0.45 μm pvdf membrane

Whole-cell lysates were prepared using RIPA containing protease and phosphatase inhibitors (Beyotime, Shanghai, China). The protein was quantified with BCA kit (Solarbio, Beijing, China), separated on a 10% SDS-polyacrylamide gel electrophoresis and transferred onto 0.45 μm PVDF membranes (Invitrogen, Carlsbad, USA). Membranes were blocked using 5% bovine serum albumin for 1 h at room temperature and incubated with primary antibodies at 4 °C overnight After washed three times by TBST for 10 min intervals, membranes were incubated with diluted secondary antibodies for 1 h at room temperature. The expression of proteins was assessed using an ECL kit (Tanon, Shanghai, China), and quantified by the image J software. The expression of β-actin did not change significantly either under hypoxia or after drug treatments, which was used as an internal parameter in our experiment accordingly.
The primary antibodies used were antibodies against α-ENaC (1:2000, Invitrogen, Carlsbad, USA), γ-ENaC (1:2000, Abcam, Cambridge, USA), β-actin (1:2000, Proteintech, Chicago, USA), HIF-1α (1:1000, Affinity Biosciences, Cincinnati, USA), ERK1/2, p-ERK1/2 (1:1000, Cell Signaling, Mass, USA), inhibitor κBα (IκBα), p-IκBα, p65, and p-p65 (1:1000, Abmart, Shanghai, China). The secondary antibodies used were goat anti-rabbit or goat anti-mouse antibodies (1:5000, ZSGB-bio, Beijing, China), respectively.

Cells were washed with PBS (Gibco) then harvested using lysis buffer (150 mM NaCl, 50 mM Tris (pH 8), 1% IGEPAL C360 and one tablet of protease inhibitors with EDTA (Roche) per 10 mL). Cell lysates were vortexed for 30 min at 4°C and then cleared by centrifugation at 15,000 g for 15 min, 4°C. Proteins were quantified by Bradford assay (Bio-Rad, Hercules, CA) using BSA as standard. Protein lysates were separated by SDS-PAGE and blotted onto 0.45 μm PVDF membranes (ThermoFisher Scientific, Rockford, IL). Membranes were immunoblotted as indicated and fat-free milk was used as blocking medium. Blots were quantified using the BioRad Image Lab (v5.2.1) software.

The expression levels of FA proteins were quantified using western blotting (WB) at room temperature (RT). Whole cell proteins were extracted using NuPAGE™ LDS Sample Buffer (Thermo Fisher, Waltham, MA, USA), loaded on Bolt™ Bis-Tris Protein Gel (Thermo Fisher), and blotted onto 0.45 μm PVDF membranes (Thermo Fisher). Proteins were quantified using a Qubit™ Protein Broad Range Assay and Qubit 4 Fluorometer (Thermo Fisher) following the manufacturer’s instruction. Membranes were finally incubated with primary antibodies: GAPDH (AM4300, Thermo Fisher), vinculin (ab129002, abcam), paxillin (ab32084, abcam), FAK (ab40794, abcam), and phospho Y397 FAK (ab81298, abcam) and secondary antibodies: Goat Anti-Rabbit IgG H&L HRP (ab205718, abcam) and Goat Anti-Mouse IgG H&L HRP (G21040, Thermo Fisher) using iBind Western System (Thermo Fisher). All membranes were imaged using a ChemiDoc Touch MP system (Bio-Rad Laboratories, Hercules, CA, USA). Bands were quantified using ImageJ, and statistical data were analyzed using GraphPad Prism 9.

After extraction of cell lysate and quantification of protein concentrations, 10% SDS-PAGE gels were used to separate proteins, and then they were transferred to 0.45 μm PVDF membranes (ThermoFisher, Waltham, MA, USA). After blocking the membranes with 5% non-fat milk, the membranes were incubated with primary antibodies of PLK1 (Abcam, Cambridge, UK, dilute 1 : 1000, ab189139) and GAPDH (Abcam, dilute 1 : 1000, ab9485) at 4 °C overnight, followed by incubation with a horseradish peroxidase-conjugated secondary antibody (Bioss, Beijing, China) at room temperature for 1 hour and visualization on a Tanon 5200 (Tanon, Shanghai, China).

Transfected SK-N-AS and HEK293 were harvested and lysed using RIPA-buffer supplemented with phosphatase- and protease- inhibitors (Thermo Fisher Scientific). Protein lysates (30 μg) were resolved on 4–20% precast gels (Bio-Rad Laboratories) and transferred onto 0.45 μm PVDF membranes (Thermo Fisher Scientific). Western blot was performed using antibodies with ECL-detection (Supersignal West Maximum Fempto, Thermo Fisher Scientific) as follows: FLAG-DDK M2 mouse mAb (1:750, #F3165, Sigma Aldrich), GAPDH rabbit Ab (1:500, sc-25778, Santa Cruz Biotechnology), MYO5B Rabbit mAb (1:250, HPA040902, Atlas Antibodies), transferrin receptor (CD71) rabbit mAb (1:500, #13208, Cell Signaling Technology). Chemiluminescent signal from membranes were imaged using a LAS-400 imaging system (Fujifilm). The experiment was performed twice and quantified using Image Studio Lite v5.2.5 (https://www.licor.com/bio/image-studio-lite/). GAPDH was used as a loading control.

Samples were resolved by SDS-PAGE and transferred to 0.45 μm PVDF membranes (Thermo Scientific, Rockford, IL). Blots were blocked and probed as previously described (Turkewitz et al. 1991 (link)). The rabbit anti-Grl1p, rabbit anti-Grl3p, rabbit anti polyG (Xie et al. 2007 (link)), and mouse monoclonal anti-GFP (Covance, Princeton, NJ) 1° antibodies were diluted 1:2000, 1:800, 1:10,000, and 1:5000, respectively. Protein was visualized with either ECL Horseradish Peroxidase-linked anti-rabbit (NA934), or anti-mouse (NA931) (Amersham Biosciences, Buckinghamshire, England) 2° antibody diluted 1:20,000, and SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, Rockford, IL).