Anti pcna

Proteins in cells were extracted using RIPA lysis buffer (Beyotime), and concentrations were determined using a protein assay kit (Beyotime). Protein bands were scanned using the Odyssey Infrared scanning system (Li-Cor, Lincoln, NE, USA). The following antibodies were used: anti-PCNA, anti-PTK2 (Abclonal), anti-SRSF1, anti-MMP2, and anti-MMP9 (Santa Cruz Biotechnology). RIP assays were performed using the BersinBio^TM RNA Immunoprecipitation (RIP) Kit (BersinBio), with anti-SRSF1 (Santa Cruz Biotechnology), anti-FLAG (Abclonal), and appropriate control IgG (BersinBio) antibodies. Subsequently, qRT-PCR and agarose gel electrophoresis assays were performed. IHC was performed using antibodies against PCNA, Ki-67, PTK2, and MMP2 (Abclonal). IF was performed using an antibody against SRSF1 (Santa Cruz Biotechnology). Images were taken using a microscope (Leica Microsystems). All assays were performed according to the manufacturer’s protocol. The catalog numbers for all antibodies used were provided in Table S2.

qRT-PCR analysis was performed in 10 μl reaction volumes containing 1 μl of cDNA, 0.5 μl of forward and reverse primers, 5 μl of TB Green^TM premix (Takara), and 3 μl of DNase/RNase Free deionized water (Tiangen, Beijing, China). The relative expressions of related genes were calculated by the 2^–ΔΔCt method, and three biological replicates were performed on each sample. The proteins were extracted on ice using commercial protein extraction kits (BestBio Biotech Co., Ltd., Shanghai, China) and adjusted to the same concentration, then placed at 95°C to denature for 5 min. The total volume of each well was 20 μl, including 16 μl of protein sample and 4 μl of reducing loading buffer (4:1). The steps and details of the Western blot analysis experiment are described in depth by Liu et al. (2019) (link). The antibodies were diluted according to the manufacturer’s instructions as follows: anti-Bcl2 (Santa Cruz Biotechnology, Santa Cruz, CA, United States), anti-Caspase 3 (Biorbyt, Cambridge, United Kingdom), anti-CDK2 (ABclonal Technology, Wuhan, China), anti-PCNA (ABclonal), and anti-CLF2 (ABclonal, Wuhan, China). The last, anti-β-actin (ZenBio, Chengdu, China; 1:2,000), was used as a loading control.

The proteins from tissues and GC cells were extracted using cell lysis buffer (Beyotime, Shanghai, China) and rationed by BCA Reagent kit (Beyotime, Shanghai, China). Protein (50 µg) was loaded into 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA, USA). The PVDF membranes were blocked using Tris-buffered saline with Tween 20 (TBST) with 5 % nonfat milk at room temperature for 2 h. Membranes were then washed with phosphate-buffered saline with Tween 20 (PBST) and incubated overnight at 4 °C with the corresponding primary antibodies: anti-DBF4 (1:2,000; ABclonal, Wuhan, China), anti-cyclin-A (1:2,000; ABclonal, China), anti-PCNA (1:2,000; ABclonal, China), and anti-β-actin (1:5,000; ABclonal, China). Membranes were washed and then incubated with matched secondary antibodies conjugated with horseradish peroxidase (1:5,000; ABclonal, China) for 1 h at room temperature. Membranes were visualized using an enhanced chemiluminescence (ECL) kit (Thermo Fisher Scientific, USA). β-actin was used as a loading control.

BC cells were collected after transfection. To get protein lysates, RIPA lysis buffer (Beyotime, China) together with 1 M PMSF (Beyotime, China) were added into collected cells. The concentration of total protein was determined by BCA protein assay kit (Beyotime, China). After separation by 10% sodium dodecyl sulfate-polyacrylamide gels, proteins were transferred to nitrocellulose membranes (Beyotime, China). The membranes were blocked with 5% non-fatty milk for 1 h at room temperature and then immunoblotted with primary antibodies at 4 °C overnight. Blots were washed with PBST (PBS together with 0.1% Tween 20) and incubated in secondary antibodies at room temperature for 1 h. After washing the membranes with PBST, the bands were detected by an Odyssey Infrared scanning system (LI-COR Biosciences, USA) and then analyzed with Image Studio. The primary antibodies and their dilutions were as follows: anti-PCNA (1:1000, Abclonal, China), anti-STAT1 (1:500, Wanleibio, China) and anti-ACTIN (1:10,000, Abclonal, China).

The total proteins were extracted from proliferating or differentiating MuSCs (1 × 10⁶ cells per well) transfected with an AgomiR-145-3p, miR-145-3p mimics, LNAantimiR-145-3p, or miR-145-3p inhibitor with their respective controls, using protein lysis radioimmunoprecipitation assay (RIPA) buffer containing 1 mM PMSF (Solarbio, Beijing, China) 72 h post-transfection. Subsequently, proteins in the supernatant were separated by SDS-PAGE, and then transferred to 0.2 mm polyvinylidene fluoride (PDVF) membranes and sealed with 5% skim milk for 2 h at room temperature. After, the membranes were incubated with primary antibodies specific for anti-COX1, anti-MYBL1, anti-PCNA, anti-Pax7, anti-beta-tubulin, anti-Myomaker, anti-MyoD, anti-MyHC, and anti-MyoG (ABclonal Biotechnology Co., Ltd., Hubei, China) at 4 °C overnight. The PVDF membranes were washed with Tris saline with Tween (TBST) buffer 3 times and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2.5 h at room temperature. The enhanced chemiluminescence luminous fluid (ECL) was applied for color development.