MA1-011

antibody from Invitrogen Antibodies

Targeting: RHOC ARH9, ARHC

Provider product page for MA1-011

Western blot

Immunocytochemistry

Immunoprecipitation

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [2]
Comments [0]
Validations
Western blot [3]
Immunocytochemistry [4]
Immunohistochemistry [3]
Other assay [3]

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Product number: MA1-011 - Provider product page
Provider: Invitrogen Antibodies
Product name: RhoA/RhoB/RhoC Monoclonal Antibody (1A11-4G10)
Antibody type: Monoclonal
Antigen: Recombinant full-length protein
Description: Western blot analysis of MA1-011 detects an ~20 kDa protein and shows reactivity to RhoA, RhoB, and RhoC.
Reactivity: Human, Mouse, Rat
Host: Mouse
Isotype: IgG
Antibody clone number: 1A11-4G10
Vial size: 100 µg
Concentration: 1 mg/mL
Storage: -20°C

RHOC protein structure - MA1-011 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of RhoA/RhoB/RhoC was achieved by transfecting SW480 with RhoA/RhoB/RhoC specific siRNAs (Silencer® select Product # s758, s1576, s1574, s97). Western blot analysis (Fig. a) was performed using whole cell extracts from the RhoA/RhoB/RhoC knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with RhoA/RhoB/RhoC Monoclonal Antibody (1A11-4G10) (Product # MA1-011, 1:1000 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Reduction in signal upon siRNA mediated knock down confirms that antibody is specific to RhoA/RhoB/RhoC .

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of HeLa (Lane 1), A-431 (Lane 2), SW 480 (Lane 3), SK-OV-3 (Lane 4), NIH/3T3 (Lane 5) and PC-12 (Lane 6). The blot was probed with Anti-STAT6 Monoclonal Antibody (Product # MA1-011, 1:1000 dilution) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). A 21 kDa band corresponding to RhoA/RhoB/RhoC was observed across the cell lines tested.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of RhoA/B/C was performed by loading 25 µg of various whole cell lysates or 0.5 µg of purified protein (full-length His-RhoA, truncated GST-RhoB or full-length GST-RhoC fusion) per well onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with a RhoA/B/C monoclonal antibody (Product # MA1-011) at a dilution of 1:1000 overnight at 4°C on a rocking platform, washed in TBS-0.1%Tween-20, and probed with a goat anti-mouse IgG-HRP secondary antibody (Product # 32430) at a dilution of 1:15,000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34075).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Rho A/B/C (green) showing staining in the in the cytoplasm of A431 cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a Rho A/B/C monoclonal antibody (Product # MA1-011) in 3% BSA-PBS at a dilution of 1:50 and incubated overnight at 4ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Rho A/B/C (green) showing staining in the in the cytoplasm of NIH-3T3 cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a Rho A/B/C monoclonal antibody (Product # MA1-011) in 3% BSA-PBS at a dilution of 1:50 and incubated overnight at 4ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. Nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of RhoA/B/C (green) in NIH3T3 cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% BSA/TBST (Product # 37525) for 15 minutes at room temperature. Cells were probed with a RhoA/B/C monoclonal antibody (Product # MA1-011) at a dilution of 1:100 for at least 1 hour at room temperature, washed with PBS, and incubated with DyLight 488-conjugated goat-anti-mouse IgG secondary antibody at a dilution of 1:400 for 30 minutes at room temperature. Nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ArrayScan at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of RhoA/RhoB/RhoC was performed using 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with RhoA/RhoB/RhoC Mouse Monoclonal Antibody (Product # MA1-011) at 5 µg/mL in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of Rho A/B/C showing staining in the cytoplasm of paraffin-embedded human hepatocarcinoma (right) compared with a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Rho A/B/C monoclonal antibody (Product # MA1-011) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of Rho A/B/C showing staining in the cytoplasm of paraffin-embedded human prostate carcinoma (right) compared with a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Rho A/B/C monoclonal antibody (Product # MA1-011) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of Rho A/B/C showing staining in the cytoplasm of paraffin-embedded mouse breast tissue (right) compared with a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Rho A/B/C monoclonal antibody (Product # MA1-011) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunoprecipitation of RhoA/B/C was performed on HeLa cells. Antigen-antibody complexes were formed by incubating 750 µg of HeLa cell lysate with 2 µg of a RhoA/B/C monoclonal antibody (Product # MA1-011) overnight on a rocking platform at 4øC. The immune complexes were captured on 50 µL Protein A/G Plus Agarose (Product # 20423), washed extensively, and eluted with 5X Lane Marker Reducing Sample Buffer (Product # 39000). Eluted sample (right lane) and 25 µg of HeLa cell lysate (left lane) were resolved on a 4-20% Tris-HCl polyacrylamide gel, transferred to a PVDF membrane, and blocked with 5% BSA/TBS-0.1%Tween for at least 1 hour. The membrane was probed with a Rho A/B/C monoclonal antibody (Product # MA1-011) at a dilution of 1:1000 overnight rotating at 4øC, washed in TBST, and probed with Clean-blot IP Detection Reagent (Product # 21230) at a dilution of 1:2500 for at least 1 hour.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 3. Effects of miR-27a on the expression of migration and invasion-related proteins in RA-FLS. RA-FLS were transfected with miR-27a mimic or miR-27a inhibitor. (A) The protein and mRNA expression of MMP2, MMP9, and MMP13 in RA-FLS was detected by western blot and qRT-PCR assay. (B) The protein and mRNA expression of Rac1, Cdc42, and RhoA in RA-FLS was detected by western blot and qRT-PCR assay. * p < 0.05, versus control mimic group. # p < 0.05, versus control inhibitor group.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4 PDE5 overexpression and fibroblast activation. ( A ) Immunoblotting for PDE5 expression in mouse embryonic fibroblasts (MEFs) stably transfected with pEGFP (enhanced-green-fluorescent-protein) vector (V) and pEGFP-PDE5A expression plasmid (PDE5 1 and PDE5 2). beta-Actin was used as a control for equal loading and transfer. Italicized numbers below blots represent the mean of the band optical density expressed as fold over V for PDE1 and PDE2. ( B ) MTT growth and ( C ) Trypan blue cell count assays in V, PDE5 1, and PDE5 2 stable clones under basal nonstimulated conditions at indicated times. ( D ) Wound healing assay in V, PDE5 1, and PDE5 2 stable clones with images captured at 0 (inset) and 12 h. Pictures are representative of three independent experiments. ( E ) Boyden chamber transmigration and ( F ) invasion assays in V, PDE5 1, and PDE5 2 stable clones under basal nonstimulated conditions. ( G ) Immunofluorescent staining of phalloidin staining of F-actin (stress fibers, red, upper panel) and alpha-SMA (lower panel) and in stable clones. 4',6-Diamidino-2-phenylindole (DAPI) staining was used for nuclei detection (inset). Pictures are representative of three independent experiments. Scale bar = 5 um. ( H ) Immunoblotting for N-cadherin, alpha-SMA, Rho A-C, Rac 1-3, and cdc42 expression levels in V, PDE5 1, and PDE5 2 stable clones. beta-Actin was used as a control for equal loading and transfer. Italicized numbers below blots represent the mean of the band opti