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. Author manuscript; available in PMC: 2021 Oct 1.
Published in final edited form as: J Physiol. 2020 Sep 11;598(20):4573–4590. doi: 10.1113/JP280111

Figure 5. Rhotekin couples RhoA to S100A4 and NM myosin II in SM cells and tissues stimulated with ACh.

Figure 5.

A, rhotekin (RTKN) was immunoprecipitated from tissue extracts and immunocomplexes were blotted for RhoA, S100A4, RTKN and NM myosin IIA. Stimulation for 5 min with 10−5 M ACh significantly increased the coprecipitation of NM myosin IIA (n = 6, p = 0.0070), RhoA (n = 3, p = 0.0359) and S100A4 (n = 9, p = 0.0001) with RTKN. B, in situ PLA was used to determine the interaction of RTKN and RhoA, S100A4 and NM myosin IIA in freshly dissociated tracheal SM cells. Stimulation with ACh caused a significant increase in the number of PLA complexes between all four proteins (RTKN-RhoA, n = 20, p = 0.0001; RTKN-S100A4, n = 20, p = 0.0001; RTKN-NM myosin IIA, n = 31, p = 0.0001). C, tracheal SM tissues were treated with RTKN siRNA. Depletion of RTKN significantly inhibited protein expression (n = 10, p = 0.0001) and also inhibited tension development in response to 10−5 M ACh stimulation (n = 16, p = 0.0001). D, PLA shows interactions between S100A4 and RhoA in cells dissociated from sham-treated tissues and from RTKN-depleted tissues. ACh stimulation of sham-treated cells resulted in a significant increase in the interactions between S100A4 and RhoA (US, n = 16; ACh, n = 20). RTKN depletion inhibited the ACh-induced increase of the interaction between S100A4 and RhoA (p = 0.0001) (US, n = 18; ACh, n = 19). Data analysed by a paired Student’s t test (A and C), an unpaired Student’s t test (B) or by one-way ANOVA (D). All values are the mean ± SD. *Significant difference between US and ACh; **Significant difference between treatment groups (P < 0.05); NS, no significant difference.