14-3-3ζ: an optimal housekeeping protein for western blot analysis in swine rotator cuff tendon studies.

Abstract

Healthy biomechanics of the shoulder involving rotator cuff muscles and rotator cuff tendon (RCT) is pivotal for joint stability, yet co-morbid conditions like hyperlipidemia and hyperglycemia can lead to degenerative changes jeopardizing tendon integrity. A change in protein expression, the functional moiety for molecular events, may result in altered healing of RCT and prolonged morbidity. Expression and activity of proteins are critical while investigating the underlying molecular and cellular changes involved in tendinopathy. While investigating the changes in the protein expression of various inflammatory mediators, we observed that the Western Blot bands for commonly used housekeeping genes (GAPDH, β-actin, and α-tubulin) were not uniform in different tendon samples. Therefore, we investigated for an optimal housekeeping gene for Western blot analysis in swine RCT under normal and hyperlipidemic conditions, as this is essential for accurate normalization of protein expression. The study evaluated several housekeeping genes-GAPDH, beta-actin, alpha and beta-tubulin, Ubiquitin C, Cyclophilin A, TATA-box binding protein, and 14-3-3ζ-to ensure robust normalization across experimental setups. The results revealed that the protein expression of 14-3-3ζ was uniform in all samples, thereby validating its suitability as a stable housekeeping protein. The findings are important while studying the RCT pathology in a clinically relevant animal model, like swine, which mimics human RCT and provides translationally significant findings. Thus, the 14-3-3ζ protein will be an ideal housekeeping gene in the design of experiments utilizing musculoskeletal tissues.