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The cells were cultured overnight as in step 2

Following washes, membranes were incubated with HRP-conjugated secondary antibodies (1:5000) for 1 h at room temperature

J Int Soc Sports Nutr 15(1):30 Inman LA, Rennie MJ, Watsford ML, Gibbs NJ, Green J et al (2018) Reference values for the creatine kinase response to professional Australian football match-play

doi: 10.3390/ijms21218068

The injection site is cleaned beforehand, and the injection is given in a single, straightforward step