Fig. 1: Fast and specific postsynaptic proximity biotinylation in genetically identified cortical layer 5 neurons.

a TurboID covalently binds a biotin molecule to nearby proteins. b A cytosolic.turboID construct labels cytosolic proteins. Fusion of TurboID to PSD95 (PSD95.turboID) or Homer1 (Homer1.turboID) directs its enzymatic activity to distinct postsynaptic compartments. c Whole-slice imaging of Tlx3-Cre mice injected with Cre-dependent TurboID constructs labelled for HA-tagged TurboID enzyme and biotinylated proteins. Scalebar is 200 μm. These experiments were independently repeated more than 3 times with similar results. d High resolution imaging shows biotinylated protein patterns from the TurboID constructs. Homer1 and PSD95 immunostaining labels postsynapses. Scalebar is 10 μm and 1 μm in the cropped region. These experiments were independently repeated more than 3 times with similar results. e Western blot quantification shows high levels of biotinylated proteins in dissected somatosensory cortices after 3 h of biotin injection. Tubulin is used as loading control. Molecular weight markers in kDa are indicated on the left (N = 2 mice per time point). Source data are provided as a Source Data file. f Western blot for TurboID (HA) confirms expression of constructs at the expected size. Both TurboID constructs produce strong biotinylated protein signal across molecular weights compared to the no.turboID sample, which shows only bands for endogenously biotinylated proteins. These experiments were independently repeated more than 3 times with similar results. g After streptavidin pulldown, postsynaptic marker proteins Gria1 and Homer1 are only detected in PSD95.turboID and in Homer1.turboID samples. Presynaptic marker Synaptophysin is not detected in any pulldown sample. These experiments were independently repeated more than 3 times with similar results.