Cells in culture were collected and lysed in RIPA buffer (Sigma, USA) containing protease (Complete, Mini, EDTA-free; Roche) and phosphatase inhibitors (phosphatase inhibitor cocktails 2, Sigma) at 4 °C for 5 min. Protein concentrations were determined using the BCA protein assay kit (Pierce) after three cycles of freeze thaw. For protein collected from tissues, the ciliary bodies from two eyes were pooled and disrupted in lysis buffer (Cell Signaling Technology, MA) containing protease (Complete, Mini, EDTA-free; Roche) and phosphatase inhibitors (phosphatase inhibitor cocktails 2, Sigma, USA), and exposed to high frequency ultra-sonication. Equal amounts of protein (10–50 μg for cell lysate, and 3–5 μg for tissue lysate) were separated by SDS–polyacrylamide gel electrophoresis (10% acrylamide) under reducing conditions and transferred to PVDF membranes (Immobilon-P, Millipore). Membranes were blocked in 1 × TBS containing 0.1% Tween, 5% non-fat milk and 2.5% BSA for 1 h, and then probed with primary antibody overnight at 4 °C. Secondary antibody incubations were performed with the corresponding horseradish peroxidase-conjugated antibody at room temperature for 1 h. Proteins were detected by chemiluminescence with SuperSignal West Pico or Dura (Thermo Scientific).
Phosphatase inhibitor cocktails 2
Manufactured by Merck Group
Sourced in United States
Phosphatase inhibitor cocktails 2 are a blend of several phosphatase inhibitors designed to inhibit the activity of various phosphatases in biological samples. These cocktails are used to preserve the phosphorylation state of proteins during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (6 mentions)
Complete mini, by Roche (2 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Nacalai Tesque (1 mentions)
T per tissue protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Ms 100r bead beating disrupter, by TOMY (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Ecl detection kit, by GE Healthcare (1 mentions)
Halt phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Anti β actin antibody, by Abcam (1 mentions)
Neutral buffered formalin solution, by Merck Group (1 mentions)
Tissue extraction reagent 1, by Thermo Fisher Scientific (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Tissuelyser, by Qiagen (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Supersignal west pico, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Protease inhibitor cocktail p8340, by Merck Group (1 mentions)
9 protocols using phosphatase inhibitor cocktails 2
1
Western Blot Analysis of Protein Samples
2
Protein Extraction and Quantification from IVD NP
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Proteins were extracted from IVD NP tissues to evaluate protein expression in IVD NP cells by western blotting. Harvested tissues were homogenized using an MS-100R bead-beating disrupter (Tomy Seiko, Tokyo, Japan) for 30 s twice at 4 °C in a T-PER tissue protein extraction reagent (78,510; Thermo Fisher Scientific, Waltham, MA, USA) containing protease and phosphatase inhibitors [protease inhibitor cocktail (25955-11; Nacalai Tesque), phosphatase inhibitor cocktails 2 (P5726; Sigma-Aldrich), and phosphatase inhibitors cocktails 3 (P0044; Sigma-Aldrich)]. Finally, soluble proteins were collected by centrifugation at 20,000× g for 15 min at 4 °C. Protein concentration was determined using a bicinchoninic acid assay (23,227; Thermo Fisher Scientific). Samples were stored at −80 °C.
3
Western Blot Analysis of Protein Targets
4
Peptidomimetics Synthesis and Reagents
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Peptidomimetics PM1 and PM2 were prepared by a solid-phase synthesis methodology involving assembly of dimeric and/or tetrameric building blocks on a Rink amide resin by using PyBOP as a coupling reagent as earlier reported (58 (link), 59 (link)). PMA (≥99% TLC), indomethacin (≥99% TLC), protease inhibitor cocktails, phosphatase inhibitor cocktails 2, and 10% neutral-buffered formalin solution were purchased from Sigma-Aldrich (St. Louis, MO, United States). Tissue Extraction Reagent I was obtained from Thermo Fisher Scientific (Waltham, MA, United States).
5
Tissue Homogenization and Protein Extraction
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Frozen muscle and liver tissues were homogenized in ice-cold lysis buffer (25 mmol/L Tris pH = 7.5, 150 mmol/L NaCl, 1 mmol/L EDTA, 1% Triton-X 100), and supplemented with phosphatase inhibitor cocktails 2 (Sigma, St. Louis, MO, P5726) and 3 (Sigma, P0044), and protease inhibitor cocktail (Sigma, P8340) at a dilution of 1:100. The tissues were homogenized using the Qiagen TissueLyser. The samples were centrifuged at 16 000 g for 10 min at 4°C, and then the supernatants were collected and stored at −80°C for further analysis. Protein assays were done using Bio-Rad DC Protein Assay (500-0116, Bio-Rad, Hercules, CA, USA) on all soleus muscle, gastrocnemius/plantaris muscle, and liver samples to determine the protein concentrations of these samples for further use in western blot analysis.
6
Evaluating PKMζ and KIBRA Protein Levels
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Plated HEK293 cells were transfected with HA-PKMζ or HA-PKMζ and CT-KIBRA-flag using Lipofectamine 2000. A day after transfection, untreated cells (0 hour time point) were harvested, while the rest of the cells were treated with 5 mg/mL of CHX. Treated cells were harvested after 24 and 48 hours of incubation with cycloheximide. Cells were resuspended in RIPA buffer containing 1 mM phenylmethyl sulfonyl fluoride, protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor cocktails 2 (Sigma-Aldrich) and 3 (Sigma-Aldrich). Resuspended cells were sonicated and the supernatant was prepared the same as mouse tissue, then Western blot was performed to detect PKMζ and KIBRA levels.
7
Serum Starvation and BMP Stimulation
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HUVECs were serum-starved for 6 hours (h) in EBM-2, after which they were stimulated or not for 30 minutes (min) with 10 ng/mL of BMP9 or BMP10. Stimulations were repeated two times with 3 biological replicates/condition in the first experiment and 2 biological replicates/condition in the second experiment generating 5 biological replicates/condition. Cells were then lysed on ice with urea lysis buffer (8 M Urea, 50 mM Tris–HCl pH 8, 75 mM NaCl, 1 mM EDTA) supplemented with protease inhibitor cocktail (P8340) and phosphatase inhibitor cocktails 2 (P5726) and 3 (P0044) purchased from Sigma. Protein concentrations were determined using a µ-BCA assay kit (Thermo Fisher Scientific).
8
Quantitative Western Blot Analysis of Signaling Proteins
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Cell lysates were harvested from culture BMDMs with 2% SDS lysis buffer containing protease inhibitor cocktail (Sigma #P8340) and phosphatase inhibitor cocktails 2 (Sigma #P5726) and 3 (Sigma #P0044). Lysates were incubated on ice for 15 min, boiled at 95–100°C for 5 min, and then stored at −20°C before use in assay. Lysates were separated by SDS-PAGE followed by transferring to methanol-soaked PVDF membranes under ice for 2 h. Membranes were blocked in 5% non-fat milk for 1 h followed by incubation with the following primary antibodies respectively: Keap1 (Cell Signaling #8047), p62 (Cell Signaling #5114), MLKL (Cell Signaling #37705), IKK-β (Novus Biologicals # NB100-56509), phospho-NF-κB p65 (Ser536) (Cell Signaling # 3031S), total NF-κB p65 (D14E12) XP (Cell Signaling #8242S), GAPDH (Cell Signaling #2118), or beta actin (Cell Signaling #4970). Blots were incubated overnight or for 3 days before detection using HRP-linked Anti-rabbit IgG antibody (Cell Signaling #7074) and Advansta ECL detection kit (VWR #490005-020). Relative protein levels were quantified using ImageJ software. Graphpad Prism 6.0 software was used for statistical analysis and figure preparation as detailed in the statistical analysis section.
9
Immunoblotting for MAPK Phosphorylation
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Vegetative hyphae of PH-1 were harvested from 12 h YEPD cultures and treated with 500 μM 2,4-D or 0.4% ethanol (v/v) for 30 min. Total proteins were then isolated with protein lysis buffer containing protease inhibitor cocktail (Sigma-Aldrich, USA), phosphatase inhibitor cocktails 2 (Sigma-Aldrich, USA) and phosphatase inhibitor cocktails 3 (Sigma-Aldrich, USA) and separated on 10% PAGE gels as described (Zhang et al. 2018 (link)). Western blots were detected with the anti-TpGY phosphorylation-specific (for detection of phosphorylated FgHog1 with the TGY dual phosphorylation site), anti-FgHog1, anti-TpEY phosphorylation-specific (for detection of phosphorylated Gpmk1 and Mgv1 MAP kinases with the TEY dual phosphorylation site), and anti-ERK2 antibodies (Cell Signaling Technology, USA) as described (Zhang et al. 2018 (link), 2017 ) for assaying the phosphorylation and expression levels of FgHog1, Gpmk1, and Mgv1 MAP kinases. Detection with an anti-β-tubulin (anti-tub2) antibody was used as the protein loading control as described (Wang et al. 2019 ).
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