695‐1‐AP; Proteintech), anti‐α‐smooth muscle actin (SMA) (1:1 000; 14395‐1‐AP; Proteintech), anti‐glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (1:1 000; 14497‐1‐AP; Proteintech), anti‐immunoglobulin heavy chain binding protein (BIP) (1:1 000; 11587‐1‐AP; Proteintech), anti‐spliced X‐box binding protein 1 (XBP1s) (1:1 000; #12782; Cell Signaling Technology), and anti‐phospho eukaryotic initiation factor 2 (eIF2) α (P‐eIF2α) (1:1 000; #3398; Cell Signaling Technology) antibodies and corresponding HRP‐conjugated secondary antibodies. A chemiluminescent system (Amersham ECL Plus) was used for detection.

Expressions of Collagen I, Collagen III, CTGF, Fibronectin, α-SMA, MMP-9, and TIMP-1 proteins were determined with using immunofluorescent staining. To observe hEnSCs or uterine fibrosis and the ratio between MMP-9 and TIMP-1, the slides were washed 3 times with PBS, after which they were embedded in PBS containing 5% donkey serum for 30 min (Santa Cruz Biotechnology, USA). The hEnSCs and uterine sections were incubated with primary antibodies consisting of anti-Collagen I (1 : 100; Proteintech, China), anti-Collagen III (1 : 100; Proteintech, China), anti-CTGF (1 : 100; Proteintech, China), anti-Fibronectin (1 : 200; ABclonal, China), anti-α-SMA (1 : 100; Abcam, UK), anti-MMP-9 (1 : 200; Affinity, China), or anti-TIMP-1 (1 : 200, Affinity, China) at 4°C overnight. On the following day, the hEnSCs or uterine sections were maintained at room temperature for 1 hour and then incubated with secondary antibodies consisting of goat anti-rabbit IgG, Alexa Fluor 549 (Invitrogen, USA), or a mixture of goat anti-rabbit IgG and Alexa Fluor 549 and goat anti-mouse IgG and Alexa Fluor 488 (Invitrogen, USA). The hEnSCs or uterine sections were then incubated with DAPI (Solarbio, China) at room temperature and away from light for 10 min. The staining of hEnSCs or uterine sections was visualized using an inverted fluorescent microscope (Leica, Germany).

Total proteins were prepared using RIPA lysis buffer containing phosphatase inhibitors. The concentration was determined by BCA assay (Bioyear Biotechnology, Wuhan, China). Equal amounts of protein were subjected to 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk and incubated with the appropriate primary antibodies: anti-HE4 (1:1000, ABclonal Technology, Wuhan, China), anti-β-actin (1:4000, Proteintech, Wuhan, China), anti-p-p65 (1:1000, Cell Signal Technology, MA, USA), anti-p65 (1:4000, Cell Signal Technology, MA, USA), anti-Flag (1:4000, Sigma-Aldrich, St. Louis, MO, United States), anti-Fibronectin (1:1000, Proteintech, Wuhan, China), anti-collagen I (1:1000, Proteintech, Wuhan, China), anti-α-SMA (1:1000, Cell Signal Technology, MA, USA), anti-p-Smad2 (1:1000, Boster Biotech, Wuhan, China), anti-Smad2 (1:2000, Cell Signal Technology, MA, USA), anti-Survivin (1:2000, Proteintech, Wuhan, China), anti-PCNA (1:4000, Proteintech, Wuhan, China), anti-GAPDH (1:4000, Proteintech, Wuhan, China). After washing with Tris-buffered saline (TBS) containing 0.1% Tween-20, blots were incubated with secondary antibody conjugated to horseradish peroxidase (HRP; 1:4000, Proteintech, Hubei, People’s Republic of China). The intensity of individual bands was quantified using ImageJ.

Total protein was extracted from cultured cells or mouse hearts using RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with proteinase inhibitor cocktail (Roche Molecular Biochemicals, Mannheim, Germany) as previously described [22]. Protein concentration was measured using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Proteins were then separated via SDS/PAGE (10%) and electrotransferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA), incubated with the indicated antibodies, and developed using chemiluminescence methods. The following antibodies were used as follows: anticollagen I (1 : 1000; catalog number: 14695‐1‐AP; ProteinTech Group, Rosemont, IL, USA), anti‐α‐SMA (1 : 1000; catalog number: 14395‐1‐AP; ProteinTech Group, Rosemont, IL, USA), anti‐STAT3 (1 : 1000; catalog number: 9139; Cell Signaling Technology, Danvers, MA, USA), anti‐p‐STAT3 (1 : 1000; catalog number: 9145; Cell Signaling Technology, Danvers, MA, USA), and anti‐GAPDH (1 : 10 000; catalog number: 60004‐1‐Ig; ProteinTech Group, Rosemont, IL, USA).