Quantification of PLP/DM20 and MBP Levels

Prefrontal cortex (frontal to bregma 5.22) and cerebellum were dissected from mice and homogenized in 1× TBS with protease inhibitor (Complete Mini, Roche). Protein concentration was measured using the DC protein assay (Bio-Rad). Immunoblotting displayed in Figure 1C and D was essentially as described (Kusch et al., 2017 (link); Schardt et al., 2009 (link)). Briefly, lysates from prefrontal cortex (3.2 µg for PLP/DM20 and 1 µg for actin) and cerebellum (0.8 µg for PLP/DM20 and 1 µg for actin) were separated on 15% SDS-polyacrylamide gels and blotted onto PVDF membranes (Hybond, Amersham) using the Novex Semi-Dry Blotter (Invitrogen). Primary antibodies were incubated in 5% milk powder in TBST over night at 4°C. Primary antibodies were specific for PLP/DM20 (A431; 1:5000; Jung et al., 1996 (link)), MBP (1:500; DAKO), and actin (1:1000 for cerebellum, 1:5000 for prefrontal cortex; Sigma). Secondary HRP-coupled antibodies (dianova) were detected using the ChemoCam system (Intas).