Rabbit anti human taz

Proteins were extracted from the cells using RIPA buffer, resolved by SDS-polyacrylamide gels, and then transferred to poly-vinylidene difluoride (PVDF) membranes. The membranes were probed with rabbit anti-human TAZ (1:1000, cat# 83669), rabbit anti-human total caspase-3 (1:1000, cat# 9662), rabbit anti-human cleaved caspase-3 (1:1000, cat# 9661), rabbit anti-human total PARP (1:1000, clone: 46D11, cat# 9532), rabbit anti-human cleaved PARP (1:1000, clone: D64E10, cat# 5625), mouse anti-human NANOG (1:1000, cat# 4893), or rabbit anti-human SOX2 (1:1000, cat# 3579) from Cell Signaling Technology, or rabbit anti-human OCT4 (1:1000, abcam, cat# ab19857), rabbit anti-human NANOG (1:1000, clone: EPR2027(2), cat# ab109250, Abcam), mouse anti-human GAPDH (1:10000, clone: 1E6D9, cat# 60004-1-Ig, Proteintech), or rabbit anti-human α-tubulin (1:10000, cat# 11224-1-AP, Proteintech) antibodies overnight at 4 °C. HRP-linked anti-rabbit IgG (1:3000, cat# 7074, Cell Signaling Technology) or anti-mouse IgG (1:3000, cat# 7076, Cell Signaling Technology) was used and the antigen-antibody reaction was visualized by an enhanced chemiluminescence assay (ECL, Thermo). Densitometry was performed using ImageJ software. GAPDH or α-tubulin run on the same blot were used as the loading controls unless otherwise indicated.

PLA was performed using the Duolink in situ PLA Detection Kit (Sigma). Briefly, human breast cancer tissue or xenograft sections were dewaxed, hydrated and antigen repaired. After washed in PBS for 3 times, sections were incubated with Duolink blocking solution for 60 min at 37 °C. Primary mouse anti-human NANOG (1:100, cat# 4893, Cell Signaling Technology) and rabbit anti-human TAZ (1:100, cat# 83669, Cell Signaling Technology) antibodies in blocking solution was incubated for overnight at 4 °C. The PLA Probe incubation, ligation, and amplification reactions were performed according to the manufacturer’s instructions. Then, tissues were washed in Wash Buffer B and incubated with goat anti-human CK antibody (1:200, cat# ab219271, Abcam) for 1 h at RT, followed by incubated with Alexa Fluor 488-conjugated donkey anti-goat IgG (1:300, cat# A-11055, Thermo Fisher Scientific) for 1 h at RT. Finally, sections were mounted in Duolink in situ mounting medium with DAPI. Images were taken as z-stacks with a 0.25 μm step size by a Dragonfly Spinning Disc Confocal (Andor Technology). Max projections of z-stack were processed and analyzed by Imaris software.