HuT78 (ATCC, TIB-161) and HuT78 PD-1 stable cell lines are serum starved for 16 h. HuT78 parental and HuT78 PD-1 stable cell lines (transduced with human PD-1) were then seeded onto separate plates at 40,000 cells per well in the presence of serially diluted antibodies in triplicate for 40 min at 37°C., 5% CO2. pSTAT3 Tyr705 levels were measured using AlphaLISA Surefire Ultra pSTAT3 (Tyr705) Assay Kit (Perkin Elmer, #ALSU-PST3-A10K).
Alphalisa surefire ultra pstat3 tyr705 assay kit
Manufactured by PerkinElmer
Sourced in United States
The AlphaLISA SureFire Ultra pSTAT3 (Tyr705) Assay Kit is a homogeneous, bead-based assay designed to detect and quantify phosphorylated STAT3 (Tyr705) in cellular samples. It utilizes AlphaLISA technology to provide a sensitive and reproducible method for measuring this specific post-translational modification.
Automatically generated - may contain errors
Lab products found in correlation
Pcdna3.1 zeo, by Thermo Fisher Scientific (1 mentions)
Opti mem reduced serum medium, by Thermo Fisher Scientific (1 mentions)
Fugene hd, by Promega (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Dulbecco s modified eagle s medium, by Merck Group (1 mentions)
Halotag alexafluor 488, by Promega (1 mentions)
Poly d lysine hydrobromide, by Merck Group (1 mentions)
Nano glo luciferase assay system, by Promega (1 mentions)
4 protocols using alphalisa surefire ultra pstat3 tyr705 assay kit
1
PD-1 Expression Effects on pSTAT3 Levels
2
Generation of Fluorescent IL-23 Probe
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Tetramethylrhodamine (TMR) tagged IL‐23 (IL23‐TMR) was generated as described in Lay et al. (2022 (link)). Recombinant IL‐23 was obtained from GlaxoSmithKline (Stevenage, UK). Fugene HD, HaloTag Alexa Fluor 488 and the Nano‐Glo luciferase assay system were purchased from Promega (Madison, USA). Fetal bovine serum, protease‐free bovine serum albumen, Dulbecco's modified Eagle's medium, poly‐d ‐lysine hydrobromide and phosphate‐buffered saline (PBS) were obtained from Sigma‐Aldrich (Gillingham, UK). Opti‐MEM reduced serum medium was purchased from Thermo Fisher Scientific (Loughborough, UK). The Alpha‐Lisa Sure Fire Ultra p‐STAT3 (Tyr705) assay kit was obtained from Perkin Elmer (Waltham, USA). HEK293T cells (female; RRID:CVCL_0063 ) were purchased from ATCC (Virginia, USA). pcDNA3.1 zeo was purchased from Thermo FIsher Scientific and the IL23R‐MycDDK, and IL12Rβ1 expression plasmids were obtained from Origene (Rockville, USA). The pcDNA3.1 vectors containing N‐terminal HiBit, SmBit and LgBit were generated as described in Soave, Heukers, et al. (2020 (link)) and Soave, Kellam, et al. (2020 (link)). The IL6SS‐NL‐IL23R, IL6SS‐HT‐TEV‐IL12Rβ1 and IL6SS‐HT‐TEV‐IL23R expression constructs were purchased from GenScript (Piscataway, USA) as described in (Lay et al., 2022 (link)).
3
Inhibitory Effect of Crystalline Form A on STAT3 Phosphorylation in Hep3B Cells
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Example 6
Effect on JAK/STAT Signaling Pathway in Hep3B Cells
After incubating the crystalline form of the compound of Formula (I) of the present invention with Hep3B cells in a 37° C., 5% CO2 incubator for 15 min, 40 ng/μL IL-6 was added. Incubation was continued for 30 min, and the cells were lysed. The lysate was added to AlphaLISA SureFire Ultra p-STAT3 (Tyr705) assay kit (PerkinElmer), incubated at room temperature in dark, and the signals emitted by the receptor microbeads after energy absorption were measured, and the half inhibitory concentration (IC50) of inhibiting STAT3 phosphorylation was calculated. The results are shown in Table 4.
The data in Table 4 indicate that crystalline form A of the compound of Formula (I) of the present invention has a good inhibitory activity against phosphorylation of STAT3, a downstream kinase of the JAK/STAT signal in Hep3B cells.
4
Quantifying Tumor pSTAT3 Levels
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pSTAT3 levels were quantified in tumor protein lysates using AlphaLISA SureFire Ultra pSTAT3 (Tyr705) Assay Kit (PerkinElmer, Cat # ALSU-PST3-A500) according to manufacturer's instructions. The assay entailed incubation of 5 μl of tumor lysate with CaptSure donor and acceptor beads coated with antibodies against the phospho-Tyr705-epitope and the distal STAT3α and STAT3β (NP_644805 and NP_998827). In the presence of pSTAT3, the proximity of the two antibodies induced a fluorescent signal measured by the EnSight Plate reader. Samples were corrected for total μg protein added to assay wells and each sample assayed in triplicate. Each data point is representative of a tumour from a separate animal.
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