Whole cell lysates or immunoprecipitated proteins were separated by SDS-PAGE electrophoresis and transferred to PVDF membranes (Westran® Clear Signal). The membranes were blocked with 5% milk or bovine serum albumin (BSA) in TBS-0.1% Tween20 (TBS-T) and subsequently immunoblotted overnight at 4°C. The membranes were then probed with horseradish peroxidase-conjugated secondary antibodies (Jackson Immunoresearch, Beckman Coulter, France) followed by SuperSignal West Pico Substrate (Thermo Scientific). Chemiluminescence was detected with a Fuji LAS-4000 luminescent image analyzer. The antibodies used in this study were as follows: anti-actin (Sigma-Aldrich), anti-HA (Covance, Eurogentec, France), anti-pan-CD44, anti-phosphoTrkA (Tyr-674/675), anti-p115-RhoGEF, anti-ROCK1, anti-RhoA, anti-RhoC, anti-phosphoAkt (Ser-473), and anti-pan-Akt (Cell Signaling Technologies).
Anti rhoc
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-RhoC is a primary antibody that specifically recognizes the RhoC protein. RhoC is a small GTPase that belongs to the Rho family of proteins, which play important roles in regulating cell cytoskeleton organization, migration, and other cellular processes. The Anti-RhoC antibody can be used to detect and analyze the expression and localization of RhoC in various cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti rhoa, by Cell Signaling Technology (4 mentions)
Anti akt, by Cell Signaling Technology (3 mentions)
Anti rock1, by Cell Signaling Technology (2 mentions)
Anti p akt, by Cell Signaling Technology (2 mentions)
Anti rock, by Cell Signaling Technology (2 mentions)
Anti rhob, by Cell Signaling Technology (2 mentions)
Anti actin antibody, by Santa Cruz Biotechnology (2 mentions)
Anti cdc42, by Cell Signaling Technology (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Supersignal west pico substrate, by Thermo Fisher Scientific (1 mentions)
Anti pan akt, by Cell Signaling Technology (1 mentions)
Anti actin, by Merck Group (1 mentions)
Anti p115 rhogef, by Cell Signaling Technology (1 mentions)
Anti phospho akt ser473, by Cell Signaling Technology (1 mentions)
Anti ha, by Fortrea (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Anti p yap, by Cell Signaling Technology (1 mentions)
Anti tlr4, by Santa Cruz Biotechnology (1 mentions)
Anti pi3k, by Cell Signaling Technology (1 mentions)
13 protocols using anti rhoc
1
Western Blot Analysis of Cellular Proteins
2
Investigating NF-κB Signaling Pathway
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PB2 (purity > 90%), DEX (used as a positive control), and LPS derived from bacteria (serotype: 0111:B4, L5293) were purchased from Sigma (St. Louis, MO, USA). Anti-NF-κB, anti-p-NF-κB, anti-Akt, anti-p-Akt, anti-PI3K, anti-p-PI3K, anti-YAP, anti-p-YAP, anti-ROCK, anti-Rhoc, and anti-β-actin antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-TLR4 and anti-p-LATS1/2 (phospho-Ser909/872) antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA) and MyBioSource (San Diego, CA, USA), respectively.
3
Comprehensive Protein Analysis of C2C12 Cells
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C2C12 cell lysates were prepared using RIPA buffer containing protease inhibitors, phosphatase inhibitors and dithiothreitol. The protein concentration was measured using the BCA Kit (Beyotime). Western blot analyses were performed with the following primary antibodies: anti-AKT (cat. #4691), anti-p-AKT 473 (cat. #4060), anti-p-AKT 308 (cat. #2965), anti-ERK1/2 (cat. #9102), anti-p-ERK1/2 (cat. #9101), anti-CCND2 (cat. #3741) and anti-JAK1 (cat. #3344) from Cell Signalling Technology (USA) (all at 1:1000), anti-RHOC (1:500, cat. #D260057) from Sangon Biotech (China), and anti-β-actin (1:5000, cat. #A5441) from Sigma-Aldrich (USA). After an overnight incubation at 4 °C, the membranes were incubated for 1 h at RT with the following secondary antibodies: anti-rabbit or anti-mouse IgG-horseradish peroxidase (HRP) (Proteintech, USA). Finally, the protein bands were detected using the Super ECL Detection Reagent (Tanon, China).
4
Western Blot Protein Quantification
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Total protein was extracted with an appropriate amount of lysis buffer (P0013; Beyotime Biosciences, Shanghai, China), and appropriate amounts of protease inhibitor and phosphatase inhibitor (B14002 and B15002, respectively; Biotool, Shanghai, China) were used to extract protein from 35 μg of samples. The ratio of protein lysate buffer, protease inhibitor, and phosphatase inhibitor was 100:1:1, and the total volume of the mixture was four times the cell volume. The following primary antibodies were used: anti-CUL3 (11107-1-AP, 1:100), anti-p27 (25614-1-AP, 1:500), anti-MMP9 (10375-2-AP, 1:1,000), and anti-FLAG (66008-2-Ig, 1:100) from Proteintech Group (Chicago, IL, USA); anti-phospho-mTOR (5536 s, 1:1,000), anti-mTOR (2983 s, 1:1,000), anti-PI3Kp85α (13666S, 1:1,000), anti-PI3Kp85α (4257S, 1:1,000), anti-AKT (4691S, 1:1,000), anti-phospho-AKT (4060, 1:1,000), anti-PD-L1 (13684S, 1:1,000), anti-RhoC (D40E4, 1:1,000), anti-N-Cadherin (13116 s, 1:1,000), anti-Snail (3879 s, 1:1,000), anti-Slug (9585 s, 1:1,000), and anti-HA-Tag (3724 s, 1:1,000) from Cell Signaling Technology (Danvers, MA, USA); and anti-GAPDH (TA319654, 1:1,000) from Origene. Proteins were visualized using enhanced chemiluminescence (34080; Thermo Fisher Scientific). ImageJ software (NIH, Bethesda, MD, USA) was used to quantitatively evaluate the grayscale integral value of each band [35 (link)].
5
Cell Culture Reagents and Antibodies
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BD Matrigel matrix was purchased from BD Bioscience (Bedford, MA). Lipofectamine 2000, fluorescein isothiocyanate–gelatin (G13187), Alexa Fluor 488–phalloidin, rhodamine–phalloidin, and Hoechst were purchased from Invitrogen (Burlington, Canada). Anti-ARF1 was obtained from ProteinTech (Chicago, IL). Anti-cortactin, anti–myosin light chain 2, anti–myosin light chain 2 Thr-18/Ser-19, anti–pan-actin, Anti-cortactin, and anti-RhoC were from Cell Signaling Technology (Danvers, MA). Anti–pro-MMP-9 (IM09L) antibody was obtained from Calbiochem (Gibbstown, NJ) and anti-MMP9 from Abcam (Cambridge, MA). Anti–major histocompatibility complex class I (H300), anti-RhoA (26C4), anti–MMP-2 (H-76), anti-Ub (P4D1), and anti-Tks5 (M-300) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All others products were from Sigma Aldrich (Oakville, Canada).
6
Molecular Regulation of Cellular Dynamics
7
Pull-down Assay for Small GTPases
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The pull down assay protocol was performed as previously described (Pellegrin & Mellor, 2008 ). Briefly, 1 × 107 bone marrow cells from WT and Arhgap21+/− mice were lysed in RIPA buffer (50 mM Tris–HCl pH 8, 150 mM NaCl, 0.1% SDS, 1% NP-40 and 0.5% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and 1 μg/mL leupeptin) at 4–6 °C. Lysates were cleared by centrifuging 20 min at 15,000g, 4 °C. The cleared lysate was incubated with Rhotekin-GST or Wasp-GST beads overnight in rotation at 4 °C. The beads were washed 4 times in 4–6 °C 1× lysis buffer. Proteins eluted from beads and the input samples were analyzed by SDS-PAGE. The blots were probed with anti-Cdc42, anti-RhoA, anti-RhoC (all from Cell Signaling) and anti-actin antibodies (Santa Cruz). Wasp-GST beads were kindly provided by Prof. Anne Ridley (King’s College London) and Rhotekin-GST beads were from Cytoskeleton.
8
Multispecific Antibody Detection
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The following antibodies were used: anti-pSer19-MLC2 (3675), anti-Ki67 (12202), anti-cleaved caspase 3 (9661), anti-CD31 (77699), anti-pT696-MYPT1 (5163), anti-MYPT1 (2634), anti-pY461-Src (6943), anti-pT202/Y204ERK1/2 (4370), anti-ERK1/2 (4695), anti-pT308-Akt1 (2965), anti-pS473-Akt1 (4060), anti-Akt (2920), anti-PP2A-C (2038), anti-PP2A-A (2041), anti-Shc (2432), anti-PI3 kinase p85 (4257), and anti-RhoC (3430) (Cell Signaling Technology); anti-β-actin (A5316) (Sigma-Aldrich); anti-PyMT (sc-53,481), anti-GST (sc-138), anti-Src (sc-8056), and normal rat IgG (sc-2026) (SantaCruz Biotechnology); and anti-RhoA (ARH03) (Cytoskeleton, Inc.).
9
CRC Cell Lines Signaling Pathway Analysis
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All four CRC cell lines (LOVO, SW620, SW480 and HCT116) and the 293T cell line were purchased from Cell Bank of Chinese Academy of Sciences (Shanghai, China). The cell lines were cultured at 37 °C in a 50 mL/L CO2-humidified atmosphere with the appropriate medium according to the requirements of the Cell Bank. Anti-(p-) Pyk2 (proline-rich tyrosine kinase 2), anti-(p-) FAK, anti-(p-) MAPK (Mitogen activated protein kinase), anti-(p-) AKT and anti-RhoC antibodies were purchased from Cell Signaling Technology. Anti-flag, anti-VEGF (vascular endothelial growth factor) and anti-FMNL3 antibodies were obtained from Abbkine, Inc (Redlands, CA, United States) and Abnova (Taiwan, China), respectively. For inhibitor treatment, 1 μmol/L TAE226 (Selleck), 20 μmol/L U0126 (Selleck) or 20 μmol/L Ly294002 (Selleck) was added to the cultured cells for 48 h, respectively.
10
Western Blot Analysis of Cell Protein Markers
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Cell protein content in the lysates was determined through the BCA protein assay (Beyotime Biotechnology, China) according to manufacturer’s instructions. Cells protein lysates were separated in 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to 0.22 μm PVDF membranes (Millipore, Massachusetts, USA). 5% skim milk containing 0.1% Tween-20 was used to block the PVDF membranes and then incubated with specific antibodies at 4 °C overnight. Horseradish peroxidase-linked secondary antibodies were added at a dilution ratio of 1:1000, and incubated at room temperature for 1 h. The immunoreactive bands were visualized using ECL Kit (Thermo Fisher). Anti-cleaved Caspase3, anti-Bcl-2, anti-Bax and anti-N-Cadherin antibodies were purchased from Abcam, USA. Anti-E-Cadherin, anti-Vimentin, anti-Cyclin D1, anti-MMP-9, anti-RhoC, anti-ROCK1, anti-p38MAPK, anti-Phospho-p38MAPK, and anti-GAPDH were purchased from Cell Signaling Technology, Inc. Anti-Twist1 and anti-AFAP1 antibodies were purchased from Proteintech Group, USA.
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