Polyvinylidene difluoride pvdf membrane

We further analyzed the effects of Endostar on Wnt/β-catenin signaling pathway. HUVECs were incubated in ECM (5% FBS, 1% ECGS) with different concentrations of Endostar (50, 100 and 150 µg/ml) for 24 hours. The whole-cell extracts and the nuclear extracts were each prepared using RIPA buffer (Beyotime, China) and NE-PER nuclear and cytoplasmic extraction reagents (Thermo Scientific, USA) respectively. Proteins were separated by polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, USA). The membranes were incubated with primary antibodies anti-β-catenin, anti-VEGF, anti-cyclin D1, anti-β-actin and anti-Histone H3 followed by the addition of secondary antibody (Goat anti-rabbit or mouse IgG (H+L) antibody, KPL, USA). Protein bands were detected with an enhanced chemiluminescent substrate (EZ-ECL, Biological Industries, Kibbutz Beit-Haemek, Israel), scanned, and quantitated by a Bio-imaging analyzer (Bio-Rad).

The protein expression levels were analyzed by western blotting as described in our previous studies (Park et al., 2020 (link)). Cells were lysed in a buffer containing 50 mM Tris, 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% NP 40, and 0.2 mM PMSF. The protein concentrations of the total cell lysates were measured by using bovine serum albumin as a standard. Samples containing equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories). The membranes were blocked with 5% skim milk in Tris-buffered saline containing Tween-20 at RT. Then, the membranes were incubated with primary antibodies against MMP-2 (Cell signaling #4022), MMP-9 (Cell Signaling #13667), phospho-PI3K (Cell Signaling #4228), total PI3K (Cell Signaling #4292), phospho-Akt (Cell Signaling #4060), total Akt (Cell Signaling #4491) and β-actin (Abcam, MA, United States, ab189073) overnight at 4°C and then with polyclonal HRP-conjugated goat anti-mouse IgG (BD Pharmingen, 554002) or goat anti-rabbit IgG (BD Pharmingen, San Diego, CA, United States, 554021) secondary antibodies at room temperature for 60 min. The antigen-antibody complexes were detected using Western blot ECL reagents (GE Healthcare, Bucks, United Kingdom).

Whole protein was extracted from K562 cells using cell lysis Radioimmunoprecipitation assay buffer (RIPA buffer including 150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS (sodium dodecyl sulphate), 50 mM Tris-HCl, pH 8.0, Protease inhibitors). For BCL11A, nuclear protein was extracted by Nuclear Extraction Kit (Abcam, UK). Approximately 30 to 50 μg of protein from samples was denatured and separated by 10% or 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Polyvinylidene difluoride (PVDF) membranes (Bio-Rad, USA). After blocking by 5% BSA in TBST buffer (Tris-buffered saline, 0.1% Tween 20), immunoblotting was performed with rabbit antibodies against STAT3, AHSP, BCL11A (-XL, -L, -S), HBG globin chains, and β-actin (Abcam, UK). Horseradish peroxidase (HRP) conjugated secondary antibodies and Enhanced Chemiluminescence (ECL) kit (Amersham Biosciences, UK) were used for detection and visualization of protein levels.

The hippocampus tissue samples were homogenized with lysis buffer containing a cocktail of phosphatase and proteinase inhibitors and PMSF (Beyotime, Shanghai, China). Following denaturation, the lysates were separated on a 10% SDS-PAGE gel and transferred to polyvinylidene difluoride PVDF membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% nonfat powdered milk in TBST (Tris-buffered saline containing 0.1% Tween 20) for 1 h at room temperature (RT) and then incubated overnight at 4°C with a monoclonal rabbit anti-TLR4 (1 : 1000, Proteintech Group), anti-TLR2 (1 : 1000, Proteintech Group), or anti-GAPDH (1 : 5000, Proteintech Group) primary antibody. After washing in TBST, the membrane was incubated for 1 h at RT with a HRP-conjugated goat anti-rabbit antibody (1 : 10000; Jackson ImmunoResearch Laboratories, West Grove, PA, USA), and the protein bands were visualized using a Immun-StarTM HRP Chemiluminescence Kit (Bio-Rad). Images of the bands were recorded using the ImageQuant LAS 4000 system (GE Healthcare, Hino, Japan), and the band intensities were quantified using ImageQuant TL software (version 7.0, GE Healthcare). The amount of proteins was quantified (Quantity One, BioRad) and reported relatively to GAPDH. Three rats in each group were randomly selected for western blotting.