Journal: The Journal of Physiology

Article Title: Endurance training increases a ubiquitylated form of histone H3 in the skeletal muscle, supporting Notch1 upregulation in an MDM2‐dependent manner

doi: 10.1113/JP288947

Figure Lengend Snippet: A , H3K27 me3 was detected at 25 kDa in whole gastrocnemius muscles and C2C12 myotubes by three α‐H3K27 me3 antibodies (polyclonal pAb‐195, polyclonal pAb‐069 and monoclonal C36B11). B , α‐H3K27 me3 (C36B11) detected a band at 25 kDa in C2C12 myotubes that was not present in mouse skeletal muscle endothelial cells (mSMEC, n = 4 per cell type). C , protein extracts from sedentary (Sed.) and trained (Tr.) gastrocnemius muscles ( n = 2 per group) were treated with a cocktail of broad‐range deubiquitinating enzymes. Western blotting detected levels of ubiquitin (P4D1) and H3K27 me3 (C36B11). D – F , after immunoprecipitation of H3K27 me3 using pAb‐195 in protein extract from gastrocnemius muscle, immunoblots detected histone H3 ( D ) and ubiquitin ( E ). F , MS analysis of the trypsin‐digested 25 kDa band; table shows mass‐to‐charge ratio ( m/z ) and abundances (grouped) of unique peptides identified.

Article Snippet: The following primary antibodies were used: rabbit polyclonal (pAb) α‐PECAM (CD31) (Invitrogen; cat. no. PA5‐16301, Burlington, Canada), rabbit pAb α‐COX IV (Cell Signaling Technology; cat. no. 4844, New England Biolabs Ltd., Whitby, ON, Canada), rabbit monoclonal (mAb) α‐MDM2 (SMP14) (Santa Cruz; cat. no. sc‐965, Santa Cruz, CA, USA) and mouse mAb (2A10) (non‐commercial, kindly provided by Dr Mary Ellen Perry, NIH), rabbit mAb α‐EZH2 (D2C9, Cell Signaling Technology, cat. no. 5246), rabbit pAb α‐NEDD4 (Cell Signaling Technology; cat. no. 2740, New England Biolabs Ltd.), mouse mAb α‐Ubiquitin (P4D1) (Cell Signaling Technology; cat. no. 3936, New England Biolabs Ltd.), mouse mAb α‐Histone H3 (C96C10) (Cell Signaling Technology; cat. no. 3638, New England Biolabs Ltd.), rabbit pAb α‐Histone H2A (Cell Signaling Technology; 2578, New England Biolabs Ltd.), α‐H3K27 me3 : rabbit pAb‐195 and pAb‐069 (cat. no. C15410195 and C15410069, respectively, both Diagenode, Denville, NJ, USA) and rabbit mAb (C36B11) (Cell Signaling cat. no. 9733, New England Biolabs Ltd.), rabbit pAb α‐H3K4 me3 (cat. no. 9727, New England Biolabs Ltd.), and rabbit mAb (D27C4) α‐H2AK119 ub (cat. no. 8240). α/β‐TUBULIN (rabbit pAb, Cell Signaling Technology; cat. no. 21 485.

Techniques: Muscles, Western Blot, Ubiquitin Proteomics, Immunoprecipitation

Journal: The Journal of Physiology

Article Title: Endurance training increases a ubiquitylated form of histone H3 in the skeletal muscle, supporting Notch1 upregulation in an MDM2‐dependent manner

doi: 10.1113/JP288947

Figure Lengend Snippet: A , C2C12 myoblasts were differentiated into myotubes over a period of 7 days. Immunoblots show level of H3K27 me3 (C36B11). α/β‐TUBULIN was used as a loading control. The relative abundance of H3K27 me3 at 17 kDa (right) and at 25 kDa (centre) is shown as mean ± SD ( n = 5, one‐way ANOVA, overall effect of differentiation P < 0.0001) For post hoc Dunnet test reaching P ≤ 0.05, exact P values are shown. B , after 4 days of differentiation, C2C12 myotubes were treated with MG132 (MG.) or DMSO (D.) as a control vehicle (7 h, 10 µM). Immunoblots show level of H3K27 me3 (pAb‐195) and histone H3. α/β‐TUBULIN was used as a loading control. The relative abundance of H3K27 me3 at 17 kDa (right) and at 25 kDa (centre) is shown as mean ± SD ( n = 5–6). C , representative immunoblots for H3K27 me3 in C2C12 myotubes following 4 days of repeated EPS (90 min/day). Repeated contractile activity in C2C12 myotubes increased the relative abundance of the H3K27 me3 mark detected at both 17 and 25 kDa. Histone H3 and α/β‐TUBULIN were used as a loading control. Data show mean ± SD ( n = 6). For t test reaching P ≤ 0.05, exact P values are shown. D , H3K27 me3 was pulled down in control (CON) and EPS‐stimulated C2C12 myotubes (EPS, 90 min/day). Immunoblots were performed for ubiquitin, histone H3 and H3K27 me3 . E , H3 was pulled down in CON and EPS‐stimulated C2C12 myotubes. Immunoblots were performed for ubiquitin, H3K27 me3 and histone H3.

Article Snippet: The following primary antibodies were used: rabbit polyclonal (pAb) α‐PECAM (CD31) (Invitrogen; cat. no. PA5‐16301, Burlington, Canada), rabbit pAb α‐COX IV (Cell Signaling Technology; cat. no. 4844, New England Biolabs Ltd., Whitby, ON, Canada), rabbit monoclonal (mAb) α‐MDM2 (SMP14) (Santa Cruz; cat. no. sc‐965, Santa Cruz, CA, USA) and mouse mAb (2A10) (non‐commercial, kindly provided by Dr Mary Ellen Perry, NIH), rabbit mAb α‐EZH2 (D2C9, Cell Signaling Technology, cat. no. 5246), rabbit pAb α‐NEDD4 (Cell Signaling Technology; cat. no. 2740, New England Biolabs Ltd.), mouse mAb α‐Ubiquitin (P4D1) (Cell Signaling Technology; cat. no. 3936, New England Biolabs Ltd.), mouse mAb α‐Histone H3 (C96C10) (Cell Signaling Technology; cat. no. 3638, New England Biolabs Ltd.), rabbit pAb α‐Histone H2A (Cell Signaling Technology; 2578, New England Biolabs Ltd.), α‐H3K27 me3 : rabbit pAb‐195 and pAb‐069 (cat. no. C15410195 and C15410069, respectively, both Diagenode, Denville, NJ, USA) and rabbit mAb (C36B11) (Cell Signaling cat. no. 9733, New England Biolabs Ltd.), rabbit pAb α‐H3K4 me3 (cat. no. 9727, New England Biolabs Ltd.), and rabbit mAb (D27C4) α‐H2AK119 ub (cat. no. 8240). α/β‐TUBULIN (rabbit pAb, Cell Signaling Technology; cat. no. 21 485.

Techniques: Western Blot, Control, Activity Assay, Ubiquitin Proteomics