The Total SHP2 cellular assay monitors the expression level of SHP2, and can be used as a normalization assay with our phospho-SHP2 Y542 kit. Because these phospho and total SHP2 assays are compatible, the two kits can be used in parallel on the same lysates.
Many cancer cells overexpress checkpoint inhibitor ligands such as PD-L1. PD-L1 binds its counterpart checkpoint inhibitor receptor PD1, present at the surface of T lymphocytes. In turn, the PD1-PDL1 complex recruits and activates inhibitory effectors, such as SHP1 or SHP2. These two phosphatases, which are phosphorylated on Tyr564 and Tyr542 respectively by the kinase Lck, trigger the dephosphorylation of signaling proteins such as ZAP-70 or SLP-76, involved in the T cell activation pathway. Finally, activated SHP1 and SHP2 participate in T cell inactivation.
Preventing the activation of SHP1 and/or SHP2 by small molecule inhibitors is believed to contribute to restoring the immune response against tumors.
Total SHP2 assay principle
The Total SHP2 assay measures the expression level of SHP2 independently of its phosphorylation state. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total SHP2 assay uses 2 labeled antibodies, one with a donor fluorophore, the other with an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of SHP2 in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein's expression under a no-wash assay format.
Total SHP2 two-plate assay protocol
The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates to a 384-well low volume detection plate before the addition of Total SHP2 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Total SHP2 one-plate assay protocol
Detection of Total SHP2 with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.
Pharmacological validation using the Lck inhibitor, saracatinib, in Jurkat T-cells
Human Jurkat suspension cells were plated at 100,000 cells/well in a 96-well half area plate, and incubated for 24 h at 37 °C, 5% CO2, with increasing concentrations of Saracatinib. Before lysis, Jurkat cells were incubated 30min with Pervanadate (30 µM), followed by the addition of 10 µL of supplemented lysis buffer 4X. After 30 min lysis at RT under gentle shaking, 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF phospho-SHP2 (Tyr542) or total SHP2 detection reagents were added. The HTRF signal was recorded after an overnight incubation.
As described elsewhere, a dose dependent inhibition of SHP2 Tyr542 phosphorylation was obtained following treatment.
Pharmacological validation on NIH-3T3 mouse cells stimulated with PDGF
Mouse NIH 3T3 adherent cells were seeded at 100,000 cells/ well in a 96-well plate and incubated at 37 °C, 5% CO2. After an overnight serum deprivation, cells were stimulated with increasing concentrations of PDGF for 20min. Cell culture medium was harvested, and cells were lysed with 50 µL of supplemented lysis buffer. After 30min lysis at RT under gentle shaking, 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF phospho-SHP2 (Tyr542) or total SHP2 detection reagents were added. The HTRF signal was recorded after a 4 hour incubation.
As shown here, PDGF stimulation induced a strong SHP2 Tyr542 phosphorylation in NIH3T3 cells, whereas the SHP2 expression remained stable under the same experimental conditions.
HTRF assay compared to Western Blot
The human Jurkat cell line was seeded in a T175 flask, and incubated a 37 °C, 5% CO2. The cells were then treated with Pervanadate (30 µM) for 30 min before lysis. Serial dilutions of the cell lysate were performed in the supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF phospho-SHP2 detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot. Using the HTRF Total-SHP2 assay, 1,250 cells/well were sufficient to detect a signal, while 20,000 cells were needed using Western Blot, relying on ECL detection. These results demonstrate that the HTRF total-SHP2 assay is 16 times more sensitive than the Western Blot.
Function and regulation of SHP2
SHP1 (also known as tyrosine-protein phosphatase non-receptor type 6, PTPN6) is a tyrosine phosphatase mainly expressed in hematopoietic cells, activated by Lck and recruited by cellular surface receptors. SHP2 (also known as tyrosine-protein phosphatase non-receptor type 11) is ubiquitously expressed in hematopoietic or non-hematopoietic cells. Although SHP2 negatively regulates T cell activation, SHP2 is positively involved in ERK activation in response to growth factors such as PDGF or FGF.
In T lymphocyte cells, SHP1 and 2 are recruited by immune checkpoint inhibitors, thereby participating in the suppression of the TCR signaling pathway. SHP1 and 2 interact with PD1 ITIM domains and are phosphorylated and activated by the Lck kinase. Activated SHP1 and 2 phosphatases lead to the dephosphorylation of key TCR signaling effectors, such as ZAP70 or SLP76, which are required for T-cell proliferation and function.
