The mature amino acid sequence of murine CXCL10 (aa 22–98) was obtained from NCBI, GeneID 15945. gBlocks were created for this sequence with a C-terminal “LPETG” Sortase recognition site and complementary 5’ and 3’ overhangs to the BamHI/HindIII double digested pCARSF63 Thioredoxin-SUMO fusion expression plasmid (Addgene #64695).89 (link) The resulting gBlocks were ligated into pCARSF63 expression plasmids using Gibson assembly (NEB) and transformed into chemically competent SHuffle T7 Express E.coli (NEB). After bacterial liquid cultures were grown to 0.6 OD600, protein expression was induced overnight at 16°C with 0.2mM IPTG. The overnight cultures were lysed and sonicated in non-denaturing conditions: B-PER Complete (Thermofisher, 89821), 10 mM imidazole, 0.1% Triton X-100 + COmplete tablet (Millipore Sigma, 11873580001) on ice. mCXCL10-LPETG SUMO fusion proteins were purified by Ni-NTA pulldown and then treated overnight with Endotoxin Removal columns (Thermofisher, 88274). Cleavage of mCXCL10-LPETG from the greater SUMO fusion proteins was carried out via ULP1 digestion (Thermofisher, 12588018) and stored for subsequent PolyG-azidoester modification (Figures 3A –B , S5 ).
Complete tablet
Manufactured by Merck Group
Sourced in United States
The COmplete Tablets are a laboratory product designed for use in various scientific and research applications. The tablets are a self-contained and pre-measured solution intended to facilitate consistent and reliable sample preparation. The core function of the COmplete Tablets is to provide a standardized and reproducible method for sample processing.
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Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Shuffle t7 express e coli, by New England Biolabs (2 mentions)
Gibson assembly, by New England Biolabs (2 mentions)
Mammalian protein extraction reagent, by Thermo Fisher Scientific (2 mentions)
Luminex magpix system rcytomag 80 k, by Merck Group (2 mentions)
Endotoxin removal column, by Thermo Fisher Scientific (1 mentions)
B per complete, by Thermo Fisher Scientific (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Thermo Fisher Scientific (1 mentions)
Cell strainer 70 m nylon, by Corning (1 mentions)
Mouse cytokine array chemokine array, by Eve Technologies (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Nonidet p 40, by Merck Group (1 mentions)
Phosstop, by Merck Group (1 mentions)
Ez link sulfo nhs biotin, by Thermo Fisher Scientific (1 mentions)
7 protocols using complete tablet
1
Murine CXCL10 Protein Expression and Purification
2
Recombinant Human CCL2 Production
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The mature amino acid sequence for human CCL2 (aa 24–99) was obtained from NCBI, GeneID 6347. GBlocks (IDT) were created for this sequence with a C-terminal “LPETG” Sortase recognition site and complementary 5’ and 3’ overhangs to the NdeI/XhoI double digested pSTEPL Sortase fusion expression plasmid. 42 (link) The resulting gBlocks were ligated into pSTEPL plasmids using Gibson assembly (NEB) and transformed into chemically competent SHuffle T7 Express E.coli (NEB). After bacterial liquid cultures were grown to 0.6 OD600, protein expression was induced overnight at 16°C with 0.2mM IPTG (Thermofisher, 15529019). The overnight cultures were lysed and sonicated in non-denaturing conditions: 20mM Tris, 125mM NaCl, 10mM imidazole, 0.1% Triton X-100 + COmplete tablet (Millipore Sigma, 11873580001), and the CCL2-LPETG STEPL fusion proteins were purified via Ni-NTA pulldown for PolyG-azidoester modification (Figures S1 –2 ).
3
Cytokine and Chemokine Analysis of PD-L1-Targeted NIR-PIT
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Tumor inoculation and the treatment were performed as described earlier. Sera were serially collected from the mice before, and at 6 and 24 hours after PD-L1-targeted NIR-PIT on MC38-luc tumors. Tumors were harvested and homogenized in 1 mL PBS supplemented with protease inhibitors (cOmplete Tablets; Sigma-Aldrich, St. Louis, Missouri, USA); then, the solution was passed through a filter (Cell Strainer 70 µm Nylon; Corning, Corning, New York, USA). Concentrations of various cytokines and chemokines in the samples were analyzed with Mouse Cytokine Array/Chemokine Array from Eve Technologies (Calgary, Canada).
4
Quantifying Inflammatory Cytokines in Tissue Samples
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Tissues were homogenized in Mammalian Protein Extraction Reagent® (Thermo Fisher Scientific, Waltham, MA) and a protease inhibitor cocktail (cOmplete® tablets, Sigma-Aldrich, St. Louis, MO), with a POLYTRON homogenizer, followed by 10 minutes disruption in a bead beater and sonication for 1 min. Samples were centrifuged at 13000 rpm for 5 min, and total protein concentration was quantified using the BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA). Samples were diluted as needed in order to standardize the amount of protein. Granulocyte-macrophage colony-stimulating factor (GM-CSF), Interleukin 1 alpha (IL-1α), IL-1β, IL-6, IL-10, keratinocyte chemoattractant (KC) and Tumor Necrosis Factor-alpha (TNFα) were simultaneously quantified in each sample using the Luminex/MAGPIX system (RCYTOMAG-80 K; Millipore, Billerica, MA).
5
Quantification of Inflammatory Mediators
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Tissues were homogenized in Mammalian Protein Extraction Reagent (Thermo Fisher Scientific) and a protease inhibitor cocktail (cOmplete tablets, Sigma-Aldrich), with a POLYTRON homogenizer, followed by 10 minutes disruption in a bead beater and sonication for 1 min. Samples were centrifuged at 13000 rpm for 5 min, and total protein concentration was quantified using the BCA protein assay kit (Thermo Fisher Scientific). Samples were diluted as needed in order to standardize the amount of protein. IL-6, keratinocyte chemoattractant (KC) and Tumor Necrosis Factor-alpha (TNFα) were simultaneously quantified in each sample using the Luminex/MAGPIX system (RCYTOMAG-80K; Millipore).
6
Kidney Protein Expression Quantification
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At the end of the infusion period, the mice were sacrificed, and kidneys from each group were homogenized in lysis buffer containing the following: 50 mmol/l Tris–HCl (pH 7.5), 1 mmol/l ethylene-bis(oxyethylenenitrilo)tetraacetic acid, 1 mmol/l ethylenediaminetetraacetic acid, 50 mmol/l sodium fluoride, 5 mmol/l sodium pyrophosphate, 1 mmol/l sodium orthovanadate, 1% (wt/vol) Nonidet P-40 (Sigma-Aldrich), 0.27 mol/l sucrose, 0.1% (vol/vol) 2-β- mercaptoethanol and protease inhibitors (Complete tablets; Co-Ro Roche, Sigma-Aldrich). Sixty micrograms from each homogenate were resolved into 10% SDS–PAGE and transferred for 1 h to polyvinylidene difluoride membranes. Membranes were blocked in 10% skim milk and incubated overnight with sheep antipNCC (T60) antibody, and after stripping, they were incubated with anti-NCC antibody, both produced by Dario Alessi from Phosphorylation Research Unit (Dundee, Scotland, United Kingdom) that were previously used and characterized by our group [7 (link),18 (link)] and anti β-actin (Santa Cruz Biotechnology Inc, Dallas, Texas, USA) antibodies. For densitometric analysis purpose, total NCC and pNCC were normalized with β-actin, then, total pNCC/NCC ratio was calculated.
7
Biotinylation of Cell Surface Proteins
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The biotinylation of cell surface proteins was done essentially as described for the dimerization assay, however, instead of 2 mM bissulfosuccinimidyl suberate (BS3), 2 mM EZ-Link™ Sulfo-NHS-Biotin (Thermo Fisher Scientific, 21,217), was used. The cells were lysed in IP lysis buffer (20 mM Tris HCl, pH 8.0, 137 mM NaCl, 10% glycerol, 1% nonidet P-40 and 2 mM EDTA), supplemented with PhosSTOP (Sigma, 04 906 837 001) and cOmplete Tablets (Sigma, 04 693 124 001), cleared by centrifugation and incubated with Streptavidin Magnetic Sepharose beads (vwr, 28-9857-38) for 2 h, end-over-end at 4 °C to pull down biotinylated cell surface proteins. The beads were then washed three times in lysis buffer and finally proteins were desorbed with sample buffer.
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