Figure 6A. Coexpression of Lrig1 (red) and TrkB (green) in primary rat hippocampal neurons. Boxed area represents a higher-magnification image showing a high colocalization between TrkB and Lrig1 in a pyramidal neuron. Yellow indicates regions of colocalization. Scale bar, 30 m. Data represent n=3 independent experiments. B. Quantitative analysis of Lrig1 mRNA expression by real-time RT-PCR. Rat hippocampal cultures (10 DIV) were treated with BDNF (50 ng/ml) during the indicated times. The levels were normalized using the expression of the housekeeping gene Tbp. The results are shown as mean SD of n=3 independent experiments. *p<0.05 vs. control group by one-way ANOVA followed by Dunnetts test. C, D. Coimmunoprecipitation between Flag-Lrig1 and HA-TrkB (C) or between Flag-Lrig3 and HA-TrkB (D) overexpressed in HEK293 cells. Cell extracts were analysed by immunoprecipitation with anti-Flag antibodies followed by immunoblot (IB) with antibodies against HA. Reprobing of the same blots with anti-Flag antibodies is shown below. The bottom panel shows HA expression in total lysates. Data represent n=3 independent experiments. E. Coimmunoprecipitation between HA-TrkB and Flag-Lrig1 or between HA-TrkB and HA-Lrig2 exogenously expressed in HEK293 cells. Cell extracts were analysed by IP with anti-pan-Trk antibodies followed by IB with antibodies against Flag or Lrig2. Reprobing of the same blots with anti-TrkB antibodies is shown below. The bottom panels show Flag and Lrig2 expression in total lysates. Data represent n=2 independent assays. F. In vivo interaction between Lrig1 and TrkB. Coimmunoprecipitation between Lrig1 and TrkB receptor endogenously expressed from P15 rat hippocampal tissue extracts. Samples were equally divided in two parts and then the analysis was done by immunoprecipitation with control (anti-HA epitope tag antibody) or anti-Lrig1 antibodies, followed by immunoblotting with anti-TrkB antibody. Reprobing of the same blot with anti-Lrig1 antibody is also shown. Expression of TrkB in one aliquot of the starting material (indicated as lysate) is included. Arrow indicates the band of TrkB coimmunoprecipitated with anti-Lrig1 antibody. Similar results were obtained in n=3 independent assays. G. Ligand-dependent activation of TrkB (p-TrkB) was evaluated by transient transfection of HA-TrkB plasmid with either a control or a Flag- Lrig1 vector into HEK cells. After 36 h cells were serum-starved and stimulated with or without BDNF (30 ng/ml) for 15 min. The level of TrkB activation (p-TrkB) was evaluated in total cell lysates by immunoblotting (IB) with a specific antibody that recognizes TrkB phosphorylated in tyrosine 705 (pY705). Reprobing of the same blot with anti-HA and anti-Flag antibodies is shown. Fold of p-TrkB change relative to total TrkB is indicated. Similar results were obtained in n=3 independent assays. H. TrkB ubiquitination was evaluated by transient transfection of HA-TrkB plasmid with either a control or a Flag-Lrig1 vector into MN1 cells. After 36 h, cells were serum-starved, pre-treated with the cell-permeable proteasome inhibitor MG-132 (20 M) and stimulated with BDNF for 15 min. Total lysates were immunoprecipitated with anti-HA antibodies followed by immunoblot (IB) with antibodies against ubiquitin. Reprobing of the same blot with anti-HA antibodies is also shown. TrkB activation (p-TrkB) was evaluated in cell lysates. Reprobing of the same blot with anti-TrkB and anti-Flag antibodies is also shown. Fold of p-TrkB (p-Y705) change relative to total TrkB is indicated. Data represent n=3 independent assays.