Ab140126

Western blot analysis was performed with following antibodies (all from Abcam)²⁴: anti‐rabbit antibodies to FOXP3 (ab215206), SCP2 (ab140126), vascular endothelial growth factor (VEGF; ab10766), Ki67 (ab92742), CyclinD1 (ab16663) and Brachyury (ab209665) as well as horseradish peroxidase (HRP)‐labelled goat anti‐rabbit antibody (1: 5000). After washing, enhanced chemiluminescence was used to develop images. The optical density (OD) value of the protein bands was measured by a gel imaging analysis system with relative protein content as the ratio of average OD value of the target to that of the internal control.

The following reagents were obtained: human pituitary gland RNA standard (Clontech, 636157), methyl-β-cyclodextrin (Mβ-CD; Sigma, C4555), methyl-β-cyclodextrin cholesterol complex (Mβ-CD/CHO; Sigma, C4951), itraconazole (MedChemExpress, HY-17514), vismodegib (MedChemExpress, HY-10440), forskolin (MedChemExpress, HY-15371), ezetimibe (MedChemExpress, HY-17376), Rapamycin (Beyotime, Nanjing, China, S1842).
Antibodies against the following proteins were used in this study: SCP2 (Abcam, ab140126), protein kinase A (PKA; Abcam, ab75991), suppressor of fused (SUFU; Proteintech, Wuhan, China, 26759-1-AP), glioma-associated oncogene 1 (GLI1; Santa Cruz, sc-515751 and sc-515781), B cell lymphoma 2 (BCL2; Santa Cruz, sc-7382), cyclin D1 (CCND1; Abcam, ab134175), phosphorylation-protein kinase B (p-AKT; Cell signaling technology, 4060), protein kinase B (AKT; Cell signaling technology, 4691), phosphorylation-Mammalian Target of Rapamycin (p-mTOR; Abcam, ab84400), Mammalian Target of Rapamycin (mTOR; Abcam, ab 32028), β-actin (Abcam, ab8226) and GH (growth hormone; Santa Cruz, sc-374266).

Approximately 40 μg protein per sample was separated on 12% SDS (sodium dodecyl sulfate) polyacrylamide gels. Proteins were transferred onto 0.45 μm PVDF membranes (IPVH00010; Millipore, Boston, Massachusetts, USA) and blocked with blocking buffer (Beyotime, Jiangsu, China). The membranes were incubated with primary antibodies to SCP2 (non-specific lipid-transfer protein 2, ab140126; Abcam, Cambridge, MA, USA), IDH2 (isocitrate dehydrogenase 2; ab131263, Abcam), SLC7A8 [(solute carrier family 7 (amino acid transporter, L system), member 8, ab75610, Abcam)], COL4A2 (collagen, type IV, alpha 2, sc-70,243, Santa Cruz biotechnology; Cambridge, MA, USA), and β-actin (Beyotime). After washing with TBST [tris-buffered saline containing 0.02% (v/v) Tween-20] three times, the membranes were incubated with goat anti-rabbit IgG or goat anti-mouse IgG secondary antibodies conjugated with horseradish peroxidase (Beyotime), incubated with ECL (electrochemiluminescence) Western Blotting Substrate Kits (Beyotime), and finally visualized with a Kodak Image Station 2000MM (Kodak Molecular Imaging Systems, New Haven, USA). The relative intensities of bands were calculated with ImagePro Plus 6.0 software (Media Cybernetics, Washington, MD, USA) using β-actin as the reference protein.