Odyssey clx infrared imager

All immunoblots were visualized by fluorescently labeled secondary antibodies, and quantified on Odyssey CLx Infrared Imager and Odyssey software (LICOR Biosciences). Signals were normalized to human GDI as the neuronal loading control.

To analyze the cleavage activity of the two ChlaDUBs, purified proteins were combined at a final concentration of 100 nM enzyme (ChlaDUB1 or ChlaDUB2), 2000 ng of diubiquitin (or 1000 ng of tetraubiquitin (Boston Biochem)) and incubated at room temperature for 2 hours in reaction buffer (50 mM Tris pH 7.4, 50 mM NaCl, 1 mM DTT). The reaction products were analyzed by SDS-PAGE and probed by immunoblotting with primary antibodies specific for Ub (Anti-ubiquitin #3933, Cell Signaling, 1:1000) and Licor IRDye® 680LT anti-mouse secondary (1:10,000). Immunoblots were imaged using a Licor Odyssey CLx infrared imager.

Cortical cultures from γ2^+/− and WT mice were generated from embryonic day 14–15 embryos (129X1/SvJ line), subject to cell surface biotinylation at 21 days in vitro (DIV) and purification using NeutrAvidin agarose beads (Thermo, Rockford, IL) as described (45 (link)). For biotinylation of brain slices we adapted the protocol of Terunuma et al. (46 (link)). The biotinylated proteins were quantitated by SDS/PAGE/western blot using an Odyssey® CLx infrared imager (LI-COR, Lincoln, NE). Amounts of cell surface biotinylated proteins were normalized to amounts of β-tubulin in total extracts quantitated on parallel gels.

Animals of the desired genotype, aged P7–P10, were sacrificed. Cortical sections were obtained and dissociated using a previously published protocol (Hempel et al., 2007 (link)). Dissociated cells from the cortical slices were then subjected to FACS (MoFlo, Beckman Coulter) to obtain EGFP+/td-Tomato interneurons. On average, the percentage of GFP+ interneurons was 2% –5% for P7–P10 ages. Collected cells were subsequently used to obtain protein by lysing in radioimmunoprecipitation assay (RIPA) buffer (50 nM Tris [pH 8.0], 150 mM NaCl, 1 % Triton X-100, 0.10% SDS, and 1 mM EDTA, water-adjusted to 100 mL). The protein was analyzed by western blot. The blots were blocked with 5% milk and probed for an antibody mix consisting of Rb-Cnb (Upstate Cell Signaling, 07-069, 1:1,000), m-α-actin (1:2,000), Rb-S774 Dynamin 1 (Abcam, ab55324, 1:1,000), total dyna-min 1 (Abcam, ab3456, 1:1,000), Rb-CNA1 (Millipore, 07-1490, 1:500), Rb- CNA2 (Abcam, ab96573, 1:500), and Rb-CNA3 (Abcam, ab154863, 1:1,000). Blots were imaged using an Odyssey CLx infrared imager and analyzed using ImageStudio software (LI-COR Biosciences) or using the enhanced chemiluminescence method. Fluorescent signaling for Cnb was normalized using β-actin.