Mab17591

Cells (n = 3 for SS-hAFSCs and n = 3 for RS-hAFSCs) were washed, fixed in 4% paraformaldehyde (PFA, Sigma) and permeabilized. Cells were then blocked for 30 min with blocking buffer (PBS supplemented with 2% bovine serum albumin (BSA) and 0.1% Tween) and incubated overnight in the dark with primary antibodies at their optimal dilution, i.e. SC-5279 (Santa Cruz, 1:200), SC-8628 (Santa Cruz, 1:200), SC-9081 (Santa Cruz, 1:200), 130-105-606 (Miltenyi Biotec, 1:100), MAB4419 (Millipore, 1:200), AB198579 (Abcam, 1:200), MAB17591 (R&D systems, 10ug/ml), IC1759P (R&D Systems, 10ug/ml), then washed and incubated with secondary antibody (Alexa Flour 488 Goat anti-rabbit IgG, Alexa Flour 488 Goat anti-mouse IgG, Alexa Flour 488 Donkey anti- goat IgG and Alexa Flour 488 Donkey anti- rat IgG (all from Invitrogen 1:500) for 1 hr at RT. Then counter-stained with 4′,6-diamidino-2-phenylindole (DAPI) and visualized immediately. Images were collected using a LeicaDM 6000 fluorescence microscope (40x PLAN APO objective) and transferred to Adobe Photoshop (Adobe Systems).

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.