Fig. 3. BDNF regulates trafficking of AMPAR to the glioma postsynaptic membrane.

a, Schematic depicting AMPAR trafficking downstream of BDNF–TrkB–CAMKII signalling⁴⁶. b, Western blot analysis of cell surface and total cell protein levels of GluA4 in SU-DIPG-VI glioma with or without BDNF treatment for 5, 15 and 30 min. c, Quantification of cell surface GluA4 in b (n = 3 independent biological replicates). d, Western blot analysis of cell surface and total cell protein levels of GluA3 in SU-DIPG-VI glioma with or without BDNF treatment for 30 min. e, Quantification of cell surface GluA3 in d (n = 3 independent biological replicates). f, Western blot analysis of cell surface and total cell protein levels of GluA4 in SU-DIPG-VI cells treated with NLGN3 for 30 min. g, Quantification of cell surface GluA4 data in f (n = 3 independent biological replicates). h, Schematic showing GluA2–SEP experiments. i,j, Validation of pHluorin approach. i, Left, representative images of a glioma cell process expressing GluA2(Q)–SEP, PSD95–RFP and whole-cell TagBFP in co-culture with neurons. Right, representative GluA2(Q)–SEP puncta. Scale bars, 5 µm (left) and 1 µm (right). Cells were exposed to pH 7.4 followed by pH 5.5 and then pH 7.4. j, Quantification of fluorescence intensity of GluA2(Q)–SEP puncta before, during and after acidic exposure (n = 4 puncta from a representative cell). k, Top, representative images of two processes from glioma cells expressing GluA2(Q)–SEP, PSD95–RFP and TAG-BFP2 in co-culture with neurons (scale bar, 5 µm). Middle and bottom, representative images of GluA2(Q)–SEP puncta at 0, 5, 15 and 20 min of BDNF incubation (scale bar=1 µm). l, Fluorescence intensity of co-localized GluA2(Q)–SEP:PSD95–RFP puncta over time with BDNF treatment (n = 8 puncta, 6 cells). m, Fluorescence intensity of co-localized GluA2(Q)–SEP:PSD95–RFP puncta after 15 min versus basal fluorescence in control (vehicle, n = 4 puncta, 2 cells) or BDNF-treated cells (n = 8 puncta, 6 cells). Data are mean ± s.e.m. Two-tailed unpaired Student’s t-test (c,e,g,m); two-tailed paired Student’s t-test (j); two-tailed one-sample t-test (l).