To examine PM2.5 effects on the expression levels of T-bet, PBMC were exposed to 5 µg/mL of PM2.5 in complete culture media for 20 h followed by M.tb infection at MOI1 or MOI5 or exposed to purified protein derivative (PPD, the antigen Gemisch used in tuberculin skin testing, Statens Serum Institute, Copenhagen, Denmark) at 10 µg/mL. Following an infection period of 18 h, PBMC were lysed with RIPA (radio immunoprecipitation assay) lysis buffer system (Santa Cruz Biotechnology, Dallas, TX) and protein content quantified by Bradford Protein Assay (Bio-Rad laboratories, Hercules, CA). Protein lysates were then analyzed by SDS/PAGE followed by transfer onto polyvinylidene difluoride (PVDF) membranes. T-bet and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)-specific proteins were analyzed by western blotting with specific antibodies (Cell Signaling Technology, Danvers, MA, USA).
Lysis buffer system
Manufactured by Santa Cruz Biotechnology
Sourced in Denmark
The lysis buffer system is a laboratory product that facilitates the disruption and solubilization of cells or tissues to extract their cellular contents, including proteins, nucleic acids, and other biomolecules. The buffer system is designed to efficiently lyse a variety of cell types while preserving the integrity and functionality of the target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Specific antibodies, by Cell Signaling Technology (2 mentions)
Radioimmunoprecipitation assay (ripa), by Santa Cruz Biotechnology (2 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Elisa kits for il 17a, by Thermo Fisher Scientific (1 mentions)
Catalase, by Santa Cruz Biotechnology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Total protein extraction kit, by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Santa Cruz Biotechnology (1 mentions)
Nuclear protein extraction kit, by Beyotime (1 mentions)
Blocking buffer, by Santa Cruz Biotechnology (1 mentions)
5 protocols using lysis buffer system
1
PM2.5 Modulates T-bet Expression in PBMC
2
T-bet Expression in PM2.5-exposed PBMC
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To identify the expression levels of T-box transcription factor (T-bet), PBMC were exposed to 0, 1 and 10 μg/ml of PM2.5 annual bulk used in Mexico City for human bronchoalveolar cell exposures [21 (link)] in culture media for 24 h followed by M.tb infection at MOI1 or MOI5 or left uninfected for an additional 24h.
Following incubation, PBMC were lysed with RIPA (radio immunoprecipitation assay) lysis buffer system (Santa Cruz Biotechnology, Dallas, TX). Protein lysates were analyzed by SDS/PAGE followed by transfer onto polyvinylidene difluoride (PVDF) membranes. T-bet and GAPDH-specific proteins were analyzed by western blotting with specific antibodies (Cell Signaling Technology, Danvers, MA).
Following incubation, PBMC were lysed with RIPA (radio immunoprecipitation assay) lysis buffer system (Santa Cruz Biotechnology, Dallas, TX). Protein lysates were analyzed by SDS/PAGE followed by transfer onto polyvinylidene difluoride (PVDF) membranes. T-bet and GAPDH-specific proteins were analyzed by western blotting with specific antibodies (Cell Signaling Technology, Danvers, MA).
3
Protein expression and cytokine analysis
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Cell lysates were prepared using the RIPA Lysis Buffer System and fractionated by SDS-PAGE. Target proteins in the lysates and in immunoprecipitation eluates were detected by rabbit polyclonal antibodies specific for Cyp1a1, Ahr, Arnt and AChE (dilution; 1:500), mouse monoclonal antibodies specific for β-actin (dilution; 1:1,000) and the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5,000). The Lysis Buffer System and antibodies were purchased from Santa Cruz Biotechnology. Band intensity was quantified by ImageJ software (version 1.48; https://imagej.nih.gov/ij/download.html ). To quantify the serum and supernatant cytokine levels, ELISA kits for IL-17a, IL-6, TNF-α, TGF-β (Invitrogen), and IL-10 (GenWay) were used following the manufacturer’s instructions.
4
Antioxidant Enzyme Expression Analysis
5
Protein Expression Analysis of Pancreatic Tissue
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The harvested pancreatic tissue was lysed on ice using a lysis buffer system (Santa Cruz) supplemented with PMSF (Santa Cruz). Total protein was extracted by using a Total Protein Extraction Kit (Beyotime). Nuclear protein was extracted by using a Nuclear Protein Extraction Kit (Beyotime). The protein concentration was measured with a BCA kit (Peirce). The protein was separated after being subjected to SDS-PAGE. Then, the separated proteins were transferred to PVDF membranes electronically. The membranes were then incubated with blocking buffer (Santa Cruz). Primary antibodies against TRAF1, MKK4, phosphorylated MKK4, MKK7, phosphorylated MKK7, JNK, phosphorylated JNK, NF-κB, IL6, TNFα, GAPDH, and Histone H3 were used to incubate the membranes at 4°C for 8 h. Then, the membranes were washed in TBST and further incubated with HRP-conjugated secondary antibodies for 2 h at room temperature. The immunoblots were developed by Lumi-Light substrate (Pierce) and visualized on X-ray films.
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