Radio Immunoprecipitation Assay (RIPA) lysis buffer, provided by Sigma (St. Louis, MO, USA), was used to collect protein lysates. BCA method was applied to quantify the total protein concentration. Proteins were added to SDS polyacrylamide gel electroelectrometry (PAGE), and electrotransferred to polyvinylidene fluoride (PVDF) membrane after electrophoresis separation. Blocking buffer (5% bovine serum albumin, BSA) was used to block the membranes at room temperature. After blocking, primary antibodies were used to incubate the membranes overnight at 4°C. Primary antibodies (Abcam, Cambridge, MA, USA) were used at the following dilutions: anti-PCNA (1:1000, ab18197), anti-cleaved-caspase-3 (1:1000, ab2302), anti-caspase-3 (1:500, ab13847), anti-Twist (1:2000, ab175430), anti-MMP-2 (1:1000, ab37150), anti-MMP-9 (1:1000, ab73734), anti-phosphorylation-p38 (p-p38) (1:1000, ab4822), anti-p38 (1:1000, ab31828), anti-p-JNK (1:1000, ab124956), anti-JNK (1:1000, ab179461), anti-p-ERK (1:1000, ab65142), anti-ERK (1:10,000, ab32537), anti-GAPDH (1:500, ab8245). After washing the membranes Tris-buffered saline containing 0.1% Tween-20 (TBS-T) for 4 times, the secondary antibody was added and incubated at 37°C for 30 min. GAPDH served as the loading control. All experiments were repeated 3 times.
Ab32537
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab32537 is a laboratory equipment product offered by Abcam. This product is a device used for a specific laboratory function. The core function of this product is to perform a specific task required in a laboratory setting. No further details about the intended use or interpretation of this product are provided.
Automatically generated - may contain errors
Lab products found in correlation
Ab201015, by Abcam (6 mentions)
Ab8245, by Abcam (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Ab7090, by Abcam (3 mentions)
Ab8226, by Abcam (3 mentions)
Ab170099, by Abcam (3 mentions)
Ab31828, by Abcam (2 mentions)
Ab2302, by Abcam (2 mentions)
Ab181602, by Abcam (2 mentions)
Ab32572, by Abcam (2 mentions)
Bca kit, by Beyotime (2 mentions)
Ab6789, by Abcam (2 mentions)
Ab205718, by Abcam (2 mentions)
Ab6721, by Abcam (2 mentions)
Ab229912, by Abcam (2 mentions)
Ab2071, by Abcam (2 mentions)
Ab193955, by Abcam (2 mentions)
Ab109332, by Abcam (2 mentions)
Ab6276, by Abcam (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
23 protocols using ab32537
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of Signaling Proteins
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After 48 hours of transfection, the cells in six-well plates were collected and placed on ice. To extract the proteins, RIPA lysate with protease inhibitor was used. BCA method was used to determine the protein concentration. Then we added about 20 μg protein to each well of a vertical electrophoresis tank after being heated at 95 °C for 5 min. Following that, the protein samples were separated by SDS-PAGE and transferred onto a PVDF membrane. The membrane was blocked in skim milk for 1 h and then incubated with primary antibodies at 4 °C overnight. The primary antibodies used in the current study were list as follows: ARHGEF39 (1:1000, cat.no. ab67211, Abcam), AKT (1:500, cat. no. ab64148, Abcam), p-AKT (1:500, cat.no. ab8932, Abcam), ERK (1:1000, cat.no. ab32537, Abcam), p-ERK (1:1000, cat.no. ab131438, Abcam). Following that, the membrane was rinsed with TBST 35 min and incubated with suitable secondary antibodies at ambient temperature. Then the protein bands were washed and developed with enhanced chemiluminescence western blot detection kit. The gray value was scanned by the QUANTITY ONE software and the relative expression of each protein was calculated with GAPDH as the internal reference.
3
Western Blot Analysis of Signaling Proteins
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Cultured cells were collected and lysed with RIPA buffer containing the protease inhibitors on ice for 30 min. Protein was separated by SDS-PAGE, transferred onto a nitrocellulose membrane (Pall Co., USA) and probed with primary antibodies, including GAPDH as the internal standard, and then with horseradish peroxidase-labeled secondary antibodies. The blots were incubated with antibodies against β-catenin (1:5000, rabbit monoclonal, ab32572, Abcam, Inc.), p-ERK (1:500, rabbit polyclonal, ab79483, Abcam, Inc.), ERK (1:1000, rabbit monoclonal, ab32537, Abcam, Inc.), p-AKT (1:5000, rabbit monoclonal, ab81283, Abcam, Inc.), AKT (1:5000, rabbit monoclonal, ab179463, Abcam, Inc.). GAPDH (1:5000, rabbit monoclonal, ab181602, Abcam, Inc.) was used as a loading control. Protein levels were detected by a chemiluminescence approach according to the protocol (ECL, Forevergen, Guangzhou, China). Images were analyzed using ImageJ software (NIH, USA). The proteins were represented by using GAPDH loading as normalization.
4
Western Blot Analysis of Protein Targets
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Total protein was extracted using a protein extraction kit (Shanghai Yeasen Biotechnology Co., Ltd.) according to the manufacturer's protocol, and the protein concentration was determined using an ultraviolet spectrophotometer (Onedrop1000; Shanghai Genechem Co., Ltd.). A total of 10 µg protein/lane was loaded on a 12% polyacrylamide gel, resolved using SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Sangon Biotech Co., Ltd.). The PVDF membrane was blocked with 5% non-fat milk for 2 h at room temperature. Subsequently, the PVDF membrane was incubated with the corresponding primary antibody against the target protein overnight at 4°C followed by incubation with the secondary antibody at room temperature for 1 h. The protein band was detected using a Beyo ECL Star kit (Beyotime Institute of Biotechnology). The antibodies used were as follows: Anti-PI3K (ab70912; 1:100), anti-MEK1 (ab32091; 1:1,500), anti-ERK1 (ab32537; 1:600), anti-cdc25 (ab111830; 1:2,000) (all Abcam), anti-C-fos (554C1a; 1:500; Santa Cruz Biotechnology, Inc.), anti-ALOX12B (PA5-23608; 1:800; Invitrogen; Thermo Fisher Scientific, Inc.), anti-GAPDH (ab181602; 1:10,000; Abcam), goat anti-mouse IgG antibody (ab97035; 1:2,000) and goat anti-rabbit IgG antibody (ab7090; 1:5,000) (both Abcam).
5
Western Blot Analysis of Signaling Proteins
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A total of 30 μg protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The membrane was incubated overnight with primary antibody after blocking with 5% skim milk. The primary antibodies were provided by Abcam Company, including anti-ERK (ab32537, 1 : 1000), anti-phospho-ERK (ab201015, 1 : 1000), anti-Nrf2 (ab62352, 1 : 1000), anti-polyadenosine-diphosphate-ribose polymerase (PARP) (ab120981, 1 : 1000), anti-GAPDH (ab8245, 1 : 1000), anti-HO-1 (ab52947, 1 : 1000), and anticatalytic subunit of glutamylcysteine ligase (GCLC) (ab207777, 1 : 1000). Membranes were subsequently incubated with appropriate HRP-conjugated secondary antibody at room temperature for 1 h. The bands were visualized using an enhanced chemiluminescence kit (PerkinElmer Life Science, Boston, MA, USA) and were scanned by a luminescence image analyzer (Fuji Film LAS-4000, Japan). The bands were measured with Image Gauge software.
6
Protein Expression Analysis by Western Blot
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Cells and tissues were lysed for 2 hours in RIPA lysis solution freshly prepared with a protease inhibitor (Roche). The concentration of extracted proteins was determined with a bicinchoninic acid protein determination kit (enhanced) (P0010, Beyotime, Shanghai, China). The extracted proteins were then separated by molecular size on SDS‐PAGE gels, followed by transfer to polyvinylidene fluoride membranes, which were incubated at 4 °C overnight with primary antibodies (1:200–1:1000) dissolved in Tris‐buffered saline containing Tween 20 containing 5% skimmed milk powder. The antibodies were as follows: anti‐TRPC1 (sc‐133076, Santa Cruz Biotechnology), anti‐HIF‐1α (abs130612, Absin), anti‐MEK (2352S, Cell Signaling Technology), anti‐p‐MEK (ab96379, Abcam), anti‐Erk (ab32537, Abcam), anti‐p‐Erk (4370S, Cell signaling technology), and anti‐GAPDH (21612, Signalway Antibody). Afterwards, the membrane was washed with Tris‐buffered saline containing Tween 20 and incubated with the corresponding secondary antibodies for 2 hours at room temperature. All the secondary antibodies were from Abcam. All blots were detected with an ECL substrate (180‐5001, Tanon, Shanghai, China), and images were captured with Image Lab Software (Bio‐Rad).
7
Western Blot Analysis of Apoptosis-Related Proteins
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The proteins were isolated from cells by using RIPA buffer, and concentrations of protein were determined by a BCA Kit (Beyotime), which further transferred to a PVDF membrane (Millipore). Then, membranes incubated overnight with antibodies against Bax (1:1,000, ab32503; Abcam), B-cell lymphoma-2 (Bcl-2) (1:1,000, ab182858; Abcam), Cleaved Caspase-3 (1:1,000, ab2302; Abcam), IGF2BP1 (1:1,000, ab100999; Abcam), IGF2 (1:1,000, ab177467; Abcam), p-MEK (1:1,000, 2338; Cell Signaling Technology, Danvers, MA, USA), mitogen-activated protein kinase (MEK) (1:1,000, 4694; Cell Signaling Technology), p-ERK (1:1,000, 4370; Cell Signaling Technology), extracellular regulated protein kinase (ERK) (1:1,000, ab32537; Abcam) and GAPDH antibody (1:10,000, SAB2701826; Sigma-Aldrich). After washed with PBS-T, membranes were then incubated with the corresponding secondary antibody (1:10,000, ab7090; Abcam) for 60 minutes. The membranes were visualized and imaged by GEL imaging system (Bio-Rad, Hercules, CA, USA). The quantification of proteins was analyzed by the software Image J.
8
Protein Extraction and Western Blotting
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Western blotting was performed as previously described [31 (link)]. Proteins were extracted from pMECs using the radio immunoprecipitation assay (RIPA) lysis buffer (#P0013B, Beyotime, Shanghai, China) and quantified using a BCA protein assay kit (#P0009, Beyotime, Shanghai, China). Proteins (10–20 μg/sample) were separated by SDS-PAGE (Invitrogen Inc.), transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA.), and then hybridized with specific antibodies.
Primary antibodies for TLR-4 (1:500, ab13556), p65 (1:1000, ab16502), phospho-p65 (1:1000, ab183559)), β-actin (1:1000, ab5694), p38 (1:1000, ab182453), phospho-p38 (1:1000, ab207483), ERK (1:1000, ab32537), phospho-ERK (1:1000, ab207470), JNK (1:1000, ab126424), and phospho-JNK (1:1000, ab279842) were from the Abcam Company Ltd. (Cambridge, MA, USA). Primary antibodies (dilution, cat. no. follow in parentheses) for IκBα (1:1000, #9242), and phospho-IκBα (1:1000, #2859) were from the Cell Signaling Technology (Woburn, MA, USA).
Primary antibodies for TLR-4 (1:500, ab13556), p65 (1:1000, ab16502), phospho-p65 (1:1000, ab183559)), β-actin (1:1000, ab5694), p38 (1:1000, ab182453), phospho-p38 (1:1000, ab207483), ERK (1:1000, ab32537), phospho-ERK (1:1000, ab207470), JNK (1:1000, ab126424), and phospho-JNK (1:1000, ab279842) were from the Abcam Company Ltd. (Cambridge, MA, USA). Primary antibodies (dilution, cat. no. follow in parentheses) for IκBα (1:1000, #9242), and phospho-IκBα (1:1000, #2859) were from the Cell Signaling Technology (Woburn, MA, USA).
9
Protein Expression Analysis Protocol
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Total tissue and cell proteins were extracted, separated, and transferred to membranes. The membrane was blocked with 5% skim milk powder and probed with primary antibodies to TRIM11 (ab111694, 1:1,000, Abcam), TonEBP (ab3446, 1:1,000, Abcam), IGHG1 (PA5‐75428, 1:500, Thermo Fisher), ERK1 (ab32537, 1:1,000, Abcam), ERK2 (ab32081, 1:1,000, Abcam), MEK1/2 (#4694, 1:1,000, CST, USA), p‐ERK1/2 (ab201015, 1:1,000, Abcam), p‐MEK1/2 (#2338, 1:1,000, CST, Danvers, MA) at 4°C overnight. After that, the membrane was re‐probed with HRP‐labeled secondary IgG antibody (Abcam; goat anti‐rabbit: ab205718, 1:20,000 or goat anti‐rat: ab6789, 1:5,000). After development, the solution was added for developing, and quantitative analysis of proteins was performed using ImageJ software, normalized to β‐actin (ab8226, 1:1,000, Abcam).
10
Erk1 Immunoprecipitation from Nuclear Extracts
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Erk1 (MAPK3) was immunoprecipitated as described previously (Naarmann et al. 2008 (link)) from nuclear extracts employing a specific Erk1 (MAPK3) antibody (Abcam, ab32537), and Dynabeads Protein A beads (Thermo Fisher Scientific).
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