Figure 4.
Figure 4. Immunostaining of permeabilized HASMC with anti–phosphorylated RLC (pRLC) antibody. Permeabilized cells were treated with TRITC-phalloidin to label actin in stress fibers and with an anti-pRLC primary antibody, and then an Alexa Fluor 488 secondary antibody (Milton et al., 2011). (A, C, and E) TRITC-actin. (B, D, and F) Alexa Fluor 488. A and B, buffer control; C and D, treated with phosphorylation buffer (150 mM KCl, 2 mM ATP, 100 nM CaM, pCa 4, and 50 nM MLCK); E and F, treated with phosphorylation buffer in the presence of wortmannin to inhibit MLCK activity. Phosphorylation was for 5 min at 25°C. Slides were observed in imaging actin buffer.

Immunostaining of permeabilized HASMC with anti–phosphorylated RLC (pRLC) antibody. Permeabilized cells were treated with TRITC-phalloidin to label actin in stress fibers and with an anti-pRLC primary antibody, and then an Alexa Fluor 488 secondary antibody (Milton et al., 2011). (A, C, and E) TRITC-actin. (B, D, and F) Alexa Fluor 488. A and B, buffer control; C and D, treated with phosphorylation buffer (150 mM KCl, 2 mM ATP, 100 nM CaM, pCa 4, and 50 nM MLCK); E and F, treated with phosphorylation buffer in the presence of wortmannin to inhibit MLCK activity. Phosphorylation was for 5 min at 25°C. Slides were observed in imaging actin buffer.

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