Anti pten

The antibodies used were as follows: anti-PTEN (Millipore), anti-Phospho-AKT (Ser473), anti-Phospho-PDK1, anti-PI3K, anti-HKII, anti-PFK1, anti-PKM2, anti-β-actin (all from Cell Signaling), and anti-GLUT1, GLUT2, GLUT3, GLUT4 (all from Cell Abcam, United Kingdom).

Western blot experiments were performed accordingly to the standard procedure38 (link). We tested the following antibodies: anti-GLUT1, anti-HKII, anti-PI3K (Cell Signaling), anti-PTEN (Millipore), anti-Phospho-AKT (Ser473) and anti-β-actin (Cell Signaling).

Immunofluorescence was performed as described [29 (link)]. Briefly, cells were grown on coverslips (Thermo Fisher Scientific), fixed with 4% paraformaldehyde and permeabilized with ice-cold methanol. Cells were rinsed with PBS, blocked with 10% goat serum and then incubated with anti-PTEN (Sigma-Aldrich, 6H2.1, 1:100) antibody overnight, followed by incubation with GFP-conjugated secondary antibodies (Thermo Fisher Scientific, 1:500). coverslips were mounted with ProLong Gold Antifade reagent with DAPI (Thermo Fisher Scientific). The stained slides were visualized using a confocal microscope.

Xenograft tumor samples were embedded in paraffin and fixed with paraformaldehyde. After being washed in PBS, the slides were blocked with protein block solution (DakoCytomation) to obstruct endogenous peroxidase activity. Samples were incubated with the following primary antibodies: rabbit polyclonal anti‐PTEN (Sigma‐Aldrich) or rabbit polyclonal anti‐Ki67 (Abcam, Cambridge, England). The dilutions of antibodies that were used for immunohistochemical localization of ghrelin, GHSR1a, PTEN, and Ki‐67 are 1:100, 1:200, 1:100, and 1:500, respectively. Normal host serum was used for negative controls, followed by staining with appropriate HRP‐conjugated secondary antibodies. The peroxidase was visualized with 3‐3'‐diaminobenzidinetetrahydrochloride solution and then counterstained with a weak hematoxylin solution stain. The stained slides were dehydrated and visualized on an Olympus microscope (Olympus, Shinjuku, Japan) by an investigator that was blinded to the xenograft tumor groups. The stained sections were counted in 10 random views at 200 × magnification. In each view, the grayscale intensity was measured at 20 randomly selected points and averaged. The value of integrated option density (IOD) was proportional to the PTEN protein level, and the proliferation index was expressed as Ki‐67‐positive cells/total cells in the view ×100%.

Western blot analysis was performed as previously described,^{33 (link)} using anti-α-tubulin, anti-Bmi-1(Millipore, Billerica, MA), anti-p21, anti-p27, anti-cyclin D1, anti-Rb, anti-phosphorylated Rb (Abcam, Cambridge, MA), anti-PTEN, anti-AKT, anti-phosphorylated AKT^Thr308 and anti-phosphorylated AKT^Ser473 (Sigma, St. Louis, MO) antibodies.^{34 (link)}