Powerpac basic power supply

Liver tissues were lysed in RIPA buffer (GenDEPOT) with protease inhibitors (P3100, GenDEPOT) and phosphatase inhibitors (P3200, GenDEPOT). Proteins (100 μg) were separated by 12% SDS-PAGE at 120 V for 150 min using Mini-Protean Tetra cell and PowerPac^TM Basic Power supply (BioRad, Hercules, CA, USA) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Darmstadt, Germany) at 0.15 A for 90 min using Novex^® semi-dry blotter (Invitrogen Co., Carlsbad, CA, USA). The membranes blocked with 5% skimmed milk in Tris-buffer including 0.1% Tween 20 were incubated with primary antibodies against interleukin 33 (IL33, R&D system, Minneapolis, MN, USA, 1:1000), major basic protein (MBP, MyBioSource, San Diego, CA, USA, 1:1000), interferon γ (IFNγ, Santa Cruz Biotechnology, 1:1000), and β-actin (Sigma-Aldrich, 1:5000) overnight at 4 °C, and incubated with secondary anti-mouse antibodies (1:3000) for 3 h at room temperature. Protein was visualized using the enhanced chemiluminescent solution and detected by a Chemi-doc (FluorChem E system, San Jose, CA, USA).

After 24 h of treatment, total protein was isolated from cells by RIPA lysate with protease inhibitor and phosphatase inhibitor. After determining the protein concentration by the BCA protein assay kit, 20 μg protein was added into each well of the vertical electrophoresis tank and separated by 10% SDS-PAGE. Subsequently, the protein was transferred onto the PVDF membrane. The Power Pac basic power supply, Wide mini-sub cell GT system, Trans-Blot SD Semi-Dry electrophoretic transfer tank were all provided by Bio-Rad (Bio-Rad Laboratories, Inc., Hercules, CA, USA). After being blocked by 5% skim-milk for 2 h, then the incubation of primary antibodies (TNF-α, p65, p-p65, IκBα, p-IκBα, GBP2, IRF1, SAMHD1, p-SAMHD1, β-actin, GAPDH, 1:2000) was performed overnight at 4 °C. After washing by TBST three times for 30 min, the secondary antibodies (anti-rabbit IgG HRP-linked antibody, 1:2000) were incubated for 1 h at room temperature. After TBST wash, the ECL chromogenic substrate was used to detect specific bands in Image Quant LAS 500 (GE HealthCare Technologies Inc., Chicago, IL, USA). The gray value was estimated by Image J software (National Institutes of Health, Bethesda, MD, USA) normalized to β-actin.

The hrCN PAGE protocol was adapted from Lemaire et al. (2018) (link). Glycerol was added to the sample at a final amount of 20% (v/v). Ponceau S at a final concentration of 0.001% (w/v) served as a marker to follow the migration. The buffer composition for the electrophoresis cathode was the following: 50 mM tricine, 15 mM Bis-Tris/HCl, pH 7.0, 0.05% (w/v) sodium deoxycholate, 2 mM DTT, and 0.01% (w/v) dodecyl maltoside, whilst the anode buffer contained 50 mM Bis-Tris/HCl, PH 7.0, 2 mM DTT. An 8–15% linear polyacrylamide gradient gel was used, and electrophoresis was run under a N₂/CO₂ (90:10%) atmosphere with a constant 40 mA current (PowerPac^™ Basic Power Supply, Bio-Rad). After electrophoresis, protein bands were visualised with Ready Blue^™ Protein Gel stain (Sigma Aldrich, Hamburg, Germany). The native protein ladder used is NativeMark^™ Unstained Protein Standard (Thermo Fischer Scientific, Driesch, Germany).
The determination of the oligomeric state by gel filtration was performed in triplicate on a Superose 6 Increase 10/300 GL (GE Healthcare, Munich, Germany) in 25 mM Tris/HCl pH 7.6, 2 mM DTT, 10% (v/v) glycerol at a flow rate of 0.4 ml/min, and in an anaerobic Coy tent containing an N₂/H₂ (97:3%) atmosphere. High molecular weight range gel filtration calibration kit (GE Healthcare, Munich, Germany) was used as the protein standard.

To assess purity, a polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate (SDS-PAGE) under reducing conditions was performed, in a Mini-PROTEAN Tetra Cell (Biorad), mixing samples with sample buffer and loading 20 µL of samples and 5 µL of the Broad Multi Color Pre-Stained Protein Standard (Genscript) into the wells of ExpressPlus PAGE Gels (Genscript) and using Tris-MOPS-SDS buffer (Genscript) as running buffer, in a two-step running starting at 60V for 30 min and then changed to 110 V for 1 hour and 30 minutes, with a PowerPac Basic power supply (Biorad). Gels were stained with 0.25% Coomassie Blue R-250 for 4 hours and distained with a solution containing 10% acetic acid, 30% methanol, and 60% water, applying four washes of 30 min each. Both staining and distaining were performed using a rocking platform settled at 14 oscillations/min.

A total of nine genes associated with virulence in P. multocida (sodA, ompH, tbpA, tadD, hsf1, hgbB, pfhA, ptfA, and toxA) were investigated. Three multiplex PCR reactions were established to detect the virulence factors sodA, ompH, and tbpA (reaction 1), virulence factors tadD, hsf1, and hgbB (reaction 2), and virulence factors pfhA and ptfA (reaction 3). The toxA gene was detected by simplex PCR reaction as previously described [18 (link)]. The PCR conditions, adapted from Gharib Mombeni et al. [37 (link)], were as follows: initial denaturation at 94 °C for 15 min; 25 cycles at 94 °C for 30 s, at 53 °C for 30 s, and at 72 °C for 60 s; with a final extension at 72 °C for 10 min. All reactions were performed using an MJ Mini Gradient Thermal Cycler (Bio-Rad, England). Simplex Taq PCR and Multiplex PCR Master Mix kits from Qiagen (Germantown, USA) were used in the reactions. PCR-generated products were detected by electrophoresis in 2.0% agarose gels supplemented with 1X SYBR safe DNA gel stain (Invitrogen SA) using a PowerPac basic power supply (Bio-Rad) at 90 V and 400 mA for 45 min. P. multocida strains NCTC 10322 (cap-A, tadD, pfhA, ptfA), NCTC 10323 (cap-B, tbpA, hgbB), NCTC 12178 (cap-D, lps-6, ompH, toxA), NCTC 10326 (cap-E, lps-2, sodA), and C104013 (cap-F, lps-3, hsf-1) were used as positive controls for the genes indicated.

Samples were mixed with 4X Laemmli buffer and boiled at 95°C for 5 min. 10% polyacrylamide gels were hand-casted and loaded with 20 μg of sample. The gels were run using a Mini-PROTEAN Tetra Cell (BioRad #1658001) and PowerPac Basic Power Supply (BioRad #1645050) at 100V for approximately 90 min. Gels were prepared for transfer to PVDF membranes, and the transfer was run using a Mini Trans-Blot Module (BioRad #1703935) at 400 mV for 60 min. The membranes were removed and blocked in 5% milk dissolved in TBST for 1 h. They were then incubated overnight with primary antibody [anti-tau (1:500,000), anti-GAPDH (1:1,000), and anti-Histone H3 (1:1,000)] overnight at 4°C on a shaker. The membranes were washed and incubated with secondary antibody [anti-rabbit AlexaFluor600 (1:5,000) and anti-mouse AlexaFluor800 (1:10,000)] for 1 h at room temperature. Imaging was done using a Licor Odyssey 9120 Imaging System. Quantification was done using ImageJ.

Liver tissues from wild-type (wt) and NASH model mice were finely minced and then subjected to protein extraction via the RIPA method. The expression levels of β-actin and tubulin were normalized using their grayscale values measured by ImageJ. Polyacrylamide gels were prepared using the One-Step PAGE Gel Fast Preparation Kit (15%) from Vazyme (catalog number E305), with the 180 kDa Prestained Protein Marker from Vazyme (catalog number MP102) used for molecular weight estimation. Electrophoresis and membrane transfer were conducted using the PowerPac Basic Power Supply from BIO-RAD. Blocking was performed with 5% BSA. Primary antibodies were diluted as follows: β-actin at 1:1000 from Servicebio (catalog number GB15001-100), tubulin at 1:5000 from Affinity Biosciences (catalog number T0023), CDKN1B at 1:1000 from BIOSS (catalog number bs-0742R), and TFAM at 1:1000 from Proteintech (catalog number 22586-1-AP). Imaging was done using the Tanon 4800 system. Grayscale values for all bands were acquired with ImageJ, and the relative protein expression levels were determined using β-actin and tubulin as standards. Statistical analysis and graphical representation were performed using GraphPad Prism 9.