We performed the PLA in O9-1 cells by using the Duolink™ In Situ Red Starter Kit according to the manufacturer’s guidelines (Sigma-Aldrich DUO92101). Briefly, O9-1 cells were plated on 24-well plates, cultured in basal medium or osteoblast differentiation medium for 48 h, and fixed with 4% paraformaldehyde for 10 min at room temperature. Then, the cells were permeabilized in PBS/Triton 0.5% for 15 min at room temperature and blocked for 1 h in Duolink® blocking solution at 37 °C. After this, the cells were incubated with primary antibodies against Yap (1:100, Santa Cruz; sc-101199) and β-catenin (1:200, Cell signaling; 9562) overnight at 4 °C. Next, the cells were incubated with the proximity ligation assay probes (1 h), ligase (30 min), and polymerase (100 min) at 37 °C. After washing the cells, they were mounted by using the Duolink® In Situ Mounting Medium with DAPI. Red fluorescence indicated the association of Yap and β-catenin in cells. To control for nonspecific fluorescence background, each primary antibody was used alone. Slides were visualized by using a LAS X imaging system (Leica).
Sc 101199
Manufactured by Cell Signaling Technology
Sc-101199 is a laboratory reagent manufactured by Cell Signaling Technology. It is a protein that is commonly used in scientific research applications.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using sc 101199
1
Yap and β-catenin Interaction Analysis
2
Immunocytochemical Analysis of Adipocyte Markers
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Cells were fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature. Samples were washed three times with 1× PBS and incubated in blocking/permeabilization buffer (5.0% goat serum, 2.0% bovine serum albumin, 0.5% Triton X-100 in PBS) overnight at 4°C. For PDGFRα staining, blocking/permeabilization buffer without the goat serum (2.0% bovine serum albumin, 0.5% Triton X-100 in PBS) was used. The following primary and secondary antibodies were used for immunocytochemistry in this study: anti-PLIN (Abcam; ab3526; 1:200); anti- αSMA (Abcam; ab7817; 1:200); anti-YAP (Santa Cruz Biotechnology; sc101199; 1:200); anti-TAZ (Cell Signaling Technology; 83669S; 1:100); anti-PPARγ (Santa Cruz Biotechnology; sc7273; 1:200); anti-PDGFRα (R&D Systems; AF1062; 1:200); anti-β-CATENIN (Cell Signaling Technology; 8480S; 1:100); goat anti-rabbit Alexa 488 (Thermo Fisher Scientific; A11008; 1:500); goat anti-mouse Alexa Fluor 546 (Thermo Fisher Scientific; A11003; 1:500); goat anti-mouse Alexa 488 (Thermo Fisher Scientific; PIA32723; 1:500); and goat anti-rabbit Alexa 647 (Thermo Fisher Scientific; PIA32733; 1:500). Hoechst 33342 (Thermo Fisher Scientific; 1:1,000) and Phalloidin-iFluor 488 (Cayman Chemicals; 1:1,000) were used to stain nuclei and F-actin, respectively.
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