G box xt4 chemiluminescence and fluorescence imaging system

Protein extracts were prepared using high-salt lysis buffer (50 mM Hepes (pH 7.5), 500 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.1% NP-40) supplemented with 1% protease inhibitor cocktail (Sigma-Aldrich). 20 μg of cell lysates were separated on 10% tris-glycine Wedge-wells gels (Invitrogen), transferred onto a nitrocellulose membrane (Invitrogen) and incubated with the following antibodies: anti-p65BTK BN49 [18 (link)]; anti-Actin (#A1978, Sigma-Aldrich); anti-vinculin (#V9131, Sigma-Aldrich); anti-pERK (#4370, Cell Signaling Technology); BTK(#611117, Becton Dickinson). Purified p77BTK (#B4312) was from Sigma-Aldrich. Images were acquired using G:BOX XT4 Chemiluminescence and Fluorescence Imaging System (Syngene) and processed with Adobe Photoshop.

To analyze the soluble factors involved in the angiogenesis process, a RayBio^® C-Series Human Angiogenesis Antibody Array C1000 (RayBiotech) was used according to the manufacturer’s instructions. This kit can detect 43 proteins involved in angiogenesis and invasiveness thanks to the antibodies spotted in duplicate onto nitrocellulose membranes. Briefly, at the end of the experiment (96 hours), the conditioned medium of MDA-MB-231 and BT-549 cells untreated or treated with STD and mCHT protocols were collected, centrifugated and stored at -20°C until further use. Membranes were incubated with blocking buffer for 30 minutes at room temperature and then incubated with conditioned medium overnight at 4°C on a rocking platform. After washing the membranes with the appropriate buffers, a biotinylated antibody cocktail was added to the well and incubated for 2 hours at room temperature. Following the washing steps, HRP-Streptavidin solution was added onto the membrane, incubated for 2 hours at room temperature and then detection buffer was added and quickly visualized afterward. The chemiluminescent signal was acquired using G:BOX XT4 Chemiluminescence and Fluorescence Imaging System (Syngene, Cambridge, UK). The signal was then quantified with ImageJ Software (Wayne Rasband National Institutes of Health, USA) and reported as the percentage of untreated control.

The PCR product from the pan-fungal PCR was then used as the template for a second (nested) PCR assay using the newly designed primers NE Fw: 5’-ATG CCT GGA AGT ATG CCT GT-3’ and NE Rev: 5’-TCA CTG CGT TCG AGC ATT AC-3’. These primers were designed to specifically amplify a product size of 191 bp of P. insidiosum rDNA. This protocol used 12.5 μL of the GoTaq^® Colorless Master Mix, 1 μL of each primer NE Fw (10 Μm) and NE Rv (10 Μm), 9.5 μL of nuclease-free water, and 1 μL of the assigned sample. The thermoprofile consisted of enzyme activation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 56 °C for 1 min, and extension at 72 °C for 1 min. One cycle of final extension at 72 °C for 5 min followed. P. insidiosum DNA isolated from a known positive clinical sample was used as the positive PCR control and nuclease-free water as the negative control. After amplification, the PCR products and molecular weight markers (GeneRuler 1 kb Plus DNA Ladder, ThermoFisher Scientific) were loaded into a 1.5% agarose gel with 0.5 μg/mL ethidium bromide and separated by electrophoresis for 1 h at 100 V. Visualization and documentation was achieved by exposing the gel to 312 nm UV light (G: BOX XT4: Chemiluminescence and Fluorescence Imaging System, Syngene, Frederick, MD, USA).