β casein

Western blotting analysis was performed as previously described [24 (link)]. Briefly, BMECs were harvested on ice using RIPA buffer (Beyotime Biotechnology, Shanghai, China) to which protease inhibitors had been added (2 mM PMSF, 5 mM Na₃PO₄, 5 mM NaF, and complete protease inhibitor) at 48 h post-transfection and then homogenized by ultrasonication. The BCA protein assay was used to measure protein concentration in the samples. Approximately 50 μg total protein was subjected to a 10% SDS-PAGE. The separated proteins were transferred to a nitrocellulose membrane and incubated in blocking buffer. Membranes were then incubated with primary antibodies overnight at 4 °C, followed by incubation with horseradish peroxidase-conjugated secondary antibodys against mouse, rabbit or goat (ZSGB-BIO, Beijing, China) at room temperature for 1 h. Protein bands were visualized using an ECL system. The following primary antibodies were purchased from Santa Cruz Biotechnology: antibodies for phosphorylated signal transducer and activator of transcription-5 (p-STAT5) (1:200 dilution, sc-11,761), signal transducer and activator of transcription-5 (STAT5) (1:200 dilution, SC-836), protein kinase B (also known as AKT1) (1:200 dilution, SC-1618), phosphorylated AKT1 (1:200 dilution, sc-135,650), phosphorylated p70-S6 Kinase (1:200 dilution, p-p706SK) (sc-11,759), p70-S6 Kinase (p706SK) (1:200 dilution, sc-230), Cyclin D1 (1:200 dilution, sc-718), and β-actin (1:400 dilution, sc-47,778); from Cell Signaling Technology: antibodies for IGF1R (1:1000 dilution, #3027), phosphorylated mammalian target of rapamycin (p-mTOR) (1:1000 dilution, #2971) and mammalian target of rapamycin (mTOR) (1:1000 dilution, #2972); and from Abbiotec Company: β-casein antibody (1:200 dilution, #251309). β-actin was used as an endogenous reference gene. All experiments were performed in triplicate. Western blotting data were scanned and quantified using Bandscan5.0 software.

Immunofluorescence assay was carried out as described previously [29 (link)]. BMEC was probed with primary antibodies against the following: β-casein (1:50, Abbiotec, 251309), cytokeratin 18 (Santa Cruz, CA, USA, sc-51582), Tudor-SN (Abcam, ab 71186), p-Stat5 (Tyr694) (Cell Signaling Technology, #9359). Alexa Fluor 488 or Alexa Fluor 647 secondary antibodies (ZSGB-BIO, Beijing, China) were used to detect primary antibodies. Following PI or DAPI staining, the cells were imaged under a TCS-SP2 AOBS confocal microscope (Leica, Heidelberg, Germany).

Western blot analysis was performed as described previously [23 (link),29 (link)]. Briefly, 30 μg of protein equivalents of whole-cell extracts was separated on 10% SDS-PAGE. Gels were transferred to nitrocellulose membranes, then the membranes were blocked in 5% skim milk in pH 7.5 Tris-buffered saline with 0.1% Tween (TBST) for 2 h at 37 °C, then incubated with primary antibodies in 5% skim milk/TBST overnight at 4 °C, and then incubated with horseradish peroxidase-conjugated IgG in 5% skim milk/TBST for 1.5 h at 37 °C. The chemiluminescence detection of HRP-conjugated secondary antibodies was performed using Super ECL plus (ApplyGEN, Beijing, China).
Primary antibodies against the following proteins were used: Tudor-SN (ab 71186), p-NFκB1 p105/p50 (S337) (ab28849), Abcam, Cambridge, MA, USA; mTOR (#2983), p-mTOR (Ser2448) (#5536), and p-Stat5 (Tyr694) (#9359), Cell Signaling Technology, Beverly, MA, USA; cyclinD1 (sc-753), Stat5 (sc-28685), SREBP-1c (the mature form, 68 kD) (sc-365513), β-actin (sc-47778), Santa Cruz, CA, USA; β-casein (Abbiotec, San Diego, CA, USA, 251309).