α actin sc 1616

MCF-7 cells were plated in T25 flask at a density of 2 × 10⁶ cells/flask. At about 70% confluence, the culture medium was removed, and cells incubated with fresh complete medium for 48 h. For abrogation of the effect of c10orf1118 protein, CM from MCF-7 cells was harvested, centrifuged to remove cell debris and incubated with gentle agitation for 1h at 37 °C with 4 µg/mL (final concentration) of anti-c10orf118 rabbit polyclonal antibody (HPA018019; Sigma). The same concentrations of α-actin (sc-1616; Santa Cruz) were used as control. NHDF cells were plated in 6-well at a density of 5 × 10⁵ cells/well. Then, 800 µL of pre-incubated CM with blocking antibodies were added. After 48h, NHDF cells were harvested for RNA in TRI Reagent^® to study HASes gene expression.

Preparation of whole cell protein extracts was performed as described previously (21). Samples equivalent to 30 μg of protein (Bio‐Rad protein assay) were resolved on 8% SDS polyacrylamide gels and transferred to enhanced chemiluminescence nitrocellulose membranes (ECL; Amersham, Little Chalfont, UK). The antibodies used were α Runx1 #8229 (Cell Signalling Technologies) and α actin sc1616 (Santa Cruz).