Fig. 2. GluN2B-NMDAR signaling is highly activated in human brain metastasis.

a-b, Representative images (a) and quantification (b) of IHC staining of pGluN2B Y1472 in paired human primary BC (primary) and brain metastases (brain mets). Mean ± s.e.m. Two sided Wilcoxon two-tail test. Scale bar, 100 μm. BC subtype: 3 Her2⁺, 1 luminal and 5 unknown.

c, Western blotting of pGluN2B Y1252, pGluN2B Y1472, GluN2B, and GluN1 in pairs of human (left column, MDA231) and mouse (right column, TS1) parental BC cell lines and their corresponding B2BM derivatives. Three independent experiments. Protein levels in B2BM cells were quantified relative to cognate parental cells, after normalization to ß-actin or GAPDH controls, for which blotted membranes were cut into two parts, of <50 K for beta-actin or GAPDH staining, and 50-250 Kd for pGluN2B Y1252/Y1472 staining. Additional, equally loaded gels were blotted and stained for GluN2B or GluN1, using the loading control from the pGluN2B Y1252 gel.

d, Schematic of assays performed with luciferase-expressing B2BM cells either inoculated orthotopically into the mammary fat pad (MFP) to develop primary tumours, or intracardiac (ICD) to seed brain mets, or intravenous (IV) into the tail vein to seed lung mets.

e,f, IHC staining of pGluN2B Y1472, total GluN2B, and luciferase (e) and quantification of pGluN2B Y1472 staining (f) in primary tumours, brain mets, and lung mets formed by MDA231-B2BM cells (day 28~35 after injection). Scale bar, 10 μm. These images correspond to the inserts shown in Extended Data Figure 2l. Two-tailed Student t-test, n = 3 mice per group, mean ± s.e.m.