Anti syndecan 1

All the antibodies were obtained from commercial sources as follows: anti-MMP-7, anti-MMP-9, anti-heparanase, anti-syndecan-1 and anti-HB-EGF (Proteintech Group, Inc., Chicago, IL, USA), anti-Ras antibody (Upstate Biotechnology, Inc., Waltham, MA, USA), anti-EGFR, anti-phosphorylated-EGFR (Tyr. 1068), anti-extracellular signal-regulated kinase (ERK)1/2, anti-phosphorylated-ERK1/2 (Thr-202/Tyr-204), anti-MAPK/ERK kinase (MEK)1/2 and anti-phosphorylated-MEK1/2 (Ser-217/221) (Cell Signaling Technology Inc., Beverly, MA, USA), and anti-GAPDH (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

Brain tissues from 2 to 4 mm posterior to the frontal pole were solubilized and immunoblotted as previously reported (28 (link)). In brief, protein extracts were separated on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gels and then transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% bovine serum albumin in Tris‐buffered saline with 0.1% Tween‐20 (TBST), and then incubated overnight at 4°C with the following primary antibodies: anti‐Cav1 (1:1000, Abcam), anti‐phospho‐Cav1 (1:1000, Cell Signaling Technology), anti‐syndecan1 (1:600, Proteintech), anti‐PY102 (1:1000, Cell Signaling Technology), and anti‐GAPDH (1:10,000, Proteintech). After washing with TBST, the blots were incubated with HRP‐conjugated secondary antibodies (1:20,000; Abcam) and were then developed by incubation with Luminata Crescendo Western HRP substrate (Millipore). Protein bands were imaged, and their densities were quantified using ImageJ after normalization to GAPDH.