Author(s): GALVÁN ÁLVAREZ, V., MARTINEZ, M.1,2, GARCIA, E.1,2, GALLEGO, A.1,2, MORALES, D.1,2, MARTIN, M.1,2, CALBET, J.A.1,2,3,4, Institution: UNIVERSIDAD DE LAS PALMAS DE GRAN CANARIA, Country: SPAIN, Abstract-ID: 2322

INTRODUCTION:
A main procedure applied to determine protein expression and translational modifications in skeletal muscle is western blotting. Nevertheless, this technique may be imprecise due to differences in protein loading. Therefore, an accurate quantification of protein concentration in lysates is necessary and the amount of protein loaded must be checked, which is usually achieved by staining methods. Another extended practice is to measure in the same gel another protein which is not varying with the intervention, generically called housekeeping (Hk) protein. The results are usually reported in arbitrary units, representing the ratio between band density of the protein and band density of the Hk (or a total protein loading control, e.g., stain free or Memcode).
METHODS:
This study examines the impact of strength training (S1), HIIT (S2), SIT (S3), SIT with post-ischaemia (S4), and acute exhaustive exercise (S5) on the commonly used Hk proteins: GAPDH, B-tubulin and Vinculin.
RESULTS:
For this purpose, we used muscle biopsies from several previous studies including a total of 44M/31W (31 obese). Basal expression of all Hk proteins was similar in both sexes (p>0.21). After normalization by total protein with Memcode, vinculin was 78% lower in males (p<0.001) and 61% lower in obese than lean participants (p<0.001). S1: 10 males underwent 8 wk strength training program eliciting a 9% hypertrophy of the v. lateralis, GAPDH was reduced by 10% (p=0.045) and B-tubulin increased by 23% (p=0.003). S2: 6M/6W underwent 6 wks of HIIT followed by a 3wk detraining, B-tubulin levels decreased non-significantly (p=0.08), mainly due to a 18% reduction after detraining. S3: 12 males performed SIT arm cranking and leg pedaling (30s sprints, 6 sessions in 2 wks). B-tubulin expression was higher in arms than legs (p=0.q004) and training reduced (11%) and augmented (25%) the levels in arms and legs, respectively (time*extremity, p=0.02). GAPDH had higher expression levels in arms (p=0.002) and was reduced by training (p=0.04) regardless of extremity. S4: 3M/7W performed SIT training (30s sprints, 6 sessions in 2 wks) with post-exercise ischaemia in one leg. GAPDH was reduced (8%) by SIT (p=0.02), similarly in both legs. B-tubulin was increased only by SIT+ischaemia (46%, p=0.05). S5: 7M/5W underwent a session of acute exhaustive exercise accompanied by ischemia in one leg: B-tubulin expression was reduced by 18% (p=0.05), only in the leg with ischaemia. GAPDH was reduced when both legs were analysed conjointly (p=0.049).
CONCLUSION:
This study shows variation in classical Hk proteins used to normalize western blot analysis, depending in the extremity analysed, sex and body composition. Exercise training changes the basal expression of GAPDH and B-tubulin. These findings have crucial relevance for the proper interpretation of studies using the western blot technique, whose results may distorted if normalized by proteins that not behave as actual Hk.
Grants: PID 2021-25354OB-C21 & CSD (EXP_75097).