The expression of RhoC and HMGA2 was detected by immunocytochemistry (IHC) at the protein level. The control and shRNA transfected CAL-27 (1 × 105) and SCC-15 (2 × 104) cells were, respectively, cultured in 24-well plates overnight. Next, 10% neutral buffer formalin fixative was used to fix cells for 30 min and cells were incubated with Triton X-100 for 10 min. Goat serum (10%) was used to block the cells for 1 h, which were subsequently washed with PBS 3 times. Next, cells were incubated with 3% goat serum containing antibody RhoC (ab180785, Abcam, 1 : 400) and HMGA2 (ab97276, Abcam, 1 : 500), respectively, overnight at 4°C. Cells were washed with PBS and were incubated with 3% goat serum containing the second antibody for 1 h and then stained with a diaminobenzidine (DAB) kit (ComWin Biotech Co., Ltd., China). The cells were then photographed under an inverted microscope.
Ab97276
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab97276 is a recombinant anti-TRPV1 antibody. It is designed for use in immunohistochemistry, western blotting, and other applications.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Ab205718, by Abcam (3 mentions)
Ab8227, by Abcam (3 mentions)
Ab6276, by Abcam (2 mentions)
Ecl reagent, by Merck Group (2 mentions)
Ab9485, by Abcam (2 mentions)
Ab109183, by Abcam (2 mentions)
Ab227198, by Abcam (2 mentions)
Ab18197, by Abcam (2 mentions)
Ab40772, by Abcam (2 mentions)
Ab180785, by Abcam (1 mentions)
Bicinchoninic acid assay, by Beyotime (1 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)
Sc 8312, by Santa Cruz Biotechnology (1 mentions)
Sc 33437, by Santa Cruz Biotechnology (1 mentions)
Sc 8319, by Santa Cruz Biotechnology (1 mentions)
Radioimmunoprecipitation buffer, by Beyotime (1 mentions)
Instat, by GraphPad (1 mentions)
T5168, by Merck Group (1 mentions)
Eif4e, by Cell Signaling Technology (1 mentions)
15 protocols using ab97276
1
Immunocytochemical Analysis of RhoC and HMGA2
2
Western Blot Analysis of Capan-2 Cells
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Capan-2 cells were collected and lysed in radioimmunoprecipitation buffer (Beyotime). The protein concentration was determined using a bicinchoninic acid assay (Beyotime). Briefly, equivalent weights of protein samples (40 μg/lane) were separated on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and subsequently electrotransferred onto polyvinylidene fluoride membranes (Bio-Rad). Subsequently, all membranes were incubated with the following primary antibodies against HMGA2 (ab97276; Abcam), AKT (sc-8312; Santa Cruz), phosphorylated AKT (sc-33437; Santa Cruz), mTOR (sc-8319; Santa Cruz), and phosphorylated mTOR (p-mTOR, sc-101738; Santa Cruz) at 4 °C overnight. After incubation with secondary antibodies for 1 hour at room temperature, all bands were determined using an enhanced chemiluminescence system kit (MultiSciences).
3
Western Blot Analysis of eIF4 Proteins
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Protein samples were fractionated on SDS-polyacrylamide gels, transferred to PVDF membrane (Millipore), and probed with the indicated antibodies. Antibodies used in this study were directed against: eIF4AI (ab31217, Abcam), eIF4AII (ab31218, Abcam), eIF4E (Cell Signaling), eIF4GI (A300-502A, Bethyl Labs), PDCD4 (cs9535, Cell Signaling), Tubulin (T5168, Sigma), Actin (A5441, Sigma), GAPDH (ab8245, Abcam), Ago2 (C34C6, Cell Signaling), and HMGA2 (ab97276, Abcam). For statistical analysis, Student t-tests were performed using GraphPad InStat version 3.10.
4
Evaluating HMGA2, PI3K, Akt Signaling in CRC
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The protein levels of HMGA2, PI3K, p‐PI3K, p‐Akt, Akt in CRC cells were measured using immunoblotting assays. Cells were lysed in RIPA lysis buffer (Cat. P0013B, Beyotime, Shanghai, China), and the protein concentration was evaluated by BCA Protein Assay Kit (Cat. P0010, Beyotime Tech.). The 20‐50 μg of protein samples were loaded onto 10% SDS‐PAGE minigel and then transferred onto a 0.2 μm PVDF membrane (Cat. 1620177, Bio‐Rad, Hercules, CA, USA) by Bio‐Rad Mini Trans‐Blot® systems (Bio‐Rad). Afterward, the membrane was probed with the antibodies listed below: anti‐HMGA2 (ab97276, Abcam, Cambridge, UK), anti‐PI3K (ab86714, Abcam), anti‐p‐PI3K (ab182651, phospho Y607, Abcam), anti‐Akt (ab32505, Abcam), anti‐p‐Akt (ab81283, phospho S473, Abcam), and anti‐β‐actin (ab6276, Abcam; 1:1000 in TBST) at 4°C overnight, and incubated with HRP‐conjugated secondary antibody for 1 hour at room temperature (1:5000 in TBST, Cat. sc‐2004 and sc‐2005, Santa Cruz, Co. Ltd, Sant Cruz, CA, USA). Signals were visualized using ECL Substrates (Cat. 345818, Millipore, Billerica, MA, USA) and exposed to X‐film (Cat. X‐OMAT, Kodak, Rochester, NY, USA). The immunoblots were quantified using imageJ software (NIH, National Institute of Health, Bethesda, MD, USA) and normalized to endogenous β‐actin. The original blots were shown in Figure S1 .
5
Protein Expression Analysis in Cells
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Details of protein extraction, Western blotting, IHC staining and IHC score obtainment referred to our previous studies [2 (link)]. Primary antibodies against GAPDH (ab9458, Abcam, UK), SMYD3 (ab187149, Abcam, UK), HMGA2 (ab97276, Abcam, UK), NANOG (ab109250, Abcam, UK), c-MYC (ab32072, Abcam, UK), BMI1 (ab126783, Abcam, UK), Histone H3 (ab1791, Abcam, UK), SOX2 (ab92494, Abcam, UK), H3K4me3 (ab8580, Abcam, UK) and Ki67 (ab15580, Abcam, UK) were utilized.
6
Protein Isolation and Western Blot Analysis
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The proteins were isolated by using a radioimmunoprecipitation assay buffer (Beyotime, Nantong, China), then protein concentration was determined by a bicinchoninic acid protein assay kit (Beyotime). Thereafter, total protein (30 μg) was separated by 10% SurePAGE (Genscript, Nanjing, China), and protein electrophoresis membrane was transferred. Next, the membranes were incubated with the primary antibodies at 4°C overnight after blocking with 5% non‐fat milk for 1.5 h. After incubation with horseradish peroxidase‐conjugated secondary antibody with a 1:2,000 dilution at 37°C for 1 h, the membranes were examined using ECL reagents (Beyotime), and bands densities were analyzed by ImageJ software (National Institutes of Health, Bethesda, MD, USA). The primary antibodies included: TSG101 (1:5000, ab125011), CD81 (1: 2000, ab109201), CD63 (1:2000, ab68418), fibronectin (FN; 1:1000 ab2413), collagen IV (Col IV; ab6586), HMGA2 (1:1000, ab97276), Bcl‐2 (1:2000), cleaved‐caspase 3 (1:2000, ab2302) and β‐actin (1:1000, ab6276) obtained from Abcam (Cambridge, MA, USA).
7
Western Blot Analysis of EMT Markers
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Protein samples extracted with RIPA buffer (Solarbio) were separated by polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After sealing with 5% skimmed milk, the membranes were incubated with primary antibodies against E-cadherin (ab15148, 1:500, Abcam, Cambridge, UK), Vimentin (ab137321, 1:2000, Abcam), N-cadherin (ab76057, 1:1000, Abcam), HMGA2 (ab97276, 1:3000, Abcam) or GAPDH (ab9485, 1:2500, Abcam) at 4°C. Afterwards, the membranes were probed with a secondary antibody (ab205718, 1:20,000, Abcam). The signal intensity was examined with ECL reagent (Millipore).
8
HMGA2 Protein Expression Analysis by IHC
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IHC was used to examine HMGA2 protein expression. IHC reactions were performed on slides following a standard 3,3′-Diaminobenzidine (DAB) staining protocol. Briefly, the slides were dewaxed, rehydrated, washed with water, and then pre-treated with 3% hydrogen peroxide. The slides were boiled in antigen retrieval solution, blocked with 1% BSA, and incubated overnight in a humidified chamber at 4°C with an anti-HMGA2 monoclonal antibody (1:50, Abcam, ab97276). After incubations with biotinylated antibody and streptavidin-peroxidase, staining was developed in a solution of 3,3′-Diaminobenzidine. Slides were then counterstained with hematoxylin, dehydrated, cleared with xylene, and mounted. Image pro plus (IPP) software was used to analyze the staining. Integral optical density (IOD) of staining was analyzed.
9
Protein Extraction and Western Blot Analysis
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The extraction of proteins from tissues or cells was implemented via radioimmunoprecipitation assay (RIPA, Sangon Biotech, Shanghai, China). Then, the protein samples were subjected to SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (ThermoFisher, Waltham, USA). After the block by 5% non-fat milk, Western blot analysis was applied to determine the protein expression [14 (link)]. The membranes were incubated with the diluted primary antibodies (1:2000) and secondary antibodies (1:5000), respectively. Finally, the protein signals were achieved using an enhanced chemiluminescence (Amersham Pharmacia, Piscataway, USA). Primary antibodies against Ras (1:1,000, ab180772), phosphor-ERK1/2 (p-ERK1/2, 1:1,000, ab47339), ERK1/2 (t-ERK1/2, 1:3,000, ab196883), PCNA (1:2,000, ab18197), OCT4 (1:2,000, ab109183), hexokinase 2 (HK2) (1:2,000, ab227198), HMGA2 (1:2,000, ab97276), β-actin (1:2,000, ab8227), GAPDH (1:2,500, ab9485) and secondary antibodies (1:2,000, ab205718) were all obtained from Abcam. ImageJ software was used to evaluate the band density by densitometric analysis.
10
Western Blot Analysis of HMGA2 Protein Expression
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RIPA lysis buffer with protease inhibitor (Beyotime, Shanghai, China) was used to extract total protein. Equal amounts of denatured protein (30 μg) were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with Tris-buffered saline (TBS), containing 5% non-fat milk powder, at 25 °C for 2 h. The membranes were then incubated at 4 °C overnight with the following diluted primary antibodies: anti-HMGA2 (dilution 1:1000; ab97276, Abcam; Danvers, MA, USA) and anti-β-actin (dilution 1:1000; ab8227; Abcam). After washing with TBS containing Tween 20 (TBST), membranes were incubated with an HRP-labeled secondary antibody (dilution 1:5000) for 2 h at 25 °C. After washing three times with TBST, the protein bands were detected using an enhanced chemiluminescence detection kit (Beyotime) and a ChemiDoc™ XRS imaging system (Bio-Rad). All experiments were repeated in independent triplicate.
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