Mini hd9

Cells were washed with cold Dulbecco’s phosphate buffered saline (DPBS, pH 7.4) and lysed with cold lysis buffer (150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 0.5% v/v NP-40, 5 mM NaF, 1 mM PMSF, 1 mM sodium orthovanadate and 1X Protease Inhibitor Cocktail) for 20 min at 4 °C. The lysates were centrifuged at 13,000× g at 4 °C. Proteins were quantified with Pierce^TM BCA protein kit according to the manufacturer’s instructions. Thirty to fifty micrograms of total proteins were separated in 12% polyacrylamide gels and transferred to a nitrocellulose membrane. Blots were blocked with 0.1% v/v Tween-20, 4% w/v non-fat milk in Tris-buffered saline (TBS: 50 mM Tris, pH 7.5, 150 mM NaCl) and then were probed with primary antibodies. Primary antibodies were detected with appropriate horseradish peroxidase-conjugated secondary antibodies. Secondary antibody was detected with SuperSignal^TM chemiluminescent substrate. Images were acquired with a miniHD9 (UVITEC, Cambridge, UK) chemiluminiscence photodocumentation system using NineAlliance software (UVITEC, Cambridge, UK). Protein levels were quantified by scanning densitometric analysis using UnScan it v6.1 software (Silk Scientific, Inc., Orem, UT, USA). Values obtained for proteins of interest were normalized to α-tubulin levels.

After 24 h of treatment, cells were lysed in a buffer (50 mM Tris–HCl pH 5.5, 150 mM NaCl, Triton X-100 0.5%) added with a complete protease inhibitor (Roche Applied Science). Equal amounts of proteins were denatured at 100 °C for 10 min and subjected to SDS-PAGE on 10% polyacrylamide gel. After the run, the proteins were electro-transferred on nitrocellulose membranes. Saturation of non-specific sites was performed for 2 h at room temperature in 5% milk in the TBST buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20). The membranes were exposed to primary antibodies directed against GAPDH (Santa Cruz sc-25778), α-SMA (Sigma A5228), VIMENTIN (VIM) (Santa Cruz 7557), FIBRONECTIN (FN) (Santa Cruz sc-9068) overnight at 4 °C and subsequently incubated with a secondary antibody conjugated with peroxidase for 1 h at room temperature. The signal was detected with Luminata™ Forte Western HRP Substrate (Millipore) according to the manufacturer’s instructions and the signal was acquired with Mini HD9 (UVItec, Cambridge). The band intensities were quantified using the UVItec Image Program.

After washing with PBS, cells were lysed with the pre-chilled lysis buffer. Later, the collected cell lysates were subjected to 15 min of centrifugation at 14,000 × g and 4°C and boiling with 5 × sample buffer (BSA; Thermo Fisher Scientific, Inc., CA, United States) after the protein content was measured. Afterward, WB assay was performed on those protein samples. To carry out WB, the 4%–20% precasting gel (Bio-Rad Laboratories) was used to transfer protein onto the nitrocellulose membranes, and then 5% skim milk was utilized to block the membranes (Bio-Rad Laboratories) for 1 h under ambient temperature. Later, specific antibodies (dilution, 1:1,000), including anti-SLC17A9, anti-VEGF, anti-Bax, anti-Ki67, anti-MTA1, anti-Bcl-2, anti-MMP-2, and GAPDH (Santa Cruz, CA, United States), were used to block membranes. Subsequently, secondary antibodies (Santa Cruz) were utilized to incubate membranes at ambient temperature for 1 h, followed by visualization using the ECL solution (Bio-Rad Laboratories). Finally, images were taken using the chemiluminescence imaging system (Mini HD9; UVitec, Cambridge, United Kingdom).

Total levels of total ubiquitinated proteins was determined by immunoblot assays as described (Cáceres et al., 2015 (link)) using anti-ubiquitin mouse monoclonal (Catalog number: AUB01, Cytoskeleton, Inc., Acoma ST, Denver, USA), anti-CUL3 rabbit polyclonal (Catalog number: TA326917, Origene) and anti-KCTD5 mouse monoclonal (Catalog number: TA501035, Origene) antibodies. Anti-Tubulin mouse monoclonal (Catalog number: T5168, Sigma-Aldrich, St Louis, Missouri, USA) and anti-CagA mouse monoclonal antibodies (Catalog number: sc-28368, Santa Cruz Biotechnology, Dallas, Texas, USA) were used as loading and H. pylori-infection controls, respectively. Immunoblots were visualized by Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific). Images were acquired with a MiniHD9 (UVITEC, Cambridge) chemoluminiscence photodocumentation system and quantified using NIH/ImageJ software.

The cells were washed once with phosphate‐buffered saline (PBS) and then lysed in cold lysis buffer [50 mm Tris/HCl (pH 7.4), 150 mm NaCl, 1% Triton X‐100, 0.1% SDS, 1 mm EDTA, 1 mm Na3VO4, 1 mm NaF, and 1X protease inhibitor cocktail). Cell lysates were centrifuged at 14 000 g for 15 min at 4 °C and boiled in 5X sample buffer after protein concentration assessment with a BSA kit (cat. no. 23208, Thermo Fisher Scientific, Inc.). Following protein sampling, nitrocellulose membranes (cat. no. 1620145, Bio‐Rad Laboratories), blocking reagent (5% skim milk, 1 h at room temperature), and a 4–20% precast gel (cat. no. 456‐1094, Bio‐Rad Laboratories) were used for western blot analysis with the following antibodies at dilutions of 1 : 1000: anti‐EHMT1 (A301‐642A, Bethyl Laboratories, Inc., Montgomery, TX, USA); anti‐PARP (cat. no. 9542, Cell Signaling Technology, Inc.); and anti‐p21 (sc‐6246) and anti‐β‐actin (sc‐47778) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Final incubation with secondary antibodies (rabbit: SC‐2357, mouse: SC‐2031, Santa Cruz Biotechnology, Inc.) was conducted at room temperature for 1 h, and ECL solution (cat. no. 170‐5060, Bio‐Rad Laboratories) was used for visualization. A chemiluminescence imaging system (Mini HD9, Uvitec, Cambridge, UK) was used for imaging.

After the treatment of cells with 6-OHDA and vitamin K2, respectively, cells were lysed with a lysis buffer (Beyotime) and centrifuged at 4 °C for 10 min at 12,000 rpm. A BCA assay kit (Sangon Biotech, Shanghai, China) was used to measure the concentration of total protein. For each group, 20 μg protein total proteins were separated by using 12% SDS–PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. After blocked with protein-free rapid blocking buffer (Epizyme Biotech, Shanghai, China) at 22 ± 3 °C for 20 min, the membranes were incubated with primary antibodies, including PGC-1α, PINK1, MFN1, MFN2, Nrf2, Bax, TFAM (Bimake, Houston, TX, USA), Bcl-2 (Cell Singling Technology, Boston, MA, USA) and β-actin (Proteintech, Wuhan, China), respectively, at appropriate dilutions, overnight, at 4 °C. After washing with TBST for 30 min, the membranes were incubated with anti-mouse or anti-rabbit HRP-conjugated secondary antibodies (Proteintech) at 22 ± 3 °C for 1 h. Finally, the PVDF membranes were then covered with a hypersensitive enhanced chemiluminescence (ECL) solution (Vazyme) and visualized by using a luminescent image analyzer (UVITEC, Mini HD9, Cambridge, UK).