Table 2.

Comparison of commonly used in vivo RLC phosphorylation methods

Techniques	Examples	Phosphorylation efficiency	Advantages	Disadvantages	References
Transgenic	Manipulate MLCK expression by cloning/RNAi, P-element-mediated germline transformation (Drosophila), in vivo pseudo-phosphorylation	Decreased by 95 % or significantly increased, constitutive phosphor-mimicking state in pseudophosphorylation	Able to modify specific gene/site of interest in physiological setting; Able to investigate the effect of phosphorylation from the molecular to the whole-organism levels	Expensive, time-consuming, and creating a transgenic model is generally risky; side effects; false positives; positional effects; not feasible to be performed in patients	Ding et al. (2010), Espinoza-Fonseca et al. (2008), Farman et al. (2009), Miller et al. (2011), Muthu et al. (2014)
Direct phosphorylation	Injection of angiotensin II/AT1R/phenylephrine/isoproterenol	Increased by 30–45 %	Physiological conditions; Easy to perform; Cost-effective	The proteins and sites on the proteins of which phosphorylation is being manipulated is not easy to control and may not be physiological in nature; The dosage needs optimization	Aoki et al. (2000), Ding et al. (2010), Huang et al. (2008), Verduyn et al. (2007)