Inhibition of USP14’s DUB activity leads to enhanced activation of the MLK3 signaling cascade. (A) Representative immunoprecipitates (IP) of MLK3 from spinal cord lysates from 4- to 6-week-old wild type and TgUsp14CA mice, immunoblotted (IB) for K63-linked ubiquitin, phospho-serine/threonine, and MLK3. (B) Quantitation of (A), K63- and pSer/Thr- modified MLK3 was normalized to total immunoprecipitated MLK3. (C) Representative immunoblots of wild type and TgUsp14CA spinal cord extracts probed for pMKK4 and total MKK4. β-tubulin was used as a loading control. (D) Quantitation of (C), pMKK4 was normalized to MKK4. (E) Representative immunoblots from spinal cords from 4- to 6-week-old wild type, TgUsp14CA, ax
J, and TgUsp14 mice, probed for pJNK1/2 and total JNK1/2. JNK 1 and 2 migrate to 46 and 54 kDa, respectively. β-tubulin was used as a loading control. (F) Quantitation of (E), pJNK was normalized to JNK and quantitation includes both the 46 and 54 kDa bands. Data in (B), (D), and (F) are shown as mean ± SEM, and n = 3 animals per genotype. Symbols represent Mann-Whitney tests compared against wild type mice and corrected for multiple comparisons with a Bonferonni adjustment where appropriate; *p < 0.05, **p < 0.01, ***p < 0.001. (G) Representative images of whole-mount immunostaining of TA muscles from 4-week-old wild type and TgUsp14CA mice using a pJNK antibody (blue). AChRs were labeled with rhodamine-conjugated α-BTX (red), and motor neuron axons were visualized via expression of YFP under the Thy1.2 promoter (green). Scale bars = 50 μm. (H) As in (G), except that scale bars = 20 μm. See also Additional file 4.