Species specific elisa

Western blot (WB) and co-immunoprecipitation (co-IP) assays were performed as described before [34 (link)]. Actin was used as a loading control for WB, whereas co-IPs were normalized with the antibody used to pull down. Non-immune serum (NIS) was used as a negative control for IPs while total cells lysate as a positive control. See Supplementary Table 1 for antibodies used. IGF-I in serum and tissues was determined using a species-specific ELISA (R&D Systems, USA), as described in detail elsewhere [35 (link)]. E2 was evaluated in serum by ELISA (Enzo Life Sciences, USA), following the manufacturer´s instructions. Blood was collected from the heart after pentobarbital anesthesia before trans-cardiac perfusion.

Western blot (WB) was performed as described58 (link). IGF-I in serum and tissues was determined using a species-specific ELISA (R&D Systems, USA), as described59 (link). Blood was collected from the heart after pentobarbital anesthesia before trans-cardiac perfusion.