Mice were anesthetized with Avertin (0.025 mg/mL) and perfused transcardially with 0.9% saline. Hemibrains were removed and drop-fixed in 4% paraformaldehyde overnight at 4 °C or flash-frozen in liquid nitrogen and stored at −80 °C. Frozen hemibrains were thawed in cold 1× PBS containing Halt Protease and Phosphatase Inhibitor Mixture (78447; Thermo Fisher Scientific) and microdissected immediately. The intact choroid plexus (CP) was removed first to avoid contaminating other tissues. Microdissected tissues were immediately frozen on dry ice and kept at −80 °C.
Halt protease and phosphatase inhibitor mixture
Manufactured by Thermo Fisher Scientific
Sourced in United States
Halt protease and phosphatase inhibitor mixture is a laboratory reagent designed to inhibit the activity of proteases and phosphatases in biological samples. It is intended to be used during protein extraction and purification procedures to prevent degradation of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (2 mentions)
Non fat dry milk, by Bio-Rad (2 mentions)
Mouse ccl21 6ckine duoset elisa, by R&D Systems (2 mentions)
Mouse ccl19 mip 3 beta duoset elisa, by R&D Systems (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (2 mentions)
T per, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (2 mentions)
Supersignal west dura chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Hrp conjugated secondary antibody, by GE Healthcare (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Cell surface protein isolation kit, by Thermo Fisher Scientific (1 mentions)
Mops buffer, by Thermo Fisher Scientific (1 mentions)
Licor odyssey tbs blocking solution, by LI COR (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
11 protocols using halt protease and phosphatase inhibitor mixture
1
Extraction and Preservation of Mouse Choroid Plexus
2
Western Blot Analysis of c-MYC Protein
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Whole-cell lysates were prepared using RIPA buffer (Sigma-Aldrich) supplemented with Halt Protease and Phosphatase Inhibitor Mixture (Thermo Scientific). Samples were resolved on SDS/PAGE and transferred to Polyvinylidene fluoride (PVDF) membranes (BioRad, 1704156). The c-Myc antibody (9E10) (NB600-302, Novus Biologicals Bio-Techne, United Kingdom 1:2000) was used to identify c-MYC, while anti-β-actin (Santa Cruz, SC47778 1:10,000) was used as loading control. HRP-conjugated secondary antibodies were from GE Healthcare. Bands were detected using SuperSignal West Dura chemiluminescent substrate (Thermo Fisher Scientific, 34075) and images were obtained in a ChemiDoc XRS + (BioRad). Image processing was performed with ImageLab (Life Science Research, BioRad). All Western blot data are the result of three independent biological replicates. The quantification was performed by normalising to β-actin and the percentage of decrease in c-MYC in treated versus control cells was calculated.
3
Cell Lysis and Protein Analysis
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Cells were seeded and treated as described above, washed in cold PBS, and then lysed using radioimmunoprecipitation assay (RIPA) buffer containing Halt Protease and Phosphatase inhibitor mixture (Thermo Fisher Scientific, Waltham, MA, USA) and 100 U/ml Benzonase. Lysates were incubated at 4 °C and then clarified by centrifugation at 13 000 × g for 10 min. The concentration was determined using Pierce BCA Protein Assay kit (Thermo Fisher Scientific). Equivalent amounts of lysate were analyzed by western blot.
4
IL-20RA/IL-10RB Expression and STAT3 Phosphorylation
5
Isolation and Analysis of Cell Surface Proteins
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Cell surface proteins were biotinylated and isolated using a cell surface protein isolation kit (Thermo Scientific, 89881). After various treatments, primary hippocampal mouse neurons (DIV 10–14) were washed once with ice-cold PBS and then incubated with sulfo-NHS-SS-biotin (sulfosuccinimidyl-2-(biotinamido)-ethyl-1,3-dithiopropionate; 0.25 mg/ml in ice-cold PBS) for 30 min at 4 °C. After quenching the biotinylation reaction, neurons were washed twice with ice-cold Tris-buffered saline (TBS) and lysed in Pierce IP Lysis Buffer (Thermo Scientific, 87788) with Halt protease and phosphatase inhibitor mixture (Thermo Scientific, 78440). Lysates were then sonicated on ice using five 1-s bursts and centrifuged at 1000 rpm for 5 min at 4 °C followed by determination of protein concentration by BCA protein assay. To isolate biotinylated surface proteins, 30 μg of biotinylated total protein was incubated with NeutrAvidin gel slurry (25 μl) at room temperature for 1 h followed by two washes with TBS and two additional washes with Pierce IP Lysis Buffer. Isolated biotinylated proteins were then solubilized in loading buffer for Western blot analyses.
6
Western Blot Quantification of Protein Expression
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To assess the loss of protein expression in KO cells, PMA-differentiated THP-1 cells were lysed in RIPA lysis buffer (89900; Pierce Biotechnology) containing Halt Protease and phosphatase inhibitor mixture (78441; Thermo Fisher Scientific). Protein concentration was determined using a BCA Assay Kit (23227; Pierce Biotechnology). Equal amount of protein was loaded onto NuPage 4–12% Bis-Tris gels (NP0335BOX; Invitrogen) and separated by SDS-PAGE using MOPS buffer (NP0001; Invitrogen). Gels were blotted at 30 V for 2 h onto Immobilon-FL PVDF membranes (IPFL00010, 0.45 μm pore size; MilliporeSigma) by wet transfer using transfer buffer (NP0006-1; Invitrogen). Membranes were blocked in LI-COR Odyssey TBS blocking solution (927-50000; LI-COR Biosciences) for 30 min. Membranes were incubated with primary Abs overnight at 4°C or for 2 h at room temperature. Membranes were washed in TBS containing 0.5% Tween 20 and incubated in secondary Abs for 1 h at room temperature. Blots were washed in TBS containing 0.5% Tween 20 and imaged using an LI-COR Odyssey CLx Imager.
7
Western Blot Analysis of Tumor Lysates
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Cells were lysed in RIPA buffer containing Halt Protease and Phosphatase Inhibitor Mixture (Thermo Scientific). Lysates were spun at 16,000×g at 4 °C for 30 min and normalized for protein concentration. Western blotting was performed as follows: total tumor lysates were separated by SDS/PAGE and electrotransferred to nitrocellulose membrane (Invitrogen). Membranes were blocked in PBS and 0.1% (vol/vol) Tween-20 (PBS-T) and 4% (wt/vol) nonfat dry milk (Bio-Rad) for 1 h on a shaker at room temperature. Primary antibodies were added to the blocking solution at 1:500 (TIGAR; Abcam, 37910), 1:500 (GSH; Abcam, 19534), 1:500 (PFKFB3; Abcam, 96699), and 1:1000 (Actin; Abcam, 3280) dilutions and incubated overnight and a rocker at 4 °C. Immunoblottings were washed three times, 5 min each with PBS-T, and secondary antibody was added at 1:10,000 dilution into PBS-T milk for 1 h on a shaker at room temperature. After several washes, enhanced chemiluminescence (ECL) reactions were performed according to the manufacturer’s recommendations (SuperSignal West Dura Extended Duration Substrate; Thermo Scientific).
8
Spleen Chemokine Quantification Protocol
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Spleens were harvested, flash frozen, and stored at −80°C. Spleens were homogenized in 1 ml of T-PER (Thermo Fisher) with 5 μM EDTA (Invitrogen) and 1× Halt protease and phosphatase inhibitor mixture (Thermo Fisher). Homogenates were centrifuged at 1000 × g for 10 min, and supernatants were aliquoted and frozen at −80°C until analysis. CCL21 was measured using mouse CCL21/6Ckine DuoSet ELISA (R&D Systems). CCL19 was measured using mouse CCL19/MIP-3 beta DuoSet ELISA (R&D Systems). Chemokine concentrations were normalized to total protein concentration, determined using Pierce BCA Protein Assay (Thermo Fisher).
9
Western Blot Analysis of Signaling Proteins
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Cell lysates were prepared by sonication in radioimmunoprecipitation assay lysis buffer (sc-24948A, Santa Cruz Biotechnology) supplemented with Halt protease and phosphatase inhibitor mixture (78443, Thermo Fisher). Protein concentrations were measured using the DC Protein Assay (5000113-115, Bio-Rad). Proteins (20 μg/lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (10600023, GE Healthcare). After transfer, membranes were blocked with 5% non-fat milk/BSA in Tris buffered saline with 0.1% Tween 20 (TBST ) and probed with primary antibodies to NOTUM (1:1500, SAB3500082, Sigma-Aldrich), HLA-G (1:1000. ab52455, Abcam), total CTNNB1 (1:1000, clone D10A8, 8480, Cell Signaling), active-CTNNB1 (1:1000, clone 8E7, 05-665, Sigma-Aldrich), phosphorylated CTNNB1 (1:1000, Ser33/37/Thr41, 9561 Cell Signaling), EPAS1 (1:300, 66731-1-Ig, Proteintech) and glyceraldehyde-3-phosphate dehydrogenase (ab9485; Abcam) overnight at 4°C. Membranes were washed three times for five min each with TBST and then incubated with secondary antibodies (goat anti-rabbit IgG HRP, A0545, Sigma Aldrich; goat anti-mouse IgG HRP, 7076; Cell Signaling) for 1 h at room temperature. Immunoreactive proteins were visualized by enhanced chemiluminescence according to the manufacturer’s instructions (Amersham).
10
Spleen Chemokine Quantification Protocol
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