Journal: The Journal of Cell Biology

Article Title: The Rho kinases I and II regulate different aspects of myosin II activity

doi: 10.1083/jcb.200412043

Figure Lengend Snippet: ROCKs can phosphorylate the same substrates. (A) ROCK I and II immunoprecipitates from REF were incubated with MBP-MYPT1 COOH-terminal fragment and γ-[ 32 P]ATP. Background radiolabel levels from control immunoprecipitates have been deducted. (B) 2 d after transfection with siRNA duplexes, cells were fixed and stained for pMLC2 (Ser19) and F-actin. ROCK I siRNA cells have depleted staining, whereas the abundant stress fibers of ROCK II siRNA stain strongly. Bar, 50 μm. (C and D) Total cell lysates from REF treated with Y-27632 in the presence of serum were analyzed by Western blotting for MLC2 phosphorylation on Ser19 (p-MLC) or Thr18/Ser19 (pp-MLC) (C) and MYPT phosphorylation on Thr853 or Thr696 (D). Although MYPT phosphorylation on Thr853 was reduced, more marked reductions were seen in MLC2 phosphorylation after Rho kinase inhibition. (E and F) Total cell lysates from cells transfected with siRNA duplexes were analyzed by Western blotting for MLC2 phosphorylation on Thr18/Ser19 (E) and MYPT phosphorylation of Thr853 or Thr696 (F). Phosphorylation is normalized to the relevant protein level in the lysates. Phosphorylation levels of MYPT were increased in the absence of ROCK I, partly due to a decrease in myosin phosphatase protein levels in these cells. Error bars show SD.

Article Snippet: After washing with buffer A three times and with phosphorylation buffer (40 mM Tris-Cl, pH 7.4, 0.1% CHAPS, 10 mM MgCl 2 , 1 mM DTT, 1 mM EDTA, 1 mM EGTA, phosphatase inhibitor cocktail [Sigma-Aldrich], 10 mM NaF, 10 mM β-glycerophosphate, 1 mM Na 3 VO 4 , EDTA free protease inhibitor cocktail [Roche], and 5 μg/ml pepstatin) once, immunoprecipitates in the same buffer were incubated with 4 μg GST-MLC or maltose binding protein (MBP)-MYPT1 (714–1004) and γ-[ 32 P]ATP (3 μCi, 0.11 TBq/mmol, 74 MBq/ml, 20 μM final concentration; GE Healthcare) at 30°C for 10 min.

Techniques: Incubation, Transfection, Staining, Western Blot, Inhibition

Journal: The Journal of Cell Biology

Article Title: The Rho kinases I and II regulate different aspects of myosin II activity

doi: 10.1083/jcb.200412043

Figure Lengend Snippet: ROCK II activity is regulated by PI-3 kinase. (A) Serum-starved REF were trypsinized and incubated with 10 μM LY294002 or DMSO for 30 min at 37°C. ROCK immunoprecipitates were incubated with MBP-MYPT1 COOH-terminal fragment and γ-[ 32 P]ATP. Background radiolabel levels from control immunoprecipitates have been deducted. ROCK activities from REF treated with DMSO was indicated as one. (B) REF was cotransfected with p110α and pEGFP-N1. 1 d after transfection, cells were fixed and stained for ROCK I or II. Double arrowheads indicate transfected cells and the single arrowhead shows the localization of ROCK II in membrane ruffles. (C) ROCK immunoprecipitates from REF transfected with p110α or control vector were incubated with GST-MLC and γ-[ 32 P]ATP, and the ROCK activities were measured. Activity of mock transfections is indicated as one. Bar, 10 μm.

Article Snippet: After washing with buffer A three times and with phosphorylation buffer (40 mM Tris-Cl, pH 7.4, 0.1% CHAPS, 10 mM MgCl 2 , 1 mM DTT, 1 mM EDTA, 1 mM EGTA, phosphatase inhibitor cocktail [Sigma-Aldrich], 10 mM NaF, 10 mM β-glycerophosphate, 1 mM Na 3 VO 4 , EDTA free protease inhibitor cocktail [Roche], and 5 μg/ml pepstatin) once, immunoprecipitates in the same buffer were incubated with 4 μg GST-MLC or maltose binding protein (MBP)-MYPT1 (714–1004) and γ-[ 32 P]ATP (3 μCi, 0.11 TBq/mmol, 74 MBq/ml, 20 μM final concentration; GE Healthcare) at 30°C for 10 min.

Techniques: Activity Assay, Incubation, Transfection, Staining, Plasmid Preparation