Fig 2: EGFP expression in the testis of pre-pubertal bucks on d21 after in vivo gene transfer. (A) Gel electrophoresis of quantitative real-time PCR products from gene transferred testis revealed an EGFP-specific band of 104 bp. The top of the gel with the wells was cropped; N: no template control (NTC), G1-G3: testicular tissue from pre-pubertal goats with gene transferred testis, S1 & S2: cultured seminiferous tubules from in vitro transfected testes, P: positive control (pIRES2-EGFP plasmid), M: 100 bp molecular marker; (B) graphical representation of the relative expression of EGFP mRNA in testicular tissues from pre-pubertal bucks at d21 post-gene transfer. G1, G2 & G3 are pre-pubertal bucks #1, #2 & #3, respectively. Data represent the mean ± s.e.m. for n = 3. Here, ‘ns’ means statistically not significant; (C) an approx. 27 kDa protein band was observed by western blotting analysis, using total protein from testicular tissue of an in vivo electroporated goat. The marker lane was cut separately before incubating the membrane with the primary antibody. The wells are labeled 1: gene transferred testis of a pre-pubertal goat, 2: testis of a non-gene transferred goat, 3: protein marker (#1610373, Bio-Rad).

Fig 3: Typical western blotting analysis illustrated for each measured protein. The muscle cells lysate was obtained from each animal of the four groups (Control, heat acclimated, trained, and heat + trained) in the presence of protease and phosphatase inhibitors. The proteins were resolved by SDS‐PAGE (7.5%–12%) and electroblotted onto a nitrocellulose membrane. Membranes were probed with primary antibody (Table 1) and then peroxidase‐tagged with secondary antibody (anti‐mouse or anti‐rabbit, 1/10,000). Membranes were soaked in ECL solution and scanned using a ChemiDoc XRS+ (Bio‐Rad). A molecular‐weight ladder (Precision Plus All Blue Prestained Protein Standards from Bio‐Rad Laboratories, no. 1610373) and an internal control (mix of all control group animals) were also loaded onto each gel. The bands corresponding to the molecular weight of the proteins of interest were quantified using Quantity One (version Windows, Bio‐Rad) and normalized to the internal control (software ImageLab). Dash line brands the delimitation between the two pictures of the same gel performed at two different development times (1 and 60 s) to get a good definition of each lane and subsequent quantification for the five OXPHOS complexes. PDH; pyruvate dehydrogenase. P‐PDH: phosphorylated PDH (E1 subunit). GSK, glycogen synthase kinase; P‐GSK, phosphorylated GSK; CPT, carnitine palmitoyltransferase/UCP‐3, uncoupling protein 3; ANT‐1, adenine nucleotide translocase 1; HSP‐70, heat shock protein 70; MF, mitofusin, PRK, parkin; DRP, dynamin‐related protein; PINK, PTEN‐induced putative kinase