Apatinib mesylate (Heng Rui) were grinded into powder and dissolved 0.5%CMC (Solrbio). Pemetrexed (Qi Lu) were dissolved 0.9% saline. Primary antibodies against AKT (ab8805), phospho‐AKT (ab8932), ERK (ab54230), phospho‐ERK (ab201015), mTOR (ab2732), phospho‐mTOR (ab84400), MEK (ab178876), phospho‐MEK (ab194754), HIF‐1α (ab51608), CD31 (ab28364), α‐SMA (ab5694), collagen IV (ab6586), MMP2 (ab37150), MMP‐9 (ab38898), and β‐actin (ab8227) were purchased from abcam. Primary antibodies against cleaved‐caspase3 (9664), ki67 (9449), and anti‐rabbit or anti‐mouse IgG horseradish peroxidase (HRP)‐linked secondary antibodies were purchased from Cell Signaling Technology.
Ab54230
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab54230 is a laboratory equipment product from Abcam. It serves as a core function in various experimental setups. The detailed description of the product's intended use or applications is not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Ab201015, by Abcam (6 mentions)
Ab31828, by Abcam (4 mentions)
Ab8245, by Abcam (4 mentions)
Ab8805, by Abcam (3 mentions)
Ab8227, by Abcam (3 mentions)
Ab170099, by Abcam (3 mentions)
Ab8932, by Abcam (2 mentions)
Ab2732, by Abcam (2 mentions)
Anti rabbit or anti mouse igg horseradish peroxidase hrp linked secondary antibodies, by Cell Signaling Technology (2 mentions)
Ab8226, by Abcam (2 mentions)
Ab81283, by Abcam (2 mentions)
Ab16502, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab37150, by Abcam (1 mentions)
Ab178876, by Abcam (1 mentions)
22 protocols using ab54230
1
Apatinib and Pemetrexed Combination Therapy
2
Protein Extraction and Western Blot Analysis
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Total protein was extracted from the tibial plateau cartilage using RIPA buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate] with centrifugation at 20,000 × g at 4°C for 15 min. The protein concentrations were determined by the BCA protein assay (Beyotime Institute of Biotechnology, Shanghai, China), and heated to 100°C for 5 min. An equal amount of protein (20 µg) per lane was separated by 12% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked for 1 h at room temperature with 5% non-fat milk (19 (link)), and incubated with primary antibodies against ERK (ab54230, 1:250), p38 (ab31828, 1:1,000), p53 (ab1431, 1:500), RAS (ab52939, 1:500) (all from Abcam, Cambridge, UK) and β-actin (8457, 1:1,000; Cell Signaling Technology, Inc., Beverly, MA, USA), then washed in TBST. The membranes were then incubated with a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody IgG (1:10,000, ZB-2301; Zhongshan Golden Bridge Biotechnology, Co., Ltd., Beijing, China) for 2 h at room temperature. Immunoreactive proteins were visualized using Western Blot Chemiluminescence Luminol Reagent (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and bands were quantitated with a Bio-Rad ChemiDoc XRS+ imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). All data were collected from at least 3 independent experiments.
3
Analyzing IL-6-induced Transcription Factor Phosphorylation
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To analyze the IL-6-induced phosphorylation of transcription factors, LF fibroblasts grown in a 6-well plate were incubated for 15 min with IL-6 (300 ng/ml) and lysed in radioimmmunoprecipitation assay buffer (Nacalai Tesque). The lysates were homogenized in sample buffer solution containing 2-mercaptoethanol (2×) (Nacalai Tesque) and proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (Invitrogen) that was blocked in blocking reagent (Block-Ace; DS Pharma Biomedical, Osaka, Japan) for 30 min at 25 °C and treated overnight at 4 °C with the following primary antibodies: anti-human STAT3 (mouse monoclonal, 1:500, SC-8019) and p-STAT3 (1:500) (both from Santa Cruz Biotechnology); and anti-human ERK1/2 (mouse monoclonal, 1:1000, AB54230), p-ERK1/2 (1:1000), p38 (rabbit monoclonal, 1:1000, AB170099), p-p38 (1:1000), JNK (rabbit polyclonal, 1:500, AB112501), and p-JNK (1:500) (all from Abcam). The membrane was then incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology) for 1 h at 25 °C and immunoreactivity was visualized with Chemi-Lumi One Super (Nacalai Tesque) and a luminescent image analyzer (EZ-capture2; ATTO, Tokyo, Japan) and quantified using ImageJ v.1.47 software (National Institutes of Health, Bethesda, MD, USA).
4
Western Blot Analysis of Protein Expression
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HT29 cells were lysed in RIPA buffer (WB-0071; Ding Guo Chang Sheng Biotech, Beijing, China), and centrifugated at 12,000 g for 1 h to harvest the supernatant. The protein concentration was calculated using the bicinchoninic acid protein assay kit (BCA1-1KT; Sigma Aldrich). Samples (40 µg) were separated using SDS-PAGE and then transferred to the PVDF membrane. The membranes were blocked with 5% non-fat milk and then probed with anti-IGHG1 (1:2,000; ab108969; Abcam, Cambridge, MA, USA), anti-ERK (ab54230) and anti-p-ERK (1:2,500; ab192591; Abcam), anti-FECH (1:3,000; ab219349; Abcam), and anti-β-actin (1:3,500; ab8227; Abcam). Following incubation with horseradish peroxidase-conjugated immunoglobulin G (1:4,000; ab6721; Abcam), the blots were detected using enhanced chemiluminescence (KeyGen, Nanjin, China).
5
Western Blot Analysis of Synaptic Proteins
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The tissue samples were lysed with RIPA lysate containing protease inhibitor and phosphatase inhibitor (Beyotime). The protein concentration was determined using a DC protein analysis kit (500-0111; Bio-Rad, Hercules, CA, USA) at 750 nm. The proteins were separated by sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene fluoride membrane. The membrane was then blocked with 5% bovine serum albumin for 1 h and probed with rabbit anti-human antibodies (1 : 1000) overnight at 4°C. Next, the membrane was reprobed with secondary antibody (1 : 5000). Luminata Western horseradish peroxidase (HRP) substrate (Millipore, Billerica, MA, USA) and Kodak XBT-1 negatives were adopted for color development. The band was quantified using Bio-Rad Quantity One software, normalized to actin level. All results were normalized with blank control values of 100%.
The antibodies used were as follows: GABAB receptor (Invitrogen, #PA1-32248), NR2B (Abcam, Cambridge, UK; ab81271), PKC (Abcam, ab31), CaMKII (Abcam, ab171095), phosphorylation (p)-mitogen-activated protein kinase (ERK1/2) (Abcam, ab54230), p-CREB (Abcam, ab32096), and beta-actin antibody (Abcam, ab8226). Secondary antibody was mouse anti-rabbit immunoglobulin G (IgG) light chain (Abcam, ab8226).
The antibodies used were as follows: GABAB receptor (Invitrogen, #PA1-32248), NR2B (Abcam, Cambridge, UK; ab81271), PKC (Abcam, ab31), CaMKII (Abcam, ab171095), phosphorylation (p)-mitogen-activated protein kinase (ERK1/2) (Abcam, ab54230), p-CREB (Abcam, ab32096), and beta-actin antibody (Abcam, ab8226). Secondary antibody was mouse anti-rabbit immunoglobulin G (IgG) light chain (Abcam, ab8226).
6
Hippocampal Neurons Protein Analysis
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Hippocampal neurons were washed twice with ice-cold phosphate-buffered saline and then lysed with ice-cold lysis buffer (20 mM Tris-HCl (pH=7.5), 150 mM NaCl, 1 mM Na3VO4, 1 mM PMSF, 1 mM EDTA, 1% NP40, 50 mM NaF) for 30 min. Cell lysates were centrifuged at 10,000 x g for 15 min at 4°C. Cell lysates were separated by sodium dodecyl sulfate-polyacrylamide 12% gel electrophoresis and transferred to polyvinylidene fluoride membranes. Following incubation with blocking buffer (Tris-buffered saline + 0.1% Tween-20 (TBST) + 5% non-fat milk), membranes were probed with rabbit anti-Nrf2 polyclonal antibody, (1:1,000, ab31163 abcam, Cambridge, UK) mouse anti-ERK1+ERK2 polyclonal antibody (1:1,000, ab54230, abcam), rabbit anti-ERK1 (phospho T202) + ERK2 (phospho T185) antibody (1:1,000, ab201015, abcam) and mouse anti-GAPDH monoclonal antibody (1:2,000, ab8245, abcam) overnight at 4°C. The membranes were then washed with TBST and incubated with a horseradish peroxide-conjugated secondary antibody (1:1,000, 00001–2, ProteinTech Group, Inc., Chicago, IL, USA), and signals were detected with an enhanced chemiluminescent reagent (EMD Millipore, Billerica, MA, USA). Images were captured using a Bio-Rad VersaDoc 3000 (Bio-Rad Laboratories, Inc., Gladesville, NSW, Australia) and protein levels were quantified using Image-pro Plus 6.0 (Media Cybernetics, MD Rockville, USA).
7
Extraction and Analysis of WK Herbal Extract
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Anti-p38 antibody (ab31828), anti-ERK1 + ERK2 antibody (ab54230), anti-PERK antibody (ab65142), β-actin Antibody (E12-051-1), anti-JNK1 + JNK2 + JNK3 antibody (ab179461), and other reagents were purchased from ABCam (United Kingdom), and fetal bovine serum was provided by GIBCO (United States).
WK consisting of Panax ginseng C. A. Mey. (10 g), Atractylodes macrocephala Koidz. (10 g), Arum ternatum Thunb. (10 g), Curcumae rhizoma (10 g), Actinidia chinensis Planch (15 g), and Rhodiola rosea L. (10 g) was provided by the traditional Chinese medicine (TCM) pharmacy of Jiangsu Province Hospital on Integration of Chinese and Western Medicine. The Chinese medicine was authenticated to be in compliance with the 2005 edition of “Chinese Pharmacopoeia”. WK crude drugs were crushed and soaked in the tenfold double distilled water for 24 h. Then, the mixture was heated, filtered, and refluxed for 2 cycles to get concentrated solution. The concentrate was evaporated at 60°C for the WK extract and the extract was stored at −20°C.
WK consisting of Panax ginseng C. A. Mey. (10 g), Atractylodes macrocephala Koidz. (10 g), Arum ternatum Thunb. (10 g), Curcumae rhizoma (10 g), Actinidia chinensis Planch (15 g), and Rhodiola rosea L. (10 g) was provided by the traditional Chinese medicine (TCM) pharmacy of Jiangsu Province Hospital on Integration of Chinese and Western Medicine. The Chinese medicine was authenticated to be in compliance with the 2005 edition of “Chinese Pharmacopoeia”. WK crude drugs were crushed and soaked in the tenfold double distilled water for 24 h. Then, the mixture was heated, filtered, and refluxed for 2 cycles to get concentrated solution. The concentrate was evaporated at 60°C for the WK extract and the extract was stored at −20°C.
8
Protein Extraction and Western Blot Analysis
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Lysis buffer RIPA (Thermo Fisher Scientific) and N-PER (Thermo Fisher Scientific) were used to extract proteins from glioma cells and tissues, respectively. Then, 50 μg total protein per sample was separated using 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Thermo Fisher Scientific). The membranes were probed with primary anti-IRAK3 antibody (ab8116, Abcam, MA, USA), anti-MEK1 (phospho S298) antibody [EPR3338] (ab96379), anti-ERK1 + ERK2 antibody [ERK-7D8] (ab54230), anti-c-Fos (phospho T232) antibody (ab17933), and anti-GAPDH antibody [6C5] (ab8245, Abcam) as control. The number of binding proteins was measured using AlphaEaseFC software (Genetic Technologies, FL, USA).
9
Western Blot Analysis of Protein Expression
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Total protein was extracted with radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with protease and phosphatase inhibitors (P0013B, Beyotime Biotechnology), and the concentrations of total cellular protein were measured using a BCA assay kit (P0010, Beyotime Biotechnology). Total protein samples (40 μg/gel) were separated via 12% SDS/PAGE gel and transferred to PVDF membranes (IPVH00010, Merck Millipore, Germany) by electroblotting. After blocking in TBST with 5% skimmed milk powder for 1 hr at room temperature (RT), the membranes were incubated with primary antibodies, including anti-Fyn (ab125016, 1 : 1000, Abcam, Cambridge, MA, USA), anti-HLA-G (79769, 1 : 1000, Cell Signaling Technology, Danvers, CO, USA), anti-ERK1/2 (ab54230, 1 : 1000, Abcam), anti-p-ERK1/2 (ab201015, 1 : 1000, Abcam), anti-STAT3 (ab68153, 1 : 1500, Abcam), anti-p-STAT3 (9145, 1 : 2000, Cell Signaling Technology), and anti-GAPDH (AB-P-R 001, 1 : 1000, Goodhere Biotechnology, Hangzhou, China), overnight at 4°C. Then, the membranes were washed and incubated with HRP-labelled goat anti-rabbit secondary antibody (BOSTER Biotechnology, Wuhan, China) for 1 hr at RT. The blot signals were detected by the Odyssey infrared imaging system (LI-COR Biosciences) and analysed by Odyssey software (LI-COR Biosciences).
10
Evaluating Cerebral Infarction Signaling
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Seven days after operation, paraffin-embedded cerebral cortex tissues in the area surrounding the infarction were sliced, fixed, cleared, and mounted. Next, the tissue slices were incubated with anti-mouse ERK1/2 monoclonal antibody (1: 100, ab54230), anti-mouse p38 monoclonal antibody (1: 200, ab31828), or anti-rabbit Src monoclonal antibody (1: 500, ab109381 (Abcam, Cambridge, UK) at 4°C. Next, the slices were incubated with secondary antibody working solution of goat anti-rabbit or anti-mouse (ZSGB-BIO, Beijing, China) at 37°C for 1 h, and then developed using 3, 3'-diaminobenzidine (DAB) (ZSGB-BIO, Beijing, China) for 3-5 min. True color multi-functional cell image analysis system (Media Cybernetics, Silver Spring, MD, USA) was used to record images. Four slices were taken from each specimen, and 6 fields were randomly selected. The percentage of positive cells was counted under a light microscope and the average value was taken to reflect the positive level of ERK1/2, Src or p38, respectively.
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