Fig 1.
UCHL1 in skeletal muscle is selectively expressed in oxidative muscle fibers.
A: Representative images and quantification of UCHL1 immunofluorescence of soleus (left) and EDL (right); B: Image and quantification of Western blots for UCHL1 and GAPDH from soleus, EDL, and gastrocnemius. The Western blot image was directly copied from the original membrane scan and each lane was as originally loaded without regrouping. * P< 0.05, n = 3; C: Images and quantification of immunofluorescence of gastrocnemius muscle with UCHL1 (red) and type I/IIa MyHC (green).
Fig 2.
Mouse model of skeletal muscle specific knockout (smKO) of the UCHL1 gene.
A: Schematic illustration of strategy of UCHL1 smKO. B: Representative gel image of PCR genotyping. C: Western blot of UCHL1 protein in samples of brain, heart, and skeletal muscles from wild type (WT) or UCHL1 smKO (KO) mice. D: Quantification as a ratio of UCHL1 protein band density in WT vs UCHL1 smKO soleus muscle. * P<0.05, n = 4. The representative Western blot images were copied from the original membranes scan and each lane was positioned as originally loaded without regrouping. E: Body weight of WT and UCHL1 smKO mice (n = 8).
Fig 3.
UCHL1 smKO reduces muscle oxidative activity.
A-B: Representative images and quantifications of SDH staining (A) and type I/IIa MyHC fiber immunofluorescent staining (B) of soleus from WT and UCHL1 smKO mice. C-D: Representative images and quantifications SDH staining (C) and type I/IIa MyHC fiber immunofluorescent staining (D) of gastrocnemius from WT and UCHL1 smKO mice. The quantifications were presented as percentages of positive fibers vs total fibers in a selected area. * P<0.05 vs WT.
Fig 4.
UCHL1 smKO reduces in situ muscle contraction endurance.
A: Contractile forces of the GPSC from WT (solid line) or UCHL1 (dash line) at various preload. B: Frequency-peak contractile force of GPSC from WT (solid curve) or UCHL1 smKO (dash curve); C-D: Representative tracings (C) and quantitative data (D) of repetitive single contraction over 4 minutes of GPSC from WT (solid curve) or UCHL1 smKO mice (dash curve). E-F: Representative tracings (E) and quantitation (F) of repetitive single contraction over 4 minutes in TA from WT (solid curve) or UCHL1 smKO mice (dash). *P<0.05 vs WT, n = 5.
Fig 5.
UCHL1 smKO alters mitochondrial OXPHOS proteins.
A-I: Western blot images (A) and quantifications of Western blot of soleus samples from WT or UCHL1 smKO for UCHL1 (B), SDHB (C), UQCR2 (D), NDUFB8 (E), SDHA (F), ATP5A (G), VDAC (H), and CPT1 (I). J: Activity of complex II/III in isolated mitochondria from soleus of WT or UCHL1 smKO. * P<0.05 vs WT, n = 4. The representative Western blot images were copied from the original membranes and each lane was positioned as the originally loaded without regrouping. The original blot images can be found in the S1 File.
Fig 6.
UCHL1 is present in cytosol near mitochondria.
A: Immunofluorescent staining of UCHL1 (red) and mitochondrial marker VDAC (green) in mouse soleus. The arrows indicate the co-localization of UCHL1 and VDAC staining. B: Western blot images of mitochondrial and cytosolic fractionation of gastrocnemius detecting mitochondrial markers SDH and CPT1 in mitochondrial fraction, and UCHL1 and tubulin in cytosolic fraction. C-E: Western blot images (C) and quantifications of phospho and total AMPKα (D), and HSP60 (E) in soleus from WT or UCHL1 smKO mice. F: Western blot image of HA immunoprecipitation of the protein extract from C2C12 cells with overexpression of HA tagged UCHL1.