Cells were serum-starved overnight and stimulated with LPA. Active small G-proteins were detected by pull-down with GST-RBD and GST-CRIB. Anti-RhoA (2117; Cell Signaling Technology), anti-Rac1 (2320346; Merck Millipore), and anti-Cdc42 (2466; Cell Signaling Technology) against the corresponding small G-protein were used for immunoblotting in this assay. The active and total GTPases were subsequently detected with horseradish peroxidase- (HRP-) conjugated secondary antibody according to the manufacturer's recommendations.
Anti cdc42
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-Cdc42 is a laboratory product that detects the Cdc42 protein, a small Rho GTPase involved in cell signaling and cytoskeleton regulation. It is designed for use in various research applications, such as Western blotting, immunoprecipitation, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Anti rhoa, by Cell Signaling Technology (15 mentions)
Anti rac1, by Cell Signaling Technology (4 mentions)
Anti gapdh, by Cell Signaling Technology (4 mentions)
Anti rac1, by Merck Group (4 mentions)
Horseradish peroxidase conjugated secondary antibody, by GE Healthcare (3 mentions)
Anti vimentin, by Cell Signaling Technology (3 mentions)
Anti pkcε, by Cell Signaling Technology (2 mentions)
Anti e cadherin, by R&D Systems (2 mentions)
Anti pkcε, by Santa Cruz Biotechnology (2 mentions)
Anti phospho smad2 3, by Cell Signaling Technology (2 mentions)
Anti phospho mypt1, by Cell Signaling Technology (2 mentions)
Anti pkcδ, by Cell Signaling Technology (2 mentions)
Anti vinculin, by Merck Group (2 mentions)
Anti β actin, by BD (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Anti actin antibody, by Santa Cruz Biotechnology (2 mentions)
Anti rhoc, by Cell Signaling Technology (2 mentions)
Cdc42, by Horizon Discovery (2 mentions)
Ecl system, by Merck Group (2 mentions)
Rho rac cdc42 assay reagent kit, by Thermo Fisher Scientific (2 mentions)
25 protocols using anti cdc42
1
Detecting Active Small G-proteins
2
Antibody Reagents for Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Anti-LC3B, anti-Bcl-xL, anti-Atg16L1, anti-Rac1, anti-Rho1, anti-Cdc42, anti-phospho-ERK, anti-EEA1 and anti-LAMP1 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Mouse monoclonal antibodies reactive with LRP1 (11H4) were a kind gift from Dr. Strickland, University of Maryland School of Medicine, Baltimore. Fibronectin (EP5) and ubiquitin (P4D1) were from Santa Cruz Biotechnologies (Santa Cruz, CA, USA); anti-Cx43, anti-ERK, anti-Bcl-2 and anti-RPTPβ antibodies were from BD Biosciences (Tokyo, Japan); anti-multi ubiquitin monoclonal antibody (FK1) was from MBL (Nagoya, Japan); anti-GAPDH antibody was from GeneTex (Irvine, CA, USA) and anti-LC3 (clone 1703) antibody was from Cosmo Bio (Tokyo, Japan). Anti-RPTPα rabbit polyclonal antibodies for immunoblotting were provided by Dr. Jan Sap; anti-α-tubulin and anti-FLAG M2 antibodies, N-acetyl-l -cysteine (NAC) and ammonium chloride were from Sigma Aldrich (St. Louis, MO, USA); ProLong Gold Antifade Reagent with DAPI and DIDS were from Invitrogen (Eugene, OR, USA); an ERK inhibitor, U0126, was from Cayman Chemical (Ann Arbor, MI, USA); Rac1 inhibitor, NSC23766 was from Wako Pure Chemical Industries (Osaka, Japan); and GSH was from Merck (Darmstadt, Germany).
3
GTPase Activation Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GTPase activation was performed according to standard procedures as described elsewhere [25 (link)]. In brief, Cells were harvested in magnesium-containing lysis buffer and the lysates were sonicated for 5 s and centrifuged for 30 min at 18,000×g and 4 °C the Rho/Rac/Cdc42 Assay Reagent Kit (Thermo Scientific, USA) according to the manufacturer’s instructions. The primary antibodies used were anti-RhoA (Cell Signaling Technology, #2117, diluted at 1:1000), and anti-Rac1 (Millipore, 05–389, clone 23A8, diluted at 1:200), anti-Cdc42 (Cell Signaling Technology, #2462, diluted at 1:1000).
4
Investigating MUC1-C Protein Interactions in Cell Lysates
5
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blots were performed as previously described (17 (link), 18 (link), 58 (link)) using NuPage 4 to 12% bis-tris gels and the following antibodies (at the manufacturer’s recommended concentrations): anti-TRPM7 (mouse, clone S74-25, Thermo Fisher Scientific MA5-27620), anti-Cdc42 (rabbit, clone 11A11, Cell Signaling Technology 2466), and anti-IQGAP1 (mouse, clone 9, Santa Cruz Biotechnology, sc-376021) with glyceraldehyde-3-phosphate dehydrogenase (rabbit, clone 14C10, Cell Signaling Technology 2118) or β-actin (mouse, clone C4, BD Biosciences 612656) as a loading control. Secondary antibodies (used at 1:1000 dilution): anti-mouse immunoglobulin G (IgG) horseradish peroxidase (HRP)–linked antibody (Cell Signaling Technologies) and anti-rabbit IgG HRP-linked antibody (Cell Signaling Technologies).
6
Immunoblotting Analysis of Cytoskeletal Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunoblotting analysis was performed according to a standard protocol with the following antibodies: anti-ARHGEF37 (HPA053487) and anti-Flag (F3165) (Sigma-Aldrich, St. Louis, MO, USA); anti-phosphorylated (p)-PAK1) (phospho S204) (ab79503) and anti-PAK1 (ab131522) antibodies (Abcam, Cambridge, MA, USA); anti-Cdc42 (CST#2466S), anti-N-cadherin (CST#13116S), and anti-Connexin-43 (Cx43) (CST#3512S) antibodies (Cell Signaling technology, Danvers, MA, USA); anti Cdc42-GTP (26905), anti Ras-GTP (26903), and anti RhoA-GTP (26904) antibodies (NewEast Biosciences; King of Prussia, PA, USA), and anti-GAPDH (60004-1-Ig) antibodies (Proteintech, Rosemont, IL, USA).
7
Rho GTPase Activation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were separated on SDS-PAGE and transferred to nitrocellulose membrane (Bio-Rad). The membrane was blocked with 5% non-fat milk and incubated with the corresponding mouse anti-ROCK2, EZH2, COX2, E-cadherin, α-catenin, β-catenin, N-cadherin, fibronectin, vimentin (BD Biosciences, 1:1000 dilution), STMN1 and α-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, 1:1000 dilution) and GAPDH (Cell signaling Technology, Beverly, MA, 1:500 dilution) monoclonal antibodies. The proteins were detected with enhanced chemiluminescence reagents.
PAK1 PBD-agarose (for isolating Rac1-GTP and cdc42-GTP) and rhotekinagarose (for isolating Rho-GTP) (Upstate Biotechnology, Lake Placid, NY) were used to pull down the GTP-bound form of Rho-GTPase according to the manufacturer’s manual. The levels of active Rac1, cdc42 and RhoA were detected by Western blot using specific polyclonal anti-Rac1 (1:1000), anti-cdc42 (1:1000) and anti-RhoA (1:1000) antibodies (Cell Signaling Technology, Beverly, MA).
PAK1 PBD-agarose (for isolating Rac1-GTP and cdc42-GTP) and rhotekinagarose (for isolating Rho-GTP) (Upstate Biotechnology, Lake Placid, NY) were used to pull down the GTP-bound form of Rho-GTPase according to the manufacturer’s manual. The levels of active Rac1, cdc42 and RhoA were detected by Western blot using specific polyclonal anti-Rac1 (1:1000), anti-cdc42 (1:1000) and anti-RhoA (1:1000) antibodies (Cell Signaling Technology, Beverly, MA).
8
Western Blot Analysis of Key Signaling Proteins
9
Antibody Generation and Validation Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rabbit anti-Robo1 and anti-Arl4A/C/D antibodies were generated as described previously (Li et al., 2007 (link)). The following antibodies were used: anti-Myc (1:1000, catalogue no. MMS-150R; Covance, Princeton, NJ), anti-HA (1:1000, catalogue no. SC-7392; Santa Cruz Biotechnology), anti-LexA (1:1000, catalogue no. 5397-1; Clontech, Mountain View, CA), anti-CD44 (1:200 for IF, catalogue no. MA4400; Invitrogen), anti-Cdc42 (1:500, catalogue no. 2462; Cell Signaling, Danvers, MA), anti-srGAP1 (1:1000, catalogue no. 76926; Abcam), and anti-α-tubulin (1:5000, catalogue no. T5168; Sigma-Aldrich, St. Louis, MO). A blue-fluorescent DNA stain 4′,6-diamidino-2-phenylindole (DAPI) solution was purchased from Millipore (1:5000, catalogue no. S7113). Horseradish peroxidase (HRP)–conjugated goat anti-rabbit and anti-mouse antibodies were purchased from GE Healthcare (1:5000, catalogue nos. NA934V and NA931V, respectively; Waukesha, WI). Alexa Fluor–conjugated secondary antibodies were purchased from Invitrogen (Grand Island, NY). Alexa Fluor 594/488–conjugated anti-rabbit/anti-mouse and Alexa Fluor 647–conjugated anti-rat secondary antibodies were from Invitrogen (1:1000, catalogue nos. A-11012 for Alexa Fluor 594-rabbit, A-11034 for Alexa Fluor 488-rabbit, A-11001 for Alexa Fluor 488-mouse, A-11032 for Alexa Fluor 594-mouse, and A-21247 for Alexa Fluor 647-rat).
10
Senescent Cell Signaling Regulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Detection of protein expression by immunoblotting was performed using anti-CDC42 (Cell Signaling Technology), anti-β-tubulin, anti-pan-Ras, and anti-p16 (Santa Cruz Biotechnology).
ON-TARGETplus SMARTpool siRNA targeting RHOA, RAC1, RAC2, CDC42, and the nontargeting (control) pool were transfected into senescent IMR-90 cells with Dharmafect 1 reagent (all from Dharmacon). Cells were washed 24 h after transfection; transfer assay, cell viability assay, and evaluation of knockdown were performed 48 h later.
For retroviral transductions, the following plasmids were used: pLPC mCherry or pLPC EGFP (mCherry or GFP fused to the gene of interest from an internal CMV promoter with a puromycin resistance gene driven by the LTR). Cells were infected with mCherry-H-Ras12V, GFP, or mCherry alone. Retrovirus-mediated gene transfer was performed as previously described.
Total RNA was isolated using a NucleoSpin kit (Macherey Nagel) and reverse-transcribed using the RevertAid H Minus first strand cDNA synthesis kit (Fermentas). The cDNA samples were used for real-time PCR (Fast SYBR Green master mix, Applied Biosystems). PCR amplifications were carried out using StepOnePlus real-time PCR systems (Applied Biosystems). The relative expression of each gene was normalized using the expression levels of GAPDH.
ON-TARGETplus SMARTpool siRNA targeting RHOA, RAC1, RAC2, CDC42, and the nontargeting (control) pool were transfected into senescent IMR-90 cells with Dharmafect 1 reagent (all from Dharmacon). Cells were washed 24 h after transfection; transfer assay, cell viability assay, and evaluation of knockdown were performed 48 h later.
For retroviral transductions, the following plasmids were used: pLPC mCherry or pLPC EGFP (mCherry or GFP fused to the gene of interest from an internal CMV promoter with a puromycin resistance gene driven by the LTR). Cells were infected with mCherry-H-Ras12V, GFP, or mCherry alone. Retrovirus-mediated gene transfer was performed as previously described.
Total RNA was isolated using a NucleoSpin kit (Macherey Nagel) and reverse-transcribed using the RevertAid H Minus first strand cDNA synthesis kit (Fermentas). The cDNA samples were used for real-time PCR (Fast SYBR Green master mix, Applied Biosystems). PCR amplifications were carried out using StepOnePlus real-time PCR systems (Applied Biosystems). The relative expression of each gene was normalized using the expression levels of GAPDH.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!