Western blot analysis was performed on whole cell extracts (30 ug lysate) of Mouse Kidney (Lane1), HeLa (Lane2), NIH/3T3 (Lane3), A-431 (lane 4), PC-12 (lane 5), Jurkat (lane 6), and HEK-293 (lane 7). The blots were probed with Anti-RhoA Mouse Monoclonal Antibody (Product# MA1134, 1:500-1:2000 ug/ml) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP conjugate (Product # 626520, 1:4000 dilution). A 21 kDa band corresponding to RhoA was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10 % Bis-Tris gel (Product # NP0301BOX), XCell SureLock™ Electrophoresis System (Product # EI0002), and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody using iBind™ Flex Western Starter Kit (Product# SLF2000S). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).
|Tested species reactivity||Human, Mouse, Rat|
|Host / Isotype||Mouse / IgG1|
|Immunogen||Full-length recombinant His-tagged human RhoA|
|Storage buffer||PBS with 1mg/ml BSA|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||3-5 µg/10^6 cells|
|Western Blot (WB)||1:500-1:2000|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
By Western blot, MA1-134 detects recombinant RhoA, but not recombinant RhoB or recombinant RhoC.
By Western blot on mouse and rat kidney whole tissue lysates, MA1-134 detects a band of ~22kD corresponding to the expected molecular weight of RhoA. A nonspecific band at ~55kD was also detected in both kidney lysates.
RhoA regulates a signal transduction pathway linking plasma membrane receptors to the assembly of focal adhesions and actin stress fibers. It is involved in a microtubule-dependent signal that is required for the myosin contractile ring formation during cell cycle cytokinesis, and plays an essential role in cleavage furrow formation. RhoA is required for the apical junction formation of keratinocyte cell-cell adhesion, and serves as a target for the yopT cysteine peptidase from Yersinia pestis, vector of the plague, and Yersinia pseudotuberculosis, which causes gastrointestinal disorders. RhoA stimulates PKN2 kinase activity and may be an activator of PLCE1. It is activated by ARHGEF2, which promotes the exchange of GDP for GTP. It is essential for the SPATA13-mediated regulation of cell migration and adhesion assembly and disassembly. The MEMO1-RHOA-DIAPH1 signaling pathway plays an important role in ERBB2-dependent stabilization of microtubules at the cell cortex. It controls the localization of APC and CLASP2 to the cell membrane, via the regulation of GSK3B activity. In turn, membrane-bound APC allows the localization of the MACF1 to the cell membrane, which is required for microtubule capture and stabilization.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.