Amicon ultracell 10 k mwco centrifugal filter devices

Four independent experiments were performed, where each experiment utilized freshly extracted HESCs from a pooled sample of n = 3 placentas. Equal aliquots of samples from in vitro decidualized cells were used. Decidualized primary HESCs either incubated under hypoxic conditions or silenced for TSC2 (siRNA) with normoxia or hypoxia were pooled and enriched (5-fold) using Amicon Ultracell 10 K MWCO centrifugal filter devices (Sigma Aldrich) and buffer-exchanged with PBS (Gibco). Samples were immunoprecipitated (IP) with well-established IGFBP-1 mouse monoclonal anti-human (mAb) 6303 IGFBP-1 antibody (Medix Biochemica) [43 (link)] using Protein A Sepharose beads (50 µL, 50% slurry; GE Healthcare Bio Sciences AB, Uppsala, Sweden), as described previously [32 (link)]. The IP samples were washed and then a small aliquot was analyzed by Western blot using anti-human IGFBP-1 polyclonal antibody or anti-human IGFBP-1 phospho Ser101, Ser119, and Ser169 antibodies. This preliminary step confirmed the validity of IP procedure to determine that IGFBP-1 was IP in the samples further used for parallel reaction monitoring mass spectrometry (PRM-MS). The remainder of the IP samples were digested in-solution as described below for PRM-MS analysis. IGFBP-1 also co-immunoprecipitated protein kinases CK2 and PKC which were monitored to determine their activation status.

Samples of CM from decidualized primary HESCs grown either under normoxia or hypoxia were collected to perform quantitation of total IGFBP-1 using IGFBP-1 using Immunoenzymometric Assay (IEMA) (Medix Biochemica, Kauniainen, Finland), against IGFBP-1 standard concentrations provided by the assay kit. Prior to IEMA quantitation, CM samples were first buffer exchanged with specialized cell media (high glucose DMEM with sodium pyruvate; Gibco). The buffer exchanged CM was enriched (10-fold) using Amicon Ultracell 10 K MWCO centrifugal filter devices (Sigma Aldrich) and used to perform IEMA as per manufacturer’s instructions.