Rabbit anti col 1

Total cellular proteins (20 μg from each sample) obtained above were separated by SDS-PAGE (Solarbio, Beijing, China) under reducing conditions and transferred onto a nitrocellulose membrane (Invitrogen, Shanghai, China). Membranes were blocked in 5% dry milk and probed overnight at 4 °C with gentle rocking with anti-Runx-2 (1:1000, Abcam, Cambridge, UK), rabbit anti-BMP-2 (1:1000, Abcam), rabbit anti-COL-1 (1:1000, Abcam), mouse anti-acetylated α-tubulin (1:1000, Abcam), rabbit anti-Dynlt1 (1:800, Abcam), rabbit anti-IFT88 (1:500, Santa Cruz Biotech, Dallas, Texas), or rabbit anti-GAPDH (1:800, Bioworld, Shanghai, China). After washes, membranes were incubated with a secondary antibody (HRP-conjugated goat anti-rabbit IgG or goat anti-mouse IgG, 1:10000, Bioworld). The proteins recognized by the antibody complexes were visualized by chemiluminescence (Solarbio, Beijing, China). The optical density of each maker band was measured using Image-Pro Plus 6.0 software.

Briefly, total protein was isolated from ground frozen atrium tissues and CFs using RIPA lysis buffer (Beyotime, China). The samples were clarified by centrifugation at 13 800 g for 15 minutes at 4°C. Protein concentrations were determined using the BCA Protein Assay Kit (Beyotime, China). A total of 20 μg of protein was separated on 10% SDS‐PAGE at 80‐100 V for 2 hours and transferred onto PVDF membranes (Millipore, USA) at 300 mA for 1.5 hours. The membranes were blocked with 5% non‐fat milk in Tris‐buffered saline with tween 20 (TBST) for 1 hour at room temperature. Each membrane was incubated with primary antibodies at 4°C overnight and then secondary antibodies at room temperature for 1 hour the next day. The following antibodies were used: rabbit anti‐TGFβRII (1:500, Santa Cruz), rabbit anti‐α‐SMA (1:1000, Abcam), rabbit anti‐Col I (1:1000, Abcam), rabbit anti‐vimentin (1:1000, Abcam), mouse anti‐Col3α1 (1:250, Santa Cruz) and mouse anti‐GAPDH (1:2000, Abcam) antibodies. All proteins were visualized using the ECL reagent Kit (Millipore Corp, USA). The values of the band intensities were quantified using Image J software.

Cells or tissues were homogenized in cold RIPA buffer containing protease and phosphatase inhibitors. The lysates were collected following centrifugation and protein concentration was determined using a BCA protein concentration determination kit (Beyotime; Cat# P0011). The supernatant was stored at −80 °C transferred to a polyvinylidene difluuntil use. The proteins were separated by electrophoresis and oride membrane (Millipore, Billerica, MA, USA). The membranes were blocked with fat-free dry milk and then incubated with indicted primary antibodies as follows: rabbit anti-Col I (Abcam; Cat# ab138492, 1:1000), rabbit anti-Col III (Abcam; Cat# ab184993, 1:1000), rabbit anti-SMA (Cell Signaling; Cat# 19245, 1:2000), rabbit anti-ALKBH5 (Proteintech; Cat# 16837-1-AP, 1:1000), rabbit anti-IL11RA1 (Abcam; Cat# ab125015, 1:1000), rabbit anti-IL11 (Proteintech; Cat# 55169-1-AP, 1:1000) and rabbit anti-GAPDH (Proteintech; Cat# 10494-1-AP, 1:5000). The immuno-positive bands were visualized by enhanced chemiluminescence reagent (Millipore, USA) following incubation with a secondary peroxidase-conjugated anti-rabbit (Beyotime; Cat# A0208, 1:10000) or anti-mouse (Beyotime; Cat# A0216, 1:10000) antibody. Image J (Vl.8.0) analysis was used to quantify the bands of western blot images and GDPDH was used as an internal reference.

pHLF cells were seeded (1 × 10⁴ cells/ml) into 24-well plates containing rounded glass cover slips. TGF-β1 (2 ng/mL) was added to each well, followed by the addition of medium containing TH5487, dexamethasone, nintedanib, medium only, or vehicle only. Following 24 h of treatment, cells were washed and fixed with ice-cold methanol, and then treated with 0.5% Triton X-100. Cells were blocked using Dako Protein Block (Agilent, CA, USA) for 1 h at room temperature and then primary antibodies, rabbit anti-COL I, rabbit anti-αSMA, rabbit anti-fibronectin, and rabbit anti-vimentin antibodies (Abcam, Cambridge, UK) were added for overnight. AlexaFluor 488-conjugated goat anti-rabbit secondary antibody (Invitrogen) was used. Glass cover slips were mounted onto glass slides, with nuclei counter-stained using DAPI-containing fluoroshield (Abcam). Images were visualized using a Nikon Confocal Microscope. Fluorescence was quantified using ImageJ software (Java 1.8.0_172).

Total proteins from cells or liver tissues were lysed in RIPA buffer, and protein concentration was determined by BCA assay. Proteins were separated by SDS-PAGE and then transferred to a PVDF membrane (Millipore, MA, USA) at 80 V for 2 h.
Membranes were blocked in 5% non-fat milk for 1 h and then incubated with primary antibodies for 16 h with diluted mouse anti-GAPDH (1:5,000; ptm-biolab, Hangzhou, China), rabbit anti-α-SMA (1:1,000, proteintech, Wuhan, China), rabbit anti-COL I (1:1,000, Abcam, USA), mouse anti- SMAD2 (1:1,000, ptm-biolab, Hangzhou, China), SMAD3 mouse monoclonal (1:1,000, proteintech, Wuhan, China). The membranes were incubated with the horseradish peroxidase-conjugated species-specific secondary antibody for 2 h and visualized using chemiluminescence reagent (Millipore, MA, USA).