The total proteins were extracted from testis tissue in lysis buffer supplemented with the Halt™ protease inhibitors cocktail and the protein concentrations were determined using BCA protein assay (Thermo Scientific). Protein samples were separated on NuPAGE 4%–12% Bis-Tris precast mini gels from Invitrogen. The separated proteins were then transferred onto a nitrocellulose membrane. For western blotting, the membranes were incubated with the following primary antibodies overnight at 4°C: rabbit anti-Far1 (Thermo Fisher; 1:500), anti-GAPDH (Sigma-Aldrich; 1:1000), goat anti-Wnt4 (Novus Biologicals; 1: 1000), anti-Timp1 (Novus Biologicals; 1:1000), anti-Tnp1 (Proteintech, 1:1000), and anti-Tnp2 (Santa Cruz, 1:1000). The secondary antibodies bound to the nitrocellulose membrane were then detected using enhanced chemiluminescence reagents (GE Healthcare) and then digitally imaged using the ChemiDoc™ MP Imaging System (Bio-Rad).
Halt protease inhibitors cocktail
Manufactured by Thermo Fisher Scientific
Sourced in United States
Halt Protease Inhibitors Cocktail is a ready-to-use solution designed to inhibit a broad spectrum of serine, cysteine, and metalloproteases. It is commonly used in protein extraction and purification protocols to prevent degradation of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc mp imaging system, by Bio-Rad (3 mentions)
Criterion tgx precast protein gel, by Bio-Rad (2 mentions)
Annexin a1 anxa1, by Cell Signaling Technology (2 mentions)
Immun blot pvdf membrane, by Bio-Rad (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (2 mentions)
Prism 8, by GraphPad (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence reagent, by GE Healthcare (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
Nlrp3, by LSBio (1 mentions)
Il 1β, by LSBio (1 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (1 mentions)
Tris glycine gel, by Bio-Rad (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Chemidoc system, by Bio-Rad (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Donkey anti mouse or donkey anti goat secondary antibodies, by LI COR (1 mentions)
Anti actin 1 19 sc 1616, by Santa Cruz Biotechnology (1 mentions)
7 protocols using halt protease inhibitors cocktail
1
Western Blot Analysis of Testis Proteins
2
Hippocampal Protein and ATP Measurement
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Mouse hippocampi were lysed in radioimmunoprecipitation assay (RIPA) buffer (R0278; Sigma) containing Halt Protease Inhibitors Cocktail (78430; Thermo Scientific). Proteins were separated on 4%-15% or 4%-20% Criterion TGX precast protein gels (64134751; Bio-Rad), and transferred to an Immun-Blot PVDF membrane (1620177; Bio-Rad). After blocking with 5% nonfat dry milk (170-6404; Bio-Rad), the blot was probed with the following antibodies (1:1000): NLRP3 (LS-C374964; LSBio), ASC (67824S; Cell Signaling), Caspase1 (Cleaved Asp210) (LS-C380449; LSBio), IL-1β (LS-C104813; LSBio), and Annexin-A1 (ANXA1) (3299S; Cell Signaling) or (71-3400; Invitrogen). The proteins were visualized using SuperSignal West Dura Extended Duration Substrate (34076; Thermo Scientific) on a ChemiDoc MP Imaging System (Bio-Rad). Protein loading was assessed using anti-GAPDH (2118S; Cell Signaling). Bands were quantified with Fiji software, and analyzed with Prism 8 (GraphPad Software). Hippocampal levels of ATP were determined using a commercially available assay kit (ab83355, Colorimetric/Fluorometric) following the manufacturer’s instructions.
3
Kidney Protein Expression Analysis
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Kidney tissue (cross-sectional cut through the center of the kidney, 200 mg) was homogenized in ice-cold lysis buffer (50 μM Tris-HCl, pH 7.4, 150 μM NaCl, 0.25% deoxycholic acid, 1% NP-40, 1 mM EDTA), containing Halt Protease Inhibitors Cocktail (Thermo Fisher Scientific). Homogenates were incubated for 10 min on ice and then centrifuged at 10,000 g for 10 min. Supernatants were divided into several aliquots and stored at −80°C. The protein concentration of the aliquots was analyzed by the BCA method. The proteins were separated via gel electrophoresis and then transferred onto PVDF membranes. The membranes were blocked in Tris-buffered saline (pH 7.4) containing 0.01% Tween-20% and 5% skim milk. Next, they were incubated with antibodies to Nrf2, HO-1, Ferritin, and 4-HNE at 4°C overnight with gentle shaking. The proteins were visualized using the ECL kit. Equal loading was confirmed by re-probing the blots with a rabbit polyclonal antibody to ß-Actin. Finally, the relative intensities were calculated as a densitometric ratio between the sample and ß-Actin by the ImageJ program (V1.8).
4
Protein Extraction and Western Blot Analysis
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Protein samples from miR-153 mimics and negative control siRNA transfected cells were obtained 48 h after transfection. RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific Inc., Rockford, IL, USA; Catalog # 89901) mixed with Halt Protease Inhibitors Cocktail (Thermo Fisher Scientific; Catalog # 78429) was added into the 12-well cell culture plate (125 μl/well). Cells were detached from the bottom of the plate using scrapers of appropriate size. The collected samples were kept on ice for 30 min and centrifuged at 16,000 rpm for 15 min (4 °C) for supernatant. The protocols of Western blot analysis were similar to those previously described62 (link). Information about antibody sources is provided in Supplementary Table 2 .
5
Quantitative Western Blot Analysis
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Western blots were performed with lysates from tightly synchronized rings harvested 0–6 hr post invasion. Parasite cultures were washed twice in ice-cold 1× phosphate-buffered saline (PBS), and parasites were isolated by treatment with 0.05% saponin in PBS. Released parasites were lysed in 4% SDS, 0.5% Triton X-100 and 0.5% PBS supplemented with 1× protease inhibitors (Halt Protease Inhibitors Cocktail, ThermoFisher). Samples were centrifuged at 14,000 rpm for 10 min to pellet cellular debris. Supernatants were collected and protein concentrations were determined using the DC protein assay kit (Bio-Rad). Laemmli Sample Buffer (Bio-Rad) was added to lysates and samples were denatured at 90°C for 10 min. Proteins were electrophoresed on precast 4–20% Tris-Glycine gels (Bio-Rad) and transferred onto nitrocellulose membranes. Western blots were probed with a 1:1000 dilution of primary antibodies to K13 (Gnädig et al., 2020 (link)) or the loading control ERD2 (BEI Resources), followed by a 1:200 dilution of fluorescent StarBright secondary antibodies (Bio-Rad). Western blots were imaged on a ChemiDoc system (Bio-Rad) and band intensities quantified using ImageJ.
6
Western Blot Analysis of ARID2 Protein
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Cells were washed twice in PBS and lysed in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 % NP-40, 1 mM Sodium Orthovanadate, 1 mM NaF) containing Halt protease inhibitors Cocktail (Thermo Scientific, ref 87786). Lysates were sonicated using the Bioruptor® (Dia-genode) and cleared by centrifugation at 16,000g for 20 min at 4 °C. Total protein lysates were separated by SDS-PAGE in 8% polyacrylamide gels and transferred to nitrocellulose membranes. Subsequently, membranes were washed with TBS-T (50 mM TRIS + 150 mM Sodium chloride + 0,1% Tween 20, pH 7,4) and blocked using 5% non-fat milk solution as blocking agent in TBS (50 mM TRIS + 150 mM Sodium chloride) for 1 h at RT. Membranes were then incubated with primary antibodies anti-ARID2 (E-3, sc-166117, Santa Cruz) and anti-Actin (I-19, sc-1616, Santa Cruz), diluted 1:200 and 1: 1,000 in TBS-T/5% (w/v) BSA at 4°C overnight, respectively. Donkey anti-mouse or donkey anti-goat secondary antibodies (LI-COR Biotechnology, Lincoln, USA) conjugated to IRDye 800CW (926-32212) or IRDye 680RD (926-68074) respectively were used as secondary antibodies and visualized using Odyssey Clx imager (LI-COR Biotechnology, Lincoln, USA).
7
Hippocampal Protein Expression and ATP
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Mouse hippocampi were lysed in radioimmunoprecipitation assay (RIPA) buffer (R0278; Sigma) containing Halt Protease Inhibitors Cocktail (78430; Thermo Scientific). Proteins were separated on 4%-15% or 4%-20% Criterion TGX precast protein gels (64134751; Bio-Rad), and transferred to an Immun-Blot PVDF membrane (1620177; Bio-Rad). After blocking with 5% nonfat dry milk (170-6404; Bio-Rad), the blot was probed with the following antibodies: NLRP3 (LS-C374964; LSBio), ASC (67824S; Cell Signaling), Caspase1 (Cleaved Asp210) (LS-C380449; LSBio), IL-1β (LS-C104813; LSBio), and Annexin-A1 (ANXA1) (3299S; Cell Signaling) or (71-3400; Invitrogen). The proteins were visualized using SuperSignal West Dura Extended Duration Substrate (34076; Thermo Scientific) on a ChemiDoc MP Imaging System (Bio-Rad). Protein loading was assessed using anti-GAPDH (2118S; Cell Signaling). Bands were quantified with Fiji software, and analyzed with Prism 8 (GraphPad Software).
Hippocampal levels of ATP were determined using a commercially available assay kit (ab83355, Colorimetric/Fluorometric) following the manufacturer's instructions.
Hippocampal levels of ATP were determined using a commercially available assay kit (ab83355, Colorimetric/Fluorometric) following the manufacturer's instructions.
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