Recombinant Anti-p53 VHH Single Domain Antibody (PNBL-072) (CAT#: PNBL-072)

Figure 1 Establishment of Nb139's inhibitory effect on the p53 transcriptional program.

The relative luciferase activity was measured in U2OS cells stably harboring a luciferase gene. This cell line was transiently transfected with (w) or without (wo) Nb3, Nb139 or GFP Nb, and treated 24 h post-transfection with nutlin-3a (5 μM) for an additional 24 h. The luciferase activity was then finally measured. Nb139 causes a significant reduction in luciferase transcription in comparison to the control GFP Nb.

Bethuyne, J., De Gieter, S., Zwaenepoel, O., Garcia-Pino, A., Durinck, K., Verhelle, A., ... & Gettemans, J. (2014). A sdAb modulates the p53 transcriptional program without perturbing its functional architecture. Nucleic acids research, 42(20), 12928-12938.

Figure 2 Nb139 reduces transcription of various p53 target genes.

U2OS cell lines were consecutively treated with doxycycline (500 ng/ml) (24 h) and etoposide (20 μM) (24 h), after which the transcription levels of p53 target genes (p21, MDM2, GADD45aand PUMA) were evaluated via RTqPCR. Irrespective of the target gene, Nb139 is able to significantly reduce mRNA levels in comparison to control conditions.

Bethuyne, J., De Gieter, S., Zwaenepoel, O., Garcia-Pino, A., Durinck, K., Verhelle, A., ... & Gettemans, J. (2014). A sdAb modulates the p53 transcriptional program without perturbing its functional architecture. Nucleic acids research, 42(20), 12928-12938.

Figure 3 U2OS or HEK293T cell lysate (500 μg) were successively incubated with recombinant V5-tagged Nb3, Nb139 or GFP Nb (2 μg) and 5 μg of p53 conformation-specific antibodies, i.e. Ab1620 (wild type) or Ab240 (mutant).

sdAb-p53 complexes were immunoprecipitated (relying on the used conformation-specific antibodies) and analysed for the presence of p53. Nb139, nor other sdAbs, influences the architecture of p53. In addition, the presence of p53-bound sdAb was also evaluated via western blot.

Bethuyne, J., De Gieter, S., Zwaenepoel, O., Garcia-Pino, A., Durinck, K., Verhelle, A., ... & Gettemans, J. (2014). A sdAb modulates the p53 transcriptional program without perturbing its functional architecture. Nucleic acids research, 42(20), 12928-12938.

Figure 4 Nb139 maintains wild type conformation of p53.

The U2OS cell lines were consecutively treated with doxycycline (500 ng/ml) (24 h) and etoposide (20 M) (24 h), after which the ChIP was performed. In the presence of Nb139 p53 is able to bind in vivo the response elements of p21 (primer set F) and MDM2 (primer set E). After taking the background signal (i.e. signal derived from the input and anti-NTF2 antibody control) into account, Nb139 differs in a similar significant fashion as GFP Nb from the reference TBP gene.

Bethuyne, J., De Gieter, S., Zwaenepoel, O., Garcia-Pino, A., Durinck, K., Verhelle, A., ... & Gettemans, J. (2014). A sdAb modulates the p53 transcriptional program without perturbing its functional architecture. Nucleic acids research, 42(20), 12928-12938.