Biotin xx phalloidin

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.

Mammalian Cell Lysis buffer (Sigma Aldrich) was used for C2C12 cells and COS7 cells. Immunoblot analysis were prepared as previously described^{15 (link)}. The following antibodies were used: anti-Histone H3 (1:10,000 dilution; ab1791, Abcam, Cambridge, UK), anti-Histone H3 acetyl K9 (1:2,000 dilution; ab4441, Abcam), anti-SIRT1 (1:1000 dilution; ab110304, Abcam), anti-CTTN (1:200 dilution, H-191, Santa Cruz Biotech, Texas, USA), anti-acetylated CTTN (1:50 dilution, 09-881, Millipore, Massachusetts, USA), anti-GFP (1:2,000 dilution, ab13970, Abcam), Anti-DYKDDDDK tag (Flag; 1:10,000 dilution, FUJIFILM Wako Pure Chemicals), anti-GAPDH (1:2000 dilution; MAB374, Sigma Aldrich), anti-β-Actin (1:10,000 dilution; 281-98721, FUJIFILM Wako Pure Chemicals) and anti-Vinculin antibody (1:10,000 dilution; V9131, Sigma-Aldrich). Following antibody was used for immunoprecipitation; anti-Flag antibody-conjugated beads (50 μl for each sample, FUJIFILM Wako Pure Chemicals). The immunoprecipitates were washed three times with TBST and analyzed by immunoblotting. For phalloidin pull-down, the lysate was incubated overnight at 4 °C with 5 units of Biotin-XX Phalloidin (B7474, Thermo Fisher Scientific), then incubated with 500 µg of Dynabeads M-280 Streptavidin (11205D, Thermo Fisher Scientific) for 1 h and washed three times with TBST. The pull-downs were analyzed by immunoblotting.

QD samples were prepared by dispersing a 4 μL drop of QDs suspended in water (525 nm emitting nanocrystals provided by QD Vision) on a microscope slide. A drop of UV-curing adhesive (Norland Products NOA 74) was placed on the dried QDs, a coverslip placed on top, and the sample cured by exposure to a UV source.
HeLa cells on a glass coverslip were fixed at room temperature using 4% paraformaldehyde (Electron Microscopy Sciences 15742-10) for 10 min, permeablized for 2 × 15 min using 0.25% Triton X-100 (Sigma Aldrich 93427) in phosphate-buffered saline, and then treated with an endogenous biotin blocker (Life Technologies E21390). This was followed by treatment with Biotin-XX Phalloidin (Life Technologies B7474) for 20 min, then a 605-nm-emission QD-streptavidin conjugate (Life Technologies Q10151MP) before sealing with a coverslip using Mount Quick medium (Electron Microscopy Sciences 18000).