Anti vinculin

Morphology and morphometry of cells on TiO₂ surfaces of all groups were examined after 24 hours following cell seeding to evaluate the cellular spreading and cytoskeletal arrangement of the cultured cells using confocal laser scanning microscopy. Cells were fixed in 10% formalin and confocal microscopic images of cells stained with 4′,6-diamidino-2-phenylindole (DAPI) to detect the nucleus (keygentec), antivinculin to detect vinculin protein within the cytoplasm (Proteintech), and rhodamine phalloidin for actin filaments (beyotime.inc).

The samples were washed with PBS, fixed with 4 % paraformaldehyde for 30 min, according to experimental requirements, were incubated with anti-Pdpn (proteintech, 1:200), anti-Grem1 (CST, 1:50), anti-Cd200 (proteintech, 1:200), anti-Ctsk (Abcam, 1:200), anti-Ptgs2 (proteintech, 1:200), anti-vinculin (proteintech, 1:200) or anti-YAP (CST, 1:200) at 4 °C for 8–12 h. After that, samples incubated with goat anti-rabbit IgG (Servicebio, Alexa Fluor 488, 1:300), goat anti-rabbit IgG (Servicebio, Cy3, 1:300) or goat anti-mouse IgG (Servicebio, Alexa Fluor 488, 1:200) for 1 h at room temperature. Nuclear staining was conducted with DAPI (Sigma), and F-actin stained by rhodamine phalloidin (Beyotime). Tissue samples were fixed in 4 % paraformaldehyde. After decalcification, all samples were embedded in paraffin and sectioned at 6-μm slices. The tissue sections were immunofluorescently stained with primary antibodies targeting Sry (Santa Cruz, 1:200), CD11b (Servicebio, 1:500), TNF-α (Uscn Life Science Inc., 1:1000), IL-1β (Servicebio, 1:1200) and IL-10 (Servicebio, 1:1000), and the goat anti-rabbit IgG (Servicebio, Alexa Fluor 488, 1:400) and goat anti-rabbit IgG (Servicebio, Cy3, 1:500). DAPI was used to stain the nuclei. The images were observed by a fluorescence microscope (Olympus CKX53) or Nikon confocal microscope (Nikon).

The total protein was extracted by protein lysis buffer (BioRad, Hercules, California) according to manufacturer's protocols. And then proteins were resolved via 10 % sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The membranes were incubated with anti-vinculin (proteintech, 1:5000), anti-phospho-YAP (CST, 1:2000), anti-YAP (CST, 1:2000), anti-COX2/Cyclooxygenase 2/Ptgs2 (proteintech, 1:1000), anti-Lamin b1 (proteintech, 1:2000) and anti-GAPDH (CST, 1:2000) at 4 °C for 8–12 h. After that, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Servicebio, 1:3000) or HRP-conjugated goat anti-mouse IgG (Servicebio, 1:3000) at room temperature for 1 h.

The Flag-tagged DTX3-expressing plasmid was purchased from Vigene Biosciences, Inc. (Shandong, China). The Myc-tagged DTX3 plasmid was generated by inserting the full-length cDNA amplified by PCR into the pcDNA3.1/Myc-His vector, using the following primers, 5′-GGAATTCATGTCGTTCGTCCTGTCC-3′ and 5′-CGGGATCCGTCATCTGTGATACCCTTCGC-3’. The plasmids encoding non-tagged mtp53 (R175H, S241F, R248W, and R273H), His-Ub and HA-MDM2 were described previously.²³ (link) The anti-Flag (Catalogue No. F1804, Sigma–Aldrich), anti-DTX3 (Catalogue No. ab197360, Abcam, Cambridge, MA, USA), anti-p53 (DO-1, Catalogue No. sc-126, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p53 (Catalogue No. ab179477, Abcam) anti-GAPDH (Catalogue No. 60004-1-Ig, Proteintech, Hubei, China), anti β-actin (Catalog No. 60008-1-Ig, Proteintech), anti-Vinculin (Catalogue No. 66305-1-Ig, Proteintech), and the secondary antibodies for rabbit (Catalogue No. ARG65351, Arigo) and mouse (Catalogue No. ARG65350, Arigo), and the light chain-specific secondary mouse antibody (Catalogue No. 115-035-174, Jackson) were commercially purchased.