Bcip nbt kit

Confluent EPCs (passage 2–4) were washed with DPBS, fixed with 4% paraformaldehyde (Sigma, USA), then incubated with Ulex Europaeus agglutinin 1(UEA-1, Vector Laboratories, USA) in the dark at 37°C for 20 min. Cells were washed twice with DPBS, fixed with 4% paraformaldehyde for 30 min, DAPI staining (Sigma, USA) was used to mark nuclei. Samples were observed under fluorescence microscopy.
Confluent EPCs (passage 2–4) were incubated with Alexa Flour 488 acetylated low-density lipoprotein (DiI-ac-LDL; Molecular Probes/Life Technologies) at 37°C for 4 h. Cells were washed twice with DPBS and fixed with 4% paraformaldehyde for 30 min; DAPI staining was used to mark nuclei. Samples were observed under fluorescence microscopy.
MSCs were induced to differentiate into adipose cells or osteocytes as previously described [15 (link)]. Briefly, the MSCs were induced in adipogenic differentiation medium or osteogenic differentiation medium for 14 days. Induced adipose cells were detected by oil red O staining (Sigma, USA) at 0, 7 and 14 days. Induced osteocytes were detected at 0, 7 and 14 days using a BCIP/NBT kit (Invitrogen, USA) to test for the expression of alkaline phosphatase.

The cells were lysed in RIPA buffer containing a phosphatase and protease inhibitor cocktail (EMD Millipore, Temecula, CA, USA). Thirty micrograms of protein was separated by 10% SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes (Millipore Corp., MA, USA), and incubated with rabbit anti-phospho-mTOR (1:1000), anti-mTOR (1:1000), anti-phospho-AKT (1:1000), anti-AKT (1:1000), anti-PI3K (1:1000), or anti-β-actin (1:1000) antibodies overnight at 4 °C. The membranes were then incubated with alkaline phosphatase-conjugated anti-rabbit IgG antibodies (1:2000; Cell Signaling Technology, Danvers, MA, USA) for 1 h and visualized using a BCIP/NBT kit (Invitrogen, Carlsbad, CA, USA).

For protein level analysis, proteins were isolated from PSC samples as described [17 (link)] and protein concentration was measured using the BCA protein assay. The proteins were separated through 12% SDS-PAGE and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore Corp., MA, USA). After blocking, the membranes were incubated with mouse anti-rEgTPx serum (1:1000) or rabbit anti-β-actin serum (Santa Cruz Biotechnology, CA, USA. 1:2000) overnight at 4 °C followed by incubation with AP-conjugated second antibody (Cell Signaling Technology, Danvers, MA, USA). Protein bands were visualised with a BCIP/NBT kit (Invitrogen, Carlsbad, CA, USA). The expression levels of the respective proteins were quantified using Quantity One software (Bio-Rad) after scanning of the membranes.