Criterion tgx 4 20 precast gel

BMB171 recombinant strains carrying pHT606:cry4Aa, pHT618:cry4Ba, pWF45:cyt1Aa:p20, pTBT02-cyt1Aa-like:p20, pTBT02:cyt1Da1-like:p20, pTBT02:cry4Aa4-like, pTBT02:cry53Ab1-like:orf2, pTBT02:orf1:cry56Aa-like and pTBT02:Tpp36Aa1-like were grown in 50 mL of CCY medium (supplemented with 20 μg/mL erythromycin) after inoculating single colonies from LB plates [81 (link)]. The strains were grown constantly at 28 °C with shaking at 200 rpm. Crystal formation was observed daily at the microscope. Once the cells lysed, after 48–72 h, spore-and-crystal mixtures were washed first with 1M NaCl and 10 mM EDTA, resuspended in 1 mL of dH₂O water and kept at 4 °C until use. The mixtures were solubilized in carbonate buffer (50 mM Na₂CO₃ and 100 mM NaCl, pH 11.3) and quantified for their total amounts of protein by using the Bradford method [82 (link)] and by using bovine serum albumin as a standard. For protein profile analysis, the washed spore-and-crystal mixtures were mixed with 2× sample buffer (Bio-Rad, Hercules, CA, USA), boiled at 100 °C for 5 min and then subjected to electrophoresis with a previously described method [83 (link)] using Criterion TGX™ 4–20% Precast Gel (Bio-Rad, Laboratories Inc., Hercules, CA, USA). Gels were stained with Coomassie brilliant blue R-250 (Bio-Rad, Laboratories Inc., Hercules, CA, USA) and then destained in a solution of 30% ethanol and 10% acetic acid.

The protein levels in the cartilage and synovial samples were estimated via BCA kit (Thermo-Fischer, United States), and equal amounts of proteins were denatured in 4 × Laemmli sample buffer (Bio-Rad, United States) and denatured at 96°C for 6 min. Equal volumes of synovial fluid samples were lyzed with 2% SDS and denatured with Laemmli buffer, such as cartilage and synovial samples. An equal amount of protein from the various experimental groups was separated on Criterion TGX 4–20% precast gels (Bio-Rad, United States) and transferred onto a PVDF membrane (Roche, Switzerland). After blocking with blocking reagent from the BM Chemiluminescence Western Blotting Kit (Roche, Switzerland), membranes were incubated overnight with primary antibodies (detailed information in the Supplementary Material) using a SignalBoost Immunoreaction Enhancer Kit (Merc, Germany). The amount of β-actin was measured on the same membrane on which the other proteins were measured by mild stripping (using a protocol published by Abcam). In control samples of synovial fluid concentration of proteins was very low, so this tissue was normalized to volume and detection of reference proteins was not possible.

Yellow field pea flour (~20% protein content) was provided by AGT Foods (Regina, SK, Canada) and commercial pea protein isolate (cPPI, 81.2% protein, 3.86% ash), PURIS™ Pea Protein, was provided by Puris Foods (Minneapolis, MN, USA). Commercial whey protein isolate (cWPI, 94.6% protein, 4.10% ash), BiPro^®, was provided by Agropur Ingredients (Eden Prairie, MN, USA). Defatted soy flour (~53% protein) and commercial soy protein isolate (cSPI, ~90.7% protein), ProFam^® 974, were provided by Archer Daniels Midland (ADM) (Decatur, IL, USA). All aforementioned samples were stored at −20 °C prior to usage. SnakeSkin™ dialysis tubing (3.5 kDa cut off) and Sudan Red 7B were purchased from Thermo Fisher Scientific™ (Waltham, MA, USA). Criterion™ TGX™ 4–20% precast gels, Laemmli 4X loading buffer, Imperial™ Protein Stain, 10X Tris/Glycine/SDS running buffer and Precision Plus molecular weight marker were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Vivaflow^® membrane ultrafiltration crossflow cassettes (3 kDa MWCO) were purchased from Sartorius™ (Gottingen, Germany). All other chemical reagents and supplies were purchased from Thermo Fisher Scientific and Sigma-Aldrich.

Cell or tissue lysates were mixed with Laemmli sample loading buffer, heated for 5 min at 95 °C, and separated on Criterion TGX 4–20% pre-cast gels (Bio-Rad) in 1 × MOPS running buffer. Proteins were transferred onto nitrocellulose membranes on Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked in TBST buffer (20 mM Tris–Cl, pH 7.6, 137 mM NaCl, 0.1% Tween-20) containing 5% non-fat dry milk for 1 h at room temperature, and incubated with anti-β-actin (Sigma), anti-ERK1/2 (Santa Cruz), anti-pERK1/2^T202Y204, anti-pSTAT1^Y701, anti-pSTAT3^Y705, anti-pSTAT5^Y694, pSTAT6^Y641 or anti-STAT3 antibodies (see Supplementary Table 4 for detailed information on antibodies) in blocking buffer at 4 °C overnight. Membranes were washed in TBST extensively, and bound proteins were detected with horseradish peroxidase-conjugated goat-anti-mouse IgG or goat-anti-rabbit IgG antibodies (GE Healthcare) for one hour at room temperature. Signals were developed with SuperSignal West Dura extended duration substrate (Thermo Scientific), and captured with a ChemiDoc imaging system (Bio-Rad).