For the immunoprecipitation experiment, D1 cells were lysed in buffer containing 200 mM Tris (pH 7.4), 1% Nonidet P-40, 0.2% SDS, protease inhibitor mix (Hoffman-La Roche Ltd., Basel, Switzerland), and phosphatase inhibitor mix (Hoffman-La Roche Ltd.). Equal amounts of total protein (500 µg) were subjected to immunoprecipitation using Catch and Release (EMD Millipore) with 2 µg of rabbit polyclonal RhoGDIα (Santa Cruz Biotechnology Inc.) according to manufacturer instructions. Immune complexes precipitated with RhoGDIα were subjected to SDS–polyacrylamide gel electrophoresis. After transferring proteins to polyvinylidene difluoride membranes, RhoA was detected using rabbit anti-RhoA (Santa Cruz Biotechnology Inc.). Immunoprecipitates were also immunoblotted, with RhoGDIα used as a loading control.
Rabbit anti rhoa
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Rabbit anti-RhoA is a primary antibody that recognizes the RhoA protein, a member of the Rho GTPase family. It is designed for use in various immunoassay techniques to detect and quantify RhoA expression in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor mix, by Roche (1 mentions)
Rho gdi α, by Santa Cruz Biotechnology (1 mentions)
Phosphatase inhibitor mix, by Roche (1 mentions)
Mouse anti gfap, by Merck Group (1 mentions)
Rabbit anti gapdh 14c10, by Cell Signaling Technology (1 mentions)
Mouse α tubulin, by Merck Group (1 mentions)
α rabbit cy5, by Jackson ImmunoResearch (1 mentions)
α mouse fitc, by Jackson ImmunoResearch (1 mentions)
α guinea pig fitc, by Jackson ImmunoResearch (1 mentions)
Chicken α gfp, by Abcam (1 mentions)
Mouse α dpmpk 1, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Polyvinylidene fluoride membrane, by Cytiva (1 mentions)
Rabbit anti bax, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti gapdh, by Bioworld Technology (1 mentions)
Rabbit anti yap, by Cell Signaling Technology (1 mentions)
Ecl reagent, by Thermo Fisher Scientific (1 mentions)
X ray film, by Kodak (1 mentions)
4 protocols using rabbit anti rhoa
1
RhoGDIα Immunoprecipitation for RhoA Detection
2
Protein Extraction and Antibody Analysis
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Total protein extraction and electrophoresis were done according to the previous protocol [27 (link)]. The following primary antibodies were used: rabbit anti-PDCD10 (Atlas; 1:400), rabbit anti-GAPDH (14C10) (Cell Signaling; 1:1000), rabbit anti-phospho-Akt (p-Akt, Cell Signaling; 1:600), rabbit anti-RhoA (Santa Cruz Technology, 1:200), rabbit anti-phospho-myosin light chain 2 (Thr18/Ser19) (p-MLC2) (Cell Signaling; 1:1000) and mouse anti-GFAP (Sigma Aldrich; 1:5000), For semi-quantification of the blot, integrated optical density (IOD) of the bands was measured by Image J software. The relative expression of a target protein was calculated by the IOD ratio of the target protein to the housekeeping protein GAPDH, and the data were presented as percentage of the control.
3
Immunofluorescence and Western Blot Protocols
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Primary antibodies were used at the following dilutions for immunofluorescence: chicken α-GFP (1:400; Abcam, Cambridge, MA), rabbit α-SYP-1 (1:200;(MacQueen et al., 2002 (link)), guinea pig α-HTP-3 (1:400; (Goodyer et al., 2008 (link)), rabbit α-AIR-2 (1:100;[de Carvalho et al., 2008 (link)]), rabbit α-LAB-1 (1:300;[de Carvalho et al., 2008 (link)]), mouse α-dpMPK-1 (1:500; Sigma, St. Louis, MO) and rabbit anti-RhoA (1:200; Santa Cruz). The following secondary antibodies from Jackson ImmunoResearch (Jackson ImmunoResearch, WestGrove, PA) were used at a 1:200 dilution: α-chicken FITC, α-rabbit Cy5, α-mouse FITC, and α-guinea pig FITC. Vectashield containing 1μg/μl of DAPI from Vector Laboratories was used as a mounting media and anti-fading agent.
Primary antibodies were used in the following dilutions for western blot analysis: chicken α-GFP (1:2000; Abcam), rabbit α-SYP-2 (1:200;[Colaiácovo et al., 2003 (link)]), rabbit α-SYP-3 (1:200; [Smolikov et al., 2007b (link)]), mouse α-dpMPK-1 (1:500; Sigma), mouse α-tubulin (1:2000; Sigma) and rabbit α-MPK-1 (1:2000; Santa Cruz, Dallas, TX). HRP-conjugated secondary antibodies, donkey anti-chicken, rabbit anti-mouse, and mouse anti-rabbit from Jackson ImmunoResearch were used at a 1:10,000 dilution.
Primary antibodies were used in the following dilutions for western blot analysis: chicken α-GFP (1:2000; Abcam), rabbit α-SYP-2 (1:200;[Colaiácovo et al., 2003 (link)]), rabbit α-SYP-3 (1:200; [Smolikov et al., 2007b (link)]), mouse α-dpMPK-1 (1:500; Sigma), mouse α-tubulin (1:2000; Sigma) and rabbit α-MPK-1 (1:2000; Santa Cruz, Dallas, TX). HRP-conjugated secondary antibodies, donkey anti-chicken, rabbit anti-mouse, and mouse anti-rabbit from Jackson ImmunoResearch were used at a 1:10,000 dilution.
4
Western Blot Analysis of Cellular Proteins
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Whole and nuclear proteins were prepared as described previously [6 (link)]. An aliquot of cell lysates containing 30 μg of protein was separated on 10 % sodium dodecyl sulfate–polyacrylamide gels, and then transferred to polyvinylidene fluoride membranes (Amersham Biosciences). After blocked by 5 % skim milk for 1 h at room temperature, membranes were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-YAP (Cell Signaling Technology, USA, 1:1000), rabbit anti-Histone (Cell Signaling Technology, USA, 1:3000), rabbit anti-RhoA (Santa Cruz, USA, 1:500), rabbit anti-Bax (Santa Cruz, USA, 1:500), rabbit anti-GAPDH (Bioworld Technology, China, 1:10000), rabbit anti-Dia (Santa Cruz, USA, 1:500), rabbit anti-Dendrin (Santa Cruz, USA, 1:500). The membranes were then incubated with anti-rabbit IgG (Jackson Immuno Research, USA, 1: 4000) at room temperature for 1 h. Finally, membranes were treated with ECL reagents (Pierce Chemical, IN, USA), followed by exposing to X-ray film (Kodak, USA). The bands of the resulting autoradiographs were quantified densitometrically using Bandscan software. Protein expression was quantified as the ratio of specific band to Histone (nuclear fractions) or GAPDH.
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