Fig. 6
Loss of ZDHHC3 virtually phenocopies the defects of myelination in Cadm4-KI. Brain lysates of WT and ZDHHC3-KO mice were evaluated for the level of palm-Cadm4 by Acyl-RAC assay (a), and quantified (b, two-tailed t-test, n = 3, **p ≤ 0.01). c, d Corpus callosum was isolated from prefixed brains of WT and ZDHHC3-KO mice and examined for myelination under electron microscopy. Abnormal myelination is pointed out by red arrows: loss of myelination, detachment of axon from myelin sheath, thickened myelin sheath/hypermyelination (c); myelinated axons per field of view (****p < 0.0001, t-test, n = 31–49), % of abnormal myelination (****p < 0.0001, t-test, n = 48–50) and g-ratio (****p < 0.0001, t-test, n = 414–476) were quantified from 4–6 mouse (d). e Lysates of corpus collosum from WT and ZDHHC3-KO mice were evaluated for MBP level by WB, and quantified (two-tailed t-test, n = 3, **p ≤ 0.01). Mice brains were processed for transparency with X-clarity and stained for MBP, NF (neurofilament) and DAPI for imaging (f), % of myelination and MBP fluorescence intensity were quantified (g, two-tailed t-test, n = 6, **p ≤ 0.01, ****p < 0.0001). Frozen coronal sections of corpus callosum were stained with Cadm4, APC and DAPI for imaging (h), the fluorescence intensity of Cadm4 and APC was quantified (i, two-tailed t-test, n = 6, **p ≤ 0.01, ****p < 0.0001). j PM fractions were prepared from corpus callosum of WT and ZDHHC3-KO mice and analyzed for the levels of Cadms and Mag by WB and quantified (two-tailed t-test, n = 3, **p ≤ 0.01). Immunoprecipitations were performed with the brain lysates of WT and ZDHHC3-KO mouse by using Cadm4 antibody, the IP eluent was analyzed for Cadm4 binding proteins (k), and quantified (l, two-tailed t-test, n = 3, *p ≤ 0.05, **p ≤ 0.01, ***p < 0.001). Data are represented as mean ± SEM
