Fig 1: Profile of Developmental Expression in the Forebrain and Subcellular Distribution of PRG2 in Neurons(A) Prg2 expression in vivo peaks at birth. Rat brain lysates isolated at different developmental stages were analyzed by WB using the indicated antibodies. E, embryonic day; P, postnatal day; W, weeks. Prg2 and Prg1 expression was quantified at three time points from five independent animals. Bars represent protein levels relative to tubulin ± SEM. ***p < 0.001, n.s. = not significant (one-way ANOVA with Bonferroni post hoc test).(B) Coronal sections through the mouse forebrain at E16.5 labeled with anti-L1 and anti-PRG2 antibodies show axonal localization of PRG2. Nuclei were visualized by Hoechst. Scale bar: 200 µm.(C) Prg2 expression in primary neurons. PRG2 and PTEN levels in cortical neurons at different days in vitro (DIV) analyzed by WB.(D) Subcellular distribution of PRG2 in hippocampal neurons cultured for 9 DIV and stained with anti-MAP2, anti-PRG2, and Phalloidin (F-actin). A single, confocal section at high magnification reveals punctate labeling of PRG2 at the membrane of axons. Scale bar: 2 µm. PRG2 membrane enrichment was measured as a percentage of processes with plasma-membrane-enriched PRG2 and the ratio of cell edge to middle intensity. Results are means ± SEM of 21 (axonal) and 27 (dendritic) measurements from 12 axonal and dendritic processes. ***p < 0.001 (t test).(E) Structured illumination microscopy (SIM) of PRG2 and F-actin in 9 DIV hippocampal neurons. Cells were stained with anti-PRG2 antibody and Phalloidin. Single sections show regular PRG2 puncta at the plasma membrane. Scale bar: 0.5 µm.(F) SIM of 9 DIV hippocampal neurons treated with 5 µm latrunculin B for 1 h or 3 h and labeled with anti-Tau and anti-PRG2 antibodies. Scale bar: 1 µm. Bar graph demonstrates the increase in mean distance between two consecutive PRG2 spots following latrunculin B treatment. Results are means ± SEM from n = 9 neurons per condition, with =10 µm axon lengths per neuron analyzed. ***p < 0.001 (one-way ANOVA with Bonferroni post hoc test).See also Figures S2 and S3.