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. 2017 May 24;37(21):5232–5249. doi: 10.1523/JNEUROSCI.2630-16.2017

Figure 11.

Figure 11.

Axonal accumulation is impaired in cleavage-resistant variants of NRG3 and CRD-NRG1. A, Schematic illustration of the strategy to render dual-pass NRGs resistant to processing near the TMC. Briefly, the area between the EGF-like and TM domains of NRG3 and CRD-NRG1 was replaced by the shedding-resistant juxtamembrane region of the JM-b isoform of ErbB4 (Elenius et al., 1997). B, Western blot analysis of whole-cell lysates from hippocampal neurons transduced with AAVs harboring cleavable (wt) or cleavage-resistant (cr) variants of CRD-NRG1/V5 and NRG3/V5. Arrowhead indicates bands representing uncleaved proforms. Arrows indicate processed proteins. C, D, Lack of axonal accumulation of cleavage-resistant NRG variants in neurons transfected with crNRG3/V5 (C) or crCRD1-NRG1 (D) along with GFP and wtCRD-NRG1/HA (included as internal reference). Top, Neurons were triple-labeled with anti-ErbB4, anti-HA to reveal sites of axonal CRD-NRG1 accumulation, and anti-V5 to detect crNRG variants. GFP-positive axonal processes forming varicosities across from ErbB4 puncta (left) harbor wtCRD-NRG1 puncta (right) but lack corresponding signals for crNRG3 or crCRD-NRG1 (middle). Bottom, In parallel experiments, transfected neurons were labeled with antibodies against the ECDs (V5) and the carboxyl-terminal ICDs of crNRG3 or CRD-NRG1, as described in Figure 10. Representative images of transfected neuron cell bodies (left) show accumulation of crNRGs with both ECD (middle) and ICD (right) antibodies. E, Representative confocal image of a crNRG3/V5-expressing hippocampal neuron labeled with anti-V5 and an antibody against the trans-Golgi marker protein TGN-38. F, Neurons were transfected with GFP and V5-tagged NRG3(Q360*) lacking the TMC and downstream sequences. Representative confocal images of GFP-positive processes from transfected neurons labeled for V5 and ErbB4 showing NRG3(Q360*)-puncta under untreated control conditions (top) and after BACE-IV treatment (bottom). Scale bars: C, Top, D, Top, 5 μm; C, Bottom, D, Bottom, E, F, 10 μm.