Anti cd146

Protein extraction and concentration determination were performed as previously described [7]. Briefly, equal amounts of total protein (30–45 μg) were prepared in 4× Laemmli buffer and heated to 95 °C for 5 min prior to being loaded on SDS‐PAGE gels. After the proteins were separated and transferred to PVDF membranes, the membranes were blocked and subsequently probed with the following antibodies: anti‐phospho‐ERK1/2 (1 : 1000) from Cell Signaling (Danvers, MA, USA), anti‐WNT5A (1 : 100) and anti‐RND3 (1 : 1000) from R&D Systems (Minneapolis, MN, USA), and anti‐MLC (phospho Ser 20) (1 : 1000), anti‐CD146 (1 : 1000), and anti‐β‐actin (1 : 30 000) from Abcam (Cambridge, UK). After washing, the membranes were incubated with either HRP‐conjugated rabbit anti‐goat secondary antibodies or HRP‐conjugated goat anti‐rabbit/mouse secondary antibodies (Dako, Glostrup, Denmark). Following a second wash, the separated protein bands were visualized using Immobilon™ Western Chemiluminescence HRP substrate (Millipore, Burlington, MA, USA) and imaged and analyzed using the ChemiDoc™ imaging system (Bio‐Rad Laboratories Inc., San Francisco, CA, USA). Densitometric quantification of relative protein expression was carried out by calculating the intensity of each band using image lab 6.0 software (Bio‐Rad Laboratories Inc.).

A total of 1 × 10⁴ hBMSCs were seeded onto 96-well plates and cultured in the presence of amino acids for 48 h in basal medium. The cells were subsequently fixed in 4% paraformaldehyde (PFA) for 15 min, permeabilized with PBS containing 0.25% Triton X-100 for 10 min, blocked with 5% normal goat serum (Invitrogen), and then incubated with primary antibody anti-CD146 (Abcam, Cambridge, MA, USA), anti-Ki-67 (Abcam) or the respective IgG (Abcam) overnight at 4 °C. Cells were then incubated with secondary antibody Alexa Fluor 488-conjugated IgG (Invitrogen) for 1 h at room temperature, and observed under a fluorescence microscope (BZ-X700, KEYENCE, Osaka, Japan). Cell nuclei were stained with 4′6-diamidino-2-phenylindole (DAPI, Invitrogen) [25 (link)].

HUVECs were exposed to the EP4 antagonist (AH23848; 100 μM, Sigma, Kanagawa Prefecture, Japan) with or without L-902,688 (1 µM) or TGF-β (5 ng/mL) for 24 h. Western blotting was performed using anti-eNOS (BD), anti-E-cadherin (E-cad) (Cell Signaling, Danvers, MA, USA), anti-CD146 (Abcam, Cambridge, UK), anti-α-SMA (Thermo, Waltham, MA, USA), and anti-Twist (Novus, Frauenfeld, Switzerkand) primary antibodies. Peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (Cell Signaling) were administered as secondary antibodies. Blots were visualized using the enhanced chemiluminescence detection system (Amersham, UK). Samples were normalized to GAPDH (Santa Cruz, CA, USA) and quantified by densitometry.

GMSCs were collected and fixed using 80% methanol. Then primary anti-CD44, anti-CD146 and anti-CD45 antibodies (1 μg/10⁶ cells, Abcam) were used to incubate the cells for 30 min at 4 °C. The samples were incubated using the secondary antibodies of PE goat anti-mouse IgG and goat antirabbit IgG (1 μg/10⁶ cells, Cell Signaling Technology) for one hour in the dark. Flow cytometry (FACSCalibur, BD Bioscience) was utilized to test the samples.

The expression of proliferation (e.g., transient receptor potential canonical 1, TRPC1), stemness (e.g., CD146), and neuronal differentiation (e.g., Nestin and MAP-2) markers was determined in d-DPSCs cultured in the different media by immunofluorescence staining as described previously (Luo et al., 2018a (link)). At the designated time points after culture, the cells were fixed in 4% paraformaldehyde for 30 min and incubated for 30 min in a blocking solution containing 5% BSA (Amresco) and 0.1% Triton X-100 (Solarbio) at room temperature. The cells were incubated with the following primary antibodies at 4°C overnight: anti-TRPC1 (1:50; Santa Cruz Biotechnology), anti-CD146 (1:200; Abcam), anti-Nestin (1:1000; Sigma-Aldrich), and anti-MAP-2 (1:500, Sigma-Aldrich). The secondary antibodies were donkey anti-rabbit IgG H&L and donkey anti-mouse IgG H&L (1:500; Abcam). Cell nuclei were stained with DAPI (Beyotime), and images were taken by fluorescence microscopy (Eclipse 80i; Nikon, Japan). Both the culture of undifferentiated DPSCs in complete α-MEM containing 10% FBS for 6 days and the culture of DPSCs in continuous neuronal differentiation medium for 12 days (the neural-induction group) served as control groups.

PDLSCs were cultured on 24-well plates with glass coverslips at a density of 10⁵ cells/well overnight. Then, PDLSCs were fixed with 4% paraformaldehyde and incubated with anti-CD146 (1:100; Abcam, USA), anti-CD105 (1:100; Abcam, USA), and anti-CD45 (1:200; Abcam, USA) primary antibodies. The samples were then treated with rhodamine/FITC-conjugated secondary antibodies (1:1000; Sigma-Aldrich, USA) and stained with 4,6-diamidino-2-phenylindole (Sigma-Aldrich, USA). Jurkat cells were stained with anti-CD45 antibody as a positive control. The images were captured with a fluorescence microscopy (OLYMPUS, Japan).