Hi, need to submit a 2000 words essay on the topic Effect of TGF- and Rock Inhibition on Phosphorylation of Myosin Light Chain.Download file to see previous pages... An impaired balance that exists be

Hi, need to submit a 2000 words essay on the topic Effect of TGF- and Rock Inhibition on Phosphorylation of Myosin Light Chain.

An impaired balance that exists between myosin phosphatase and Rho-kinase activities induces the abnormal sustained MLC phosphorylation which contributes to particular vascular disease pathogenesis such as hypertension and vasospasm. However, the system dynamic principle underlying MLC phosphorylation regulation remains to be clarified. The main purpose of this experiment is to look at phosphorylation of myosin light chain and the way TGF-? and Rock inhibition affects phosphorylation in the MG63 cells using western blotting technique. Introduction Myosin consists of 6 polypeptide chains: two heavy chains that are identical and two light chains pairs. Myosin light chain 2 (MLC2), also referred to as myosin regulatory light chain (MRLC), RLC, has several isoforms that depends on its distribution. (Dan et al., 2002). In smooth muscle, there is phosphorylation of MLC2 by myosin light chain kinase at Ser19 and Thr18 in a Ca2+/calmodulin-dependent pathway. The phosphorylation is associated with the activity of myosin ATPase and contraction of smooth muscle. Smooth muscle MLC2 Ser19 is also phosphorylated by ROCK, it regulates stress fibers assembly. ROCK (Rho-kinase), an effector RhoA molecule, inhibits the activity of phosphatase and phosphorylates the myosin binding subunit (MBS) of myosin phosphatase. The inhibition increases myosin light chain (MLC) phosphorylation of myosin II, which seems to induce RhoA-mediated stress fibers assembly and focal adhesions. ROCK is as well known to phosphorylate directly MLC in vitro. smooth muscle MLC2 Phosphorylation at Ser9 and Ser1/Ser2 by cdc2 and PKC inhibits myosin ATPase activity. Phosphorylation by cdc2 controls the cytokinesis timing. The pathway of RhoA/Rho-kinase is a crucial component of TGF-beta-induced effects on MLC phosphorylation of endothelial cells (Harden et al., 1996). Both non-muscle and smooth muscle myosin II activity is controlled by phosphorylation with myosin regulatory light chain (MRLC, MLC20,MLC, Myl9). MLC Phosphorylation at Ser-19 and Thr-18 results to activation of myosin II motor activity and myosin filament stability increase. (Daniels et al., 1998). The activation has critical roles in several processes of cell motile. On the other hand, other sites of phosphorylation on MLC might inhibit the activity of myosin II. In MLC, PKC phosphorylates Thr-9 and Ser-1/Ser-2, the phosphorylation leads to decrease in the activated myosin II interaction with actin, and inhibits interaction of MLC with the myosin light-chain kinase activation site. Thus, myosin II activity inhibition through Ser-1/Ser-2 phosphorylation may have crucial roles in growth factor-induced actomyosin filaments reorganization. (Dechert et al., 2001). Materials and methods After the gel was transferred to the tank, it was filled with 1x running buffer:100ml 10x Trisglycine/SDS(10xrunning buffer)900ml dH2O.The samples were loaded,5ul Bio-Rad precision markers(kaleidoscope).The samples were boiled in 2x sample buffer at 95oc for 5minutes. Samples total volume per well were 30 ul, for protein, it was 40 ug of per well 7 ul for molecular weight marker Transfer of SDS gel onto the membrane First the blotting paper pieces were cut (7.5x9.5cm).Then the pieces of the membrane 7x9cm(PVDF membrane) were cut.The PVDF membrane was soaked in methanol for 10-20 seconds.