Sc 9095

Cells were lysed by sonication in ice-cold RIPA buffer (20 mM Tris-HCl pH 7.6 containing 150 mM NaCl, 2 mM EDTA, 1% NP40, 0.5% Na-deoxycholate) supplemented with protease inhibitor cocktail (Sigma-Aldrich P2714). Equal amounts of proteins were resolved on 8-10% SDS polyacrylamide gel. After being transferred onto PVDF membranes (Amersham GE Healthcare, Buckinghamshire, UK), proteins of interest were detected by using anti-CD34 (sc-9095 Santa Cruz, 1 : 500), anti-myosin (MF20 1 : 50), anti-MyoD (sc-377460 Santa Cruz, 1 : 1000), anti-myogenin (sc-12732 Santa Cruz, 1 : 300), anti-hif-1α (NB100-105 Novus Biologicals, Segrate, Italy, 1 : 1000), and anti-caveolin-1 (ab17052 Abcam, Cambridge, UK 1 : 1000) antibodies and anti-α-tubulin (T5168, Sigma, 1 : 2000) or anti-GAPDH (Santa Cruz, sc-25778, 1 : 1000) antibodies as a normalization control.

Cells were seeded on gelatin-coated circular micro cover glass (d = 18 mm, Matsunami, Japan) submerged in a 60-mm dish (TPP, Switzerland). After 7 days of treatment with/without 200 µM CoCl₂, cells were washed with phosphate buffered saline (PBS) 3 times and then fixed with 4% paraformaldehyde (Nacalai-Tesque) at room temperature for 20 min followed by blocking with 10% FBS in PBS containing 0.05% Tween 20 (Nacalai-Tesque) for 1 h at room temperature. The cells were then incubated with anti-CD34 rabbit polyclonal antibody (H-140) (1:50, sc9095, Santa Cruz, CA) or anti-mouse TER-119/erythroid cells rat monoclonal IgG (1:100, 116,201, BioLegend, CA) diluted with blocking solution overnight at 4 °C. After PBS washes, anti-rabbit IgG linked with Alexa Fluor 555 (1:1000, A21427, Invitrogen, CA) was used to detect anti-CD34 antibody and anti-rat IgG linked with Texas red (1:1000, T6392, Invitrogen) was used to detect anti-TER-119 antibody. Incubation with the secondary antibody was performed for 1 h at room temperature. After PBS washes, the samples were prepared on glass slides (Matsunami) using VECTASHIELD Mounting Medium with 4', 6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, CA). Images were taken using an inverted light microscope equipped with a fluorescence light device (IX-80, Olympus, Japan).