Pe labeled anti mouse

We applied quantitative FRET analysis by flow cytometry, in order to study cell-by-cell the molecular association of E7 oncoprotein with GSN in different experimental conditions in entire cells. As previously reported [32 (link)] cells were fixed and permeabilized and then labeled with antibodies tagged with donor (Phycoerythrin, PE) or acceptor (Cy5) dyes. The following antibodies were used: anti-E7 mouse MAb antibody (Cervimax), anti-gelsolin rabbit PAb antibody (Novus Biologicals), PE-labeled anti-mouse (Sigma), and Cy5-labeled anti-rabbit (Termo Fisher Scientific, Waltham, MA USA). In particular, E7 protein was detected in the FL2 channel (PE), GSN in FL4 channel (Cy5), and FRET in FL3 channel.
FRET efficiency (FE) was calculated according to Riemann et al. [24 (link)] by using the following algorithm:
FE = [FL3DA – FL2DA/a – FL4DA/b]/FL3DA in which A is the acceptor and D the donor and where a = FL2D/FL3D and b = FL4A/FL3A.

We applied quantitative FRET analysis by flow cytometry to C33A cells transiently transfected with HPV16E7wt or HPV16E7-mutant constructs. FRET analysis was restricted to cells positive to FL-1 (corresponding to pAmCyan1-C1 fluorescence emission). Cells were fixed, permeabilized and labeled as previously reported [24 (link)]. For FRET analyses, we used: anti-AMOT1 (Santa Cruz Biotechnology, Dallas, TX, USA), anti-p-YAP, anti-PTPN14 (Invitrogen, Carlsbad, CA, USA; MA5-31871), HPVE16-E7 polyclonal antibody (Bioss Antibodies, Woburn, MA, USA; bs-10446R) and Biotin-XX Phalloidin (Thermo Fisher Scientific, Waltham, MA, USA), PE-labeled anti-mouse (Sigma-Aldrich, St Louis, MO, USA), and Cy5-labeled anti-rabbit (Thermo Fisher Scientific, Waltham, MA, USA). AMOT1 protein was detected in the FL2 channel (PE, donor), p-YAP and F-actin in FL4 (Cy5, acceptor), and FRET in FL3 channel.
Quantification of protein−protein interaction was obtained by calculating FRET efficiency (FE) by using the following Riemann algorithm [25 (link)]:
FE = (FL3DA − FL2DA/a − FL4DA/b)/FL3DA in which A is the acceptor and D the donor and where a = FL2D/FL3D and b = FL4A/FL3A.