Western blot analyses were performed as previously described [17 (link), 26 (link), 39 (link)]. Briefly, 90 % confluent NIH3T3 cells were serum-starved for 22 h. Cells were counted and lysed in 1× SDS running buffer, heated at 100 °C for 10 min, and centrifuged to remove debris. Lysates were resolved on 10 % polyacrylamide gels, transferred to nitrocellulose membranes, and blotted with the following primary antibodies: anti-KRAS4A specific antibody (1:500; Santa Cruz Biotechnology, sc-522), anti-RAS antibody (1:2000; Cell Signaling Technology, #3965), anti-actin (1:5000; Sigma-Aldrich, A1978), anti-GFP (1:2000; Cell Signaling Technology, #2555), and phospho-c-RAF, c-RAF, phoshpo-MEK1/2, MEK1/2, phospho-ERK1/2, ERK1/2, phospho-AKT, AKT, phospho-S6RP, and S6RP (All 1:2000; Cell Signaling Technology). HRP-labeled goat anti-mouse IgG or goat anti-rabbit IgG was used as a secondary antibody.
Anti ras antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti-Ras antibody is a laboratory reagent used to detect and quantify Ras proteins, which are important signaling molecules involved in cellular processes. The antibody is designed to specifically bind to Ras proteins, allowing researchers to identify and measure their presence in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti actin, by Merck Group (1 mentions)
Phospho s6rp, by Cell Signaling Technology (1 mentions)
Phospho c raf, by Cell Signaling Technology (1 mentions)
C raf, by Cell Signaling Technology (1 mentions)
Phospho akt, by Cell Signaling Technology (1 mentions)
Anti gfp, by Cell Signaling Technology (1 mentions)
Anti craf antibody, by Cell Signaling Technology (1 mentions)
Anti p44 42 mapk erk1 2 antibody, by Cell Signaling Technology (1 mentions)
Anti phospho p44 42 mapk erk1 2 thr202 tyr204 antibody, by Cell Signaling Technology (1 mentions)
Anti mek1 2 antibody, by Cell Signaling Technology (1 mentions)
Anti phospho mek1 2 ser217 221 antibody, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Anti tubulin antibody, by Merck Group (1 mentions)
Anti erk antibody, by Cell Signaling Technology (1 mentions)
Anti egfr antibody, by Cell Signaling Technology (1 mentions)
Anti actin antibody, by Merck Group (1 mentions)
4 protocols using anti ras antibody
1
Western Blot Analysis of Cellular Signaling
2
Protein Immunoblotting with Epitope Detection
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Proteins were blotted onto a polyvinylidene difluoride membrane, which was then probed with the appropriate antibodies. Signals were visualized by chemiluminescence (Millipore), and images were acquired with ImageQuant LAS (GE Healthcare). A monoclonal anti-M2 antibody (Sigma-Aldrich, A8592, 1:25,000) was used to detect the Flag epitope. An anti-Ras antibody (#3965, 1:1,000) was purchased from Cell Signaling Technologies, USA. Rabbit polyclonal anti-Gβ (a.a. 35–51, 1:5,000) and -Gip1 (a.a. 96–110, 1:1000) antibodies were made in-house20 (link). Rabbit polyclonal anti-Gα2 antibody (1:5000) was kindly provided by Dr. H. Kuwayama (Tsukuba University). Uncropped images are presented in Supplementary Figure 11 .
3
Western Blot Analysis of MAPK Signaling Pathway
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Cells were lysed with SDS lysis buffer (0.1 M Tris‐HCl at pH 8.0, 10% glycerol, 1% SDS) and immediately boiled for 10 minutes to obtain clear lysates. Protein concentrations were measured using the BCA method (Pierce). Lysates containing equal amounts of proteins were separated by SDS‐PAGE and transferred to PVDF membranes (Merck) for Western blot analysis using the appropriate antibodies. Immunoreactive proteins were visualized using the Immobilon Western chemiluminescent HRP substrate (Merck) or Clarity Western ECL substrate (Bio‐Rad); light emission intensity was quantified using an LAS‐3000 lumino‐image analyzer equipped with Image Gauge v2.3 software. The antibodies used in this study were: anti‐BRAF antibody (14814; Cell Signaling Technology), anti‐CRAF antibody (53745; Cell Signaling Technology), anti‐MEK1/2 antibody (8727; Cell Signaling Technology), anti‐phospho‐MEK1/2 (Ser217/221) antibody (9121; Cell Signaling Technology), anti‐p44/42 MAPK (ERK1/2) antibody (4695; Cell Signaling Technology), anti‐phospho‐p44/42 MAPK (ERK1/2) (Thr202/Tyr204) antibody (4377; Cell Signaling Technology), anti‐RAS antibody (8955; Cell Signaling Technology), anti‐CRBN antibody (71810; Cell Signaling Technology), and anti‐β‐actin antibody (A5316; Merck).
4
Evaluating ERK Signaling in Glioblastoma
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To determine ERK signaling pathway in 25 glioblastoma specimens, protein lysates were extracted with NP-40 buffer (1% NP-40, 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 10% glycerol,2 mM EDTA, 1 mM sodium orthovanadate, 10 μg/mL Aprotinin, 10 μg/mL Leupeptin, and 10 μg/mL Pepstatin). 50 μg of the protein lysate was fractionated by SDS-PAGE following the Western blotting using a phospho-specific anti-pERK antibody (Cell Signaling Technology, 1:1,000), anti-ERK antibody (Cell Signaling Technology, 1:2,000), anti-actin antibody (Sigma, 1:10,000), anti-Tubulin antibody (Sigma, 1:10,000), anti-Ras antibody (Cell Signaling Technology, 1: 1,000), anti-EGFR antibody (Cell Signaling Technology, 1:1,000). Band intensities were analyzed using Image J software (Bethesda, MD). The levels of pERK and ERK were quantitated based on methods previously described [24 (link), 25 (link)]. In brief, levels of pERK and ERK were normalized to actin. Ratio of normalized pERK to ERK was then determined and compared. Statistical comparisons of the averaged scores were performed using unpaired t-test.
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