G box chemi xrq

Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) was performed to identify recombinant 30Kc19α‐RUNX2 and HAB‐30Kc19α‐RUNX2. Purified proteins were mixed with 2× Laemmli sample buffer (Bio‐Rad, USA), and loaded to 10% SDS‐PAGE gel. For Coomassie blue staining, gels were stained with Coomassie Blue R‐250 (Sigma). For western blot analysis, the proteins on the gel were transferred to the nitrocellulose membrane (Cytiva). Anti‐RUNX2 antibody (Santa Cruz Biotechnology, USA) was used as a primary antibody, and goat anti‐mouse IgG‐HRP antibody (AbFrontier, Korea) was used as a secondary antibody. The membrane was treated with TOPview™ ECL pico plus western substrate (Enzynomics, Korea) to detect the protein of interest, and images were obtained by G:BOX Chemi XRQ (Syngene, UK).

Lung cancer cells were seeded at the density of 1 × 10³ cells in 6-well plates, were incubated for 24 h, and then treated with vehicle (Ø) or different concentrations of NT157 (3.2, 6.4 and 12.5 µM). Alternately, H1299 and H460 cells were treated with vehicle (Ø) or SP600125 20 µM for 1 h before the addition of NT157 at 12.5 µM for 6 h and replacement by drug-free medium. Colonies were detected after 7 days of culture by adding 1% crystal violet (Sigma-Aldrich) to a 10% ethanol solution for 15 min at room temperature. Images were acquired using the G:BOX Chemi XRQ (Syngene, Cambridge, UK) and analyzed using ImageJ 1.45 s software (http://imagej.nih.gov/ij; U.S. National Institutes of Health, Bethesda, MD, USA).

Cells were centrifuged, resuspended in phosphate-buffered saline (PBS) and then an equal volume of 2× Laemmli buffer was added. Samples were then boiled for 10 min and stored at −20 °C. Proteins were separated by SDS PAGE and then transferred onto PVDF membrane (Immobilon-P) at 15 V for 1 h. Membranes were blocked overnight at 4 °C in PBS + 0.1% (v/v) Tween (PBST) with 5% (v/v) dry milk powder. After blocking, membranes were washed once with PBST and then incubated with the appropriate primary antibody diluted in PBST + 3% (v/v) BSA (Sigma) for 2 h at RT. Membranes were washed 3 × 5 min in PBST and then incubated with HRP-conjugated secondary antibodies in PBST + 1% BSA for 1 h at room temperature. After further 3 × 5 min washes in PBST, the signals were detected using the ECL West Pico substrate (Thermo Fisher) and chemiluminescent signals were acquired below saturation levels using a G:BOX Chemi XRQ (Syngene, Cambridge, UK) and quantified using Fiji [22 (link)].

After various treatments for 24 h, cells were lysed, and protein quantification of the lysates was estimated using the BCA™ kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Proteins samples (30 µg) were resolved on a 10% SDS-PAGE and transferred onto a PVDF membrane and blocked with 5% nonfat milk in TBST (tris-buffered saline with Tween-20) buffer for 1 h followed by washing in TBST buffer. The membrane was then probed with primary antibodies (GAPDH, HDAC8, p21^Waf1/Cip1, and acetyl-histone H3[Ac-Lys⁹]) and incubated overnight at 4°C. The membrane was washed with TBST buffer and incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Signal was captured using SuperSignal™ West Pico Plus chemiluminescent substrate (Thermo Fischer Scientific Inc., Waltham, MA, USA) on gel documentation system (G : BOX Chemi XRQ, Syngene). The protein band densities were estimated using the software ImageJ (version 1.46r, NIH, Bethesda, MD, USA) [21 (link)].

a)
DNA extractionCommercial microbial DNA isolation kits were used to extract DNA from both the biopsy samples (Roche high pure PCR Template Preparation Kit, Mannheim, Germany) and the
H. pyloriisolates obtained from culture (DNeasy UltraClean Microbial Kit, Qiagen, Hilden, Germany). The extraction procedures were performed in accordance with the manufacturer’s instructions.
b)
Polymerase chain reaction (PCR)The PCR method was used to demonstrate the presence of
H. pyloriin the gastric biopsy specimens and to confirm the identification of the isolates recovered from the selective agar as
H. pyloriby phenotypic tests. For this purpose, primers (glmM-F 5’-AAGCTTTTAGGGGTGTTAGGGGTTT-3’ and glmM-R 5’-AAGCTTACTTTCTAACACTAAC GC-3’) specific to the phosphoglucosamine mutase (glmM) gene of
H. pyloriwere used, and the test was performed by a method of Lu et al. [18]. The amplification cycles consisted of denaturation at 93 °C for 1 min, primer annealing at 58 °C for 1 min, and extension at 72 °C for 1 min. Samples were amplified through 35 cycles. Amplified products were resolved by gel electrophoresis on 1.5% agarose (Biomax, Agarose, Lot no. 124543PR, from Prona, European Economic Community) and visualized under a UV transilluminator (G:BOX Chemi XRQ; Syngene, Cambridge, UK). Bands, which were 294-bp in size, were considered as a positive result.

To ascertain the solution stability of mRNA in the presence of PVP, 1.0 μg of mRNA was incubated in PVP solution at 800 g/L at room temperature for 24 hours. To evaluate whether the fabrication process affected the mRNA integrity, mRNA was recovered from tips of RNApatches 5, 10, 15 days after fabrication was complete. mRNA for all samples including the control were drawn from the same tube to ensure consistency. All samples were analyzed by gel electrophoresis for 30 minutes on a 1.2% agarose gel containing ethidium bromide, and evaluated using G:Box Chemi XRQ (Syngene) imaging system. mRNA recovered from RNApatch after 0, 5, 10 and 15 days were also injected subcutaneously at base of tail and luciferase expression was assayed 6 h post administration.