Total p300 assay principle

The Total p300 assay quantifies the expression level of p300 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based, and does not require gels, electrophoresis, or transfer. The Total p300 assay uses two labeled antibodies, one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of p300 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 p300 two-plate assay protocol

The 2-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of the HTRF Total p300 detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Total p300 one-plate assay protocol

Detection of Total p300 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.

Characterization of the mechanism of action of 3 CREBBP/p300 PROTACs

The experiments were carried out using the two-plate assay protocol for adherent cells. HeLa cells were plated in 96-well culture plates (70,000 cells/well in complete culture medium) and cultured overnight at 37°C, 5% CO2. The day after, the cells were pre-treated for 1 hour with 1 µM Epoxomicin, and then treated during an additional 5h-incubation with 3 doses of each CREBBP/p300 PROTAC degrader (PROTAC CBP/p300 Degrader-1, Thalidomide-NH-CBP/P300 ligand 2, or dCBP-1), as well as with the warhead GNE-781 used as negative control (the same for the three PROTACs). The cells were lysed with 50 µL of supplemented lysis buffer #1, and 16 µL of lysate were transferred into a low volume white microplate (ProxiPlate-384 Plus, Cat # 6008280/9) before adding 4 µL of the HTRF Total p300 or Total CREBBP kit detection antibodies (HTRF Total CREBBP kit, Cat # 64CREBBPTPEG/Y). The HTRF signal was recorded after both assays were incubated overnight. Cell viability was also assessed by transferring 5 µL of the same lysate into an HTRF 96-well low volume white plate (Cat # 66PL96005/025/100) and then adding 25 µL of ATPlite 1step detection reagent (ATPlite 1step Luminescence Assay System, Cat # 6016736/1/9). The luminescence signal was measured after a 10-min incubation in the dark.

After 5 hours of treatment, the three CREBBP/p300 PROTAC degraders induced a dose dependent signal decrease in both p300 and CREBBP, whereas the warhead GNE-781 caused no change. This was as expected. As indicated on the ATPlite graph, no cytotoxic effect was observed in presence of the compounds. Finally, the HTRF signals were recovered in presence of the proteasome inhibitor Expoxomicin. Taken together, our results unambiguously demonstrated PROTAC-induced p300/CREBBP degradation which is mediated by the ubiquitin proteasome system.

Assessment of Total p300 levels in various human and mouse cell lines

The experiments were carried out using the two-plate assay protocol for adherent cells. The human cell lines HeLa, SH-SY5Y, MCF7, A549, and A431, as well as the mouse cell lines NIH-3T3 and Neuro-2a, were plated in 96-well culture plates (100,000 cells/well in complete culture medium) and cultured at 37°C-5% CO2 for 24 hours. The day after, the cells were lysed with 50 µL of supplemented lysis buffer #1, and 16 µL of lysate were transferred into a low volume white microplate (ProxiPlate-384 Plus, Cat # 6008280/9) before the addition of 4 µL of HTRF Total p300 kit detection antibodies. The HTRF signal was recorded after an overnight incubation.

The HTRF Total p300 kit is compatible with human and mouse cell models and efficiently detects p300 in cell lines containing variable expression levels of the protein.

HTRF Total p300 assay compared to Western Blot

HeLa cells were seeded in a T175 flask and cultured at 37°C-5% CO2 in complete culture medium until ~ 90% confluency was reached. The cells were then lysed with 3 mL of supplemented lysis buffer #1 and serial dilutions of the lysate were performed using the supplemented lysis buffer. To detect Total p300, 16 µL of each dilution were transferred into a low volume white microplate (ProxiPlate-384 Plus, Cat # 6008280/9) and 4 µL of HTRF Total p300 detection reagents were added. Equal amounts of lysates were loaded on a gel for a side-by-side comparison with Western Blot.

Using the HTRF Total p300 assay, 1,000 cells/well were enough to detect a significant signal, while 8,000 cells were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore, in these conditions the HTRF Total p300 assay was 8 times more sensitive than the Western Blot technique.

CREBBP/p300 Signaling Pathway

The transcriptional co-activators CREBBP and p300 regulate gene expression by facilitating the assembly of transcriptional machinery and by acetylating core histones and other transcriptional regulators such as the transcription factors (TFs) c-Myc, p53, and FoxO1.

CREBBP and p300 are central to many important signaling pathways activated by intracellular or extracellular signals. They regulate growth factors and hormone signaling such as that mediated by the nuclear hormone receptors AR (Androgen Receptor) and ER (Estrogen Receptor). Other examples of signals are hypoxia, cellular stress (involving the MAP kinases p38, JNK, and ERK), high glucose, GPCR agonists (inducing cAMP signaling), and cytokines (mediated by the JAK/STAT pathway). The TLR/IRF-3 pathway is also regulated by CREBBP/p300 to control innate immune responses. Both CREBBP and p300 are required for histone acetylation at BRD4-occupied sites (e.g. H3K27ac) and for BRD4 binding at gene promoters and enhancers to regulate associated gene transcription.

All these pathways ultimately use CREBBP and p300 as downstream effectors in the nucleus to control key cellular functions such as cell cycle control and differentiation, embryonic development, and homeostasis.