Table 3.

Comparison of commonly used in vitro RLC phosphorylation methods

Techniques	Examples	Phosphorylation efficiency	Advantages	Disadvantages	References
Directly associated kinases of RLC	Targeting MLCK, Rho-Kinase PKC	Increased by 100–300 %	Easy to perform; low cost	Low specificity of some broad spectrum kinase; variable target sites; Requires condition optimization	Kampourakis and Irving (2015); Kaneko-Kawano et al. (2012); Venema et al. (1993)
Genetic engineering	Engineering Rho or Rac pathway via mutation/RNAi, in vitro pseudo-phosphorylation	Increased by about 58 %, constitutive phosphor-mimicking state in pseudophosphorylation	Specific to RLC; Controllable phosphorylation site	Mainly for non-muscle cells; Imprecise controlled expression; may not mimic in vivo physiological environment	Brzeska et al. (2004); Koga and Ikebe (2008
Chemical/biological	Calyculin A and enzymes such as Thrombin	Increased up to 400 %	Easy to perform; High phosphorylation level	Side effects; non-specific phosphorylation of RLC/other proteins; need dosage optimization	Amerongen et al. (1998); Mills et al. (1998)
Chemical genetic approach	Specifically inhibit or activate only one type of kinase that phosphorylates RLC	100 %	Can be highly specific for a particular kinase; specific to target RLC phosphorylation condition	Some kinases (~30 %) lose function due to bulky amino acid substitution in active site; some ligands might not be very effective in inhibition of kinase due to non-covalent nature of action	Garske et al. (2011); Moffat et al. (2011)
Physico-chemical	Microinjection; osmotic delivery; exchange method; stretch; stimulation frequency	Increased by 40 %	Avoids the side effects; mimic “endogenous” level	Troponin C will be lost during exchange and needs replacing; temperature-dependent efficiency	Dias et al. (2006); Monasky et al. (2010); Silver et al. (1986); Takemoto et al. (2015); Toepfer et al. (2013; Zeng et al. (2000)