Mini protean 2 electrophoresis system

Nuclear and cytoplasmic lysates containing equal amounts of protein (25 μg) were equally loaded on 12% SDS–polyacrylamide gel and transferred to PVDF membranes (Millipore) using a Mini-Protean 2 electrophoresis system (Bio-Rad Laboratories). After blocking with 5% skimmed milk in TBS plus 0.1% Tween-20, the membranes were incubated with NF-κB p65 rabbit polyclonal antibody (Proteintech) overnight at 4 °C followed by HRP-conjugated goat anti-rabbit IgG (Trans Gen Biotech). Detection was visualized using an ECL assay kit (Thermo Fisher Scientific, Inc.). Images were subsequently analyzed using Image J software to quantify the protein expression (National Institutes of Health, USA). All blots were repeated in triplicate.

EV samples were treated with RIPA (cat. no. P0013C, Beyotime Institute of Biotechnology, Shanghai, China) lysis buffer to obtain EV-proteins. The protein concentration was quantified using a standard BCA protein assay kit (cat. no. P0012S, Beyotime Institute of Biotechnology, Shanghai, China). EV-derived proteins (15 μg) were loaded onto SDS‒PAGE (3% stacking gel, 12% running gel, cat. P0012A, Beyotime Institute of Biotechnology, Shanghai, China) and running in a Mini Protean 2 electrophoresis system (Bio-Rad) then transferred to PVDF membranes (cat. no. IPVH00010, Merck Millipore, MA, USA). PVDF membranes were blocked with 5% BSA at room temperature for 1 h and incubated with primary antibodies CD81(cat. no. 10037, Cell Signaling Technology, MA, USA, 1:1000 dilution) and TSG101(cat. no. ab133586, Abcam, MA, USA, 1:1000 dilution) at 4 °C overnight. After washing with TBST, the membranes were incubated with HRP-conjugated secondary antibodies (cat. no. SA00001-2, Proteintech, Wuhan, China, 1:1000 dilution) at room temperature for 1 h. Again, the membrane was rinsed with 1 × TBST and this was repeated 3 times (5 min per rinse). The proteins transferred onto the PVDF membrane were finally detected by chemiluminescence using the BIO-RAD Chemidoc XRS system with enhanced chemiluminescence reagent (cat. no. WBULS0100, Merck Millipore, MA, USA).

The EVs’ protein extracts (30 μg of each) were denatured by heating for 5 min at 99°C in SDS-containing buffer (4% SDS, 100 mM Tris HCl, pH 6.8, 20% glycerol, 0.2% bromophenol blue, and 20% DTT). Protein samples were separated in 10% SDS–polyacrylamide gels using the Mini-Protean II electrophoresis system (Bio-Rad). The gel was stained with a fixative solution of 40% methanol (MeOH), 10% acetic acid (vol/vol), and Coomassie brilliant blue G-250 (Bio-Rad). For Western blotting, 30 μg of EV protein extracts were separated in 10% SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and blocked in 5% milk–PBS. Western blots were probed with sera from patients suffering invasive candidiasis at a dilution of 1:3,000 (72 (link)). After an overnight incubation with the sera, membranes were washed five times with 0.1% Tween 20 containing PBS and then incubated with fluorescently labeled secondary antibodies at a dilution of 1/1,000 (IR dye 800-labeled goat anti-human IgG; LI-COR Biosciences). The Western blotting was performed with the Odyssey system (LI-COR Biosciences, NE, USA).

Denatured proteins were electrophoresed using the vertical Bio-Rad Mini-Protean II electrophoresis system. One hundred nanograms of protein sample was loaded in 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE). The electrophoresed proteins were then transferred onto nitrocellulose membranes (Millipore, MA, United States), followed by 2 h incubation at room temperature (RT) with rabbit polyclonal anti-Legionella (Invitrogen, United States), or rabbit monoclonal anti-LC3 (Sana Cruz, United States) or rabbit monoclonal anti-Atg5–Atg12 complex, which were individually diluted 1:1000 in the milk-blocking buffer. After washing, the membranes were incubated for another 2 h at RT with diluted secondary antibody donkey anti-rabbit that conjugated with horseradish peroxidase. The enhanced chemiluminescence system (ECL, Amersham) was used to detect the protein band signals, after soaking the membrane for almost 1 min with the Western blotting detection reagents (the chemiluminescence solutions 1 and 2) that were mixed 1:1 (Amersham, Piscataway, NJ, United States). Housekeeping protein β-actin protein was used as a loading control (Khalil et al., 2016 (link), 2017b (link)). The quantification of Western blot patterns was done using ImageJ software, which indicated the relative band intensity that was normalized to the intensity of housekeeping protein band.

SDS-PAGE of the WPH samples was carried out using Mini-PROTEAN^® TGX™ Precast Gels with a polyacrylamide gradient of 4–20% (Bio-Rad Laboratories Inc., CA, USA) under reducing conditions as described by O’Loughlin et al. [29 (link)]. The denatured samples, containing 25 µg protein/protein equivalent, along with the broad range (6.5–200 kDa) molecular mass standard (Bio-Rad) were separated using a mini Protean II electrophoresis system (Bio-Rad) at 150 V for 1 h.

The protein samples were applied (20 µg/well) and separated on a 4%–20% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, miniPROTEAN II electrophoresis system, Bio-Rad) and next transferred to a 0.2 µm polyvinylidene fluoride (PVDF) membrane (Bio-Rad Inc., Hercules, CA, USA). After 2 h blocking in 3% BSA at RT conditions, the membrane was probed with a specific primary antibody as follows: Anti-Neurogenin2 (at 1:1000 dilution) (Abcam, Cambridge, UK; cat. number ab109172); anti-Cdk6 (at 1:750 dilution) (Abcam, Cambridge, UK; cat. number ab124821); anti-SOX4 (at 1:100 dilution) (Abcam, Cambridge, UK; cat. number ab80261); anti-Caspase-7 (at 1:750 dilution) (Abcam, Cambridge, UK; cat. number ab69540) and incubated overnight at 4 °C. To visualize the bands secondary antibody (specific to the primary antibody used in the previous step) conjugated with horseradish peroxidase (HRP) were used. Chemiluminescence detection was performed using the ECL Advance Detection Kit (Amersham Life Sciences, Buckinghamshire, UK), and the bands were visualized with a UVP camera (Gel DOC-It Imaging system, Bio-Rad). The ImageJ software ver. 1.8.0 (NIH, WI, USA) was used to determine the background-subtracted density of the bands. The relative protein expression levels of Neurog2, SOX4, Caspase7, and CDK6 were quantified in comparison to those of BMG.