PMC

Post-exercise activation of S6K1 and AKT are augmented by PRO/CHO ingestion. Resistance exercise increased S6K1 activity, with this response significantly higher in the FED group 1 h post exercise (A). In parallel, AKT kinase activity was increased post exercise, with this response significantly higher in the FED group 1 h post exercise (B). Data presented as mean ± SEM (n = 7/group). *Significantly different to PRE-CON, ^§Significantly different to PRE-FED, ^ΦSignificantly different to 1h-CON (P < 0.05).

TSC2 dissociates from the plasma membrane following resistance exercise. Immunofluorescence quantification of TSC2 (Red) and the plasma membrane marker WGA (Blue). Each panel represents one subject from CON and FED across the experimental time course (A). Group data is quantified and reported in (B). Circles represent CON, squares represent FED. All image processing was kept constant between images, including exposure time, gain, and image frame. All data presented relative to the Pre exercise CON. Data presented as mean ± SEM (n = 7/group). *Significantly different to PRE-CON (P < 0.05).

TSC2 and Rheb dissociate following resistance exercise. Immunofluorescence quantification of TSC2 (Red) and Rheb (Green) interaction, displayed as a composite image (Merge) and WGA (Blue). Yellow regions represent TSC2 and Rheb interaction. Each panel represents one subject from CON and FED across the experimental time course (A). Group data is quantified and reported in (B). Circles represent CON, squares represent FED. All image processing was kept constant between images, including exposure time, gain, and image frame. All data presented relative to the Pre exercise CON. Data presented as mean ± SEM (n = 7/group). *Significantly different to PRE-CON (P < 0.05).

mTOR, Rheb and TSC2 antibody validation. Immunofluorescence labeling of endogenous proteins was performed as described in the Methods section. Cross-sections were stained with the antibodies relating to mTOR, Rheb and TSC2 in wild-type mice (WT – Left panel) or in muscle from mTOR, Rheb and TSC2 knockout mice (mKO). Regionality of staining is provided through co-staining with WGA or Dystrophin as a marker of the cell membrane. All image processing was kept constant between images, including exposure time, gain, and image frame. GS – Grey scale image *Significantly different to WT (P < 0.05).

mTOR translocates to the microvasculature following resistance exercise. Immunofluorescence quantification of mTOR (Red) and UEA-I (Green) interaction, displayed as a composite image (Merge) and WGA (Blue). Yellow regions represent mTOR and UEA-I interaction. Each panel represents one subject from CON and FED across the experimental time course (A). Group data is quantified and reported in (B). Circles represent CON, squares represent FED. All image processing was kept constant between images, including exposure time, gain, and image frame. All data presented relative to the Pre exercise CON. Data presented as mean ± SEM (n = 7/group). *Significantly different to PRE-CON (P < 0.05).

Resistance exercise increases mTOR and Rheb co-localisation at the plasma membrane. Immunofluorescence quantification of mTOR (Red) and Rheb (Green) interaction, displayed as a composite image (Merge) and WGA (Blue). Yellow regions represent mTOR and Rheb interaction. Each panel represents one subject from CON and FED across the experimental time course (A) Group data is quantified and reported in (B) Circles represent CON, squares represent FED. All image processing was kept constant between images, including exposure time, gain, and image frame. All data presented relative to the Pre exercise CON. Data presented as mean ± SEM (n = 7/group). *Significantly different to PRE-CON (P < 0.05).

mTOR/LAMP2 complex translocation to the cell membrane following resistance exercise. Representative image of mTOR (Red), LAMP2 (Green), composite image of mTOR and LAMP2 (Merge) and WGA (Blue) for one subject from CON across the experimental time course. Yellow regions represent mTOR and LAMP2 interaction. Immunofluorescence quantification of mTOR and LAMP2 Co-localization (A), quantification of mTOR associated with the cell membrane marker WGA (B) and quantification of LAMP2 associated with WGA (C). Circles represent CON, squares represent FED. All image processing was kept constant between images, including exposure time, gain, and image frame. All data presented relative to the Pre exercise CON. Data presented as mean ± SEM (n = 7/group). *Significantly different to PRE-CON (P < 0.05).

Resistance exercise increases mTOR and eIF3F co-localisation at the plasma membrane. Immunofluorescence quantification of mTOR (Red) and eIF3F (Green) interaction, displayed as a composite image (Merge) and WGA (Blue). Yellow regions represent mTOR and eIF3F interaction. Each panel represents one subject from CON and FED across the experimental time course (A). Group data is quantified and reported in (B). Circles represent CON, squares represent FED. All image processing was kept constant between images, including exposure time, gain, and image frame. All data presented relative to the Pre exercise CON. Data presented as mean ± SEM (n = 7/group). *Significantly different to PRE-CON (P < 0.05). ^§Significantly different between groups (P < 0.05).

Rheb, TSC2 and eIF3F peptide competition assays. Immunofluorescence labeling of endogenous proteins was performed in human basal samples (CON – Left panel) as described in the Methods section. Regionality of staining is provided through co-staining with WGA or Dystrophin as a marker of the cell membrane. Competition (COMP) assays were performed to demonstrate antibody specificity of TSC2, eIF3F and Rheb. Non-specific binding/antibody efficiency was examined through imaging samples in which secondary antibody was used without prior primary antibody incubation (No Ab). All image processing was kept constant between images, including exposure time, gain, and image frame. *Significantly different to CON (P < 0.05).