Mab4419

Protein lysates (n = 3 for SS-hAFSCs and n = 3 for RS-hAFSCs) were generated using RIPA lysis buffer (150 mM sodium chloride, 1.0%Triton X-100, 0.5% sodium deoxycholate, 50 mMTris, pH 8.0, 1:100) containing 0.1% SDS. 25 μg of β-mercaptoethanol. Denatured lysates were then separated on an 8% -PAGE gel and blotted onto a Protran nitrocellulose transfer membrane (Whatman, Life Sciences). The membrane was blocked in 5% milk PBS-T (phosphate-buffered saline with 0.1% Tween-20) and immunoprobed with antibodies raised against different peptides containing primary antibody overnight at 4 °C: SC-5279 (Santa Cruz, 1:200), SC-8628 (Santa Cruz, 1:200), SC-9081 (Santa Cruz, 1:200), MAB4419 (Millipore, 1:200), AB198579 (Abcam, 1:200), MAB17591 (R&D systems, 0.5ug/ml), IC1759P (R&D Systems, 0.5ug/ml). The secondary antibodies used were Anti–mouse IgG HRP linked antibody (Cell signaling, 1:1000), Rabbit anti-rat HRP Conjugated (Thermo Fisher, 1:1000), Donkey anti-rabbit IgG HRP-linked (VWR, 1:1000), Donkey anti-goat IgG HRP-linked (Santa Cruz, 1:500). The loading control was β -actin (Abcam, 1:1000). The experiments were performed in triplicate.

Lin⁻/Sca-1⁺/CD45⁻ cells (VSELs) were sorted, plated on 22-mm diameter plates coated with poly-L-lysine (P9155, Sigma-Aldrich, St. Louis, MO, USA), and incubated for 24 hrs. Subsequently, cells were fixed in 3.5% paraformaldehyde for 15 min., permeabilized by 0.1% Triton X100, washed in PBS, and pre-blocked with 2.5% BSA for 2.5 hrs at RT to avoid nonspecific binding of antibodies. The cells were then stained overnight at 4°C for Oct-4 (mAb, 1:150, MAB4419; Millipore, Billerica, MA, USA) and 1 hr at 37°C with an antibody to nestin (mAb, 1:150, MAB6767; Abnova, Atlanta, GA, USA). Appropriate secondary Alexa Fluor 488 goat antimouse IgG and Alexa Fluor 594 goat anti-rat IgG antibodies were used (1:500; Molecular Probes, Grand Island, NY, USA, A11001 and A11007 respectively) at 37°C for 1 hr. Nuclei were stained with DAPI (Invitrogen, Foster City, CA, USA) for 20 min. at 37°C. All images were captured with an Olympus FV1000 confocal microscope.