(A) ROCK1 and 2 were immunoprecipitated from A7r5 cells in serum-free (arrested) conditions or following cell stimulation with either 1μM LPA or 10% serum. (B) A time-course of serum stimulation of A7r5 cells followed by ROCK2 immunoprecipitation is shown (Top). Pooled results from four experiments are shown on the bottom. (C) A time-course of serum stimulation of A7r5 cells followed by measurement of MBS phosphorylation at Thr850 by immunoblot (Top). Pooled results from four experiments (Bottom).

KD= kinase domain, PH=pleckstrin homology domain, vertical hatched box=RhoA binding domain, horizontal hatched box=cysteine rich domain. On MBS, vertical lines indicate ankyrin repeats, diagonal hatched box=central insert, 696 and 850 refer to the inhibitory phosphorylation sites, solid box is the leucine zipper domain. The blow-up region depicts the ROCK2 interacting domain, including both inhibitory phosphorylation sites, and a predicted coiled coil region.

(A) Immunofluorescence microscopy of representative primary rat aortic VSMCs plated on fibronectin coated coverslips and serum-deprived for 48 hours following scrambled negative control (Scr, top row), ROCK1 (middle row) and ROCK2 (bottom row) silencing and immunofluorescence labeling with phalloidin (left column) to image actin fibers and vinculin (right column) to image focal adhesions. (B) Cell area measurements of scrambled negative control (Scr), ROCK1 and ROCK2 silenced unstimulated VSMCs. (C) Stress fiber number in scrambled negative control (Scr), ROCK1 and ROCK2 silenced unstimulated VSMCs. (D) Focal adhesion number in scrambled negative control (Scr), ROCK1 and ROCK2 silenced unstimulated VSMCs.

(A) ROCK1 and ROCK2 were immunoprecipitated from A7r5 cells using 1.4μg of either goat anti-ROCK1 (gαROCK1), goat anti-ROCK2 (gαROCK2) or goat non-immune (NI IP) antibodies. Immunoblots were performed with mouse anti-ROCK1 (mαROCK1) or mouse anti-ROCK2 (mαROCK2) antibodies. These conditions were used for all subsequent immunoprecipitations of ROCK1 and 2. (B) ROCK 1 and 2 were immunoprecipitated from A7r5 cells, followed by immunoblot to detect the MBS and MP-RIP proteins. A sample of the input protein for ROCK 1 and 2, 25, 12.5 and 6.25μg, is shown by immunoblot below. (C) Non-immune, ROCK1 and ROCK2 immunoprecipitations were performed from primary rat aortic smooth muscle cells. The immunopellets were probed by immunoblot with MBS, ROCK1 and ROCK2 antibodies. (D) HEK293 cells were transfected with empty plasmid (Mock), and Myc-tagged MBS, ROCK1 or ROCK2. Immunoprecipitations were performed with anti-Myc antibodies, followed by immunoblotting for ROCK1 and ROCK2.

(A) GST, and GST-fusion proteins (amino acid numbers noted, − and + indicate absence and presence of central insert splice variant) were tested for binding to ROCK2 and MP-RIP from A7r5 cell lysates. The input lysate is shown on the left for each protein. (B) GST and GST-MBS683–866 binding to mycROCK1 and mycROCK2 expressed in HEK293 cells. Input mycROCK1 and ROCK2 is shown on the left. Mean data from three experiments is shown. (C) Schematic diagram depicting the full-length MBS molecule, and each of the MBS peptides tested for ROCK binding. The amino acid residues are shown on the left, ROCK binding is shown on the right. The vertical lines in the MBS molecule represent the ankyrin repeats, the shaded box represents the central insert and 696 and 850 are the inhibitory phosphorylation sites. (D) Purified 6xHis-tagged ROCK2 (R2, numbers refer to amino acid residues) and ROCK1 (R1) domains were tested for binding to purified GST or GST-MBS683–866 using anti-6xHis antibodies.

(A) A7r5 cells transfected with Myc-ROCK1 or Myc-ROCK2 and immunostained with both anti-phospho-MLC-Cy3 (left panels) and anti-Myc-FITC (right panels). The arrowheads indicated transfected cells. The scale bar is 30μm. (B) Immunoblot showing specific silencing of ROCK1 (R1) expression, ROCK2 (R2a and R2b, two separate oligonucleotides) expression or both ROCK1 and ROCK2 (R1+R2) expression in A7r5 cells using dsRNA oligonucleotides. Scr indicates scrambled negative control dsRNA (100nM). 2xScr indicates scrambled control dsRNA concentration adjusted to match R1+R2 (200nM). GAPDH is shown as a loading control. (C) Phosphorylation of MBS at the known inhibitory site Thr850 in A7r5 cells, following the silencing conditions described in 5B (Top). Total MBS is shown as a loading control. Pooled data from 4 experiments of MBS phosphorylation at Thr850 following ROCK isoform silencing (Bottom). The values are represented as Thr850 phosphorylation normalized to MBS expression for each sample. (D) Phosphorylation of MLC following silencing of ROCK isoforms in A7r5 cells as described in 5B (Top). Total MLC is shown as a loading control. Pooled data from 4 separate experiments of MLC phosphorylation following ROCK isoform silencing (Bottom). The values are represented as MLC phosphorylation normalized to total MLC expression for each sample. (E) Immunofluorescence microscopy of primary rat aortic smooth muscle cells following control (Scr) or ROCK isoform silencing. The cells were labeled with phalloidin to identify actin fibers (left column) and phospho-MLC (same antibody used for immunoblotting above) (right column). The scale bar is 30μm.

(A) Contractility and force production in representative scrambled negative control (Scr), ROCK1 and ROCK2 silenced primary rat aortic VSMCs. For each silencing condition, the phase-contrast image of the cell is shown before and 30 minutes after administration of 1μM LPA. Adjacent to the cell is the displacement map of the microfabricated posts. The displacement map was used to generate the force map shown to the far right of each image, as described in Methods. (B) Contraction, as measured by the change in cell length, in scrambled negative control (Scr), ROCK1 and ROCK2 silenced VSMCs treated with LPA, N=60, 69, 54 cells, respectively. (C) Force production in 20 scrambled negative control (Scr), ROCK1 and ROCK2 silenced cells treated with LPA. (D) Plot of force versus contraction for scrambled negative control (Scr, black squares), ROCK1 (red triangles) and ROCK2 (blue circles) silenced VSMCs. (E) Detail of scrambled negative control (Scr), ROCK1 and ROCK2 silenced unstimulated VSMCs labeled with phalloidin for actin filaments and phospho-MLC as in 5E. The scale bar is 30μm. (F) Plot of force direction analyzed in the longitudinal and transverse axes for scrambled negative control (black squares), ROCK1 (red triangles) and ROCK2 (blue circles) silenced LPA stimulated VSMCs. Force direction was determined by the direction in which the posts were bent and the average longitudinal and transverse components were plotted.