RIPA (GBCBIO, G3424) was used to lyse cells and extract total protein to prepare protein samples. The protein lysates were separated by conventional electrophoresis, transferred onto the membrane, and washed with PBS, followed by blocking with 1% BSA for 2 h. Then, primary antibodies Box (ab32503, Abcam, Cambridge, UK), Bcl-2 (ab32124, Abcam, Cambridge, UK), Caspase-1 (ab32503, Abcam, Cambridge, UK), Cleaved Caspase-3 (ab32503, Abcam, Cambridge, UK), Caspase-8 (ab207802, Abcam, Cambridge, UK), Caspase-9 (ab2302, Abcam, Cambridge, UK), Caspase-11 (ab25901, Abcam, Cambridge, UK), Cyclin D1 (ab202068, Abcam, Cambridge, UK), ERK 1/2 (ab180673, Abcam, Cambridge, UK), p-ERK 1/2 (ab16663, Abcam, Cambridge, UK), WNT (ab17942, Abcam, Cambridge, UK), β-catenin (ab223500, Abcam, Cambridge, UK), GSDMD (ab15251, Abcam, Cambridge, UK) and β-actin (ab32572, Abcam, Cambridge, UK) (abcam, ab32503, ab32124, ab207802, ab2302, ab25901, ab202068, ab180673, ab16663, ab17942, ab223500, ab15251, ab32572, ab219800, ab8227) each at 1:1000 dilution were incubated with the membrane overnight at 4°C. The next day, HRP (ab32572, Abcam, Cambridge, UK) and labeled IgG secondary antibody (ab150077, Abcam, Cambridge, UK) each at 1:5,000 were further incubated with the membrane at room temperature for 1 h. ECL Luminescence Kit (Thermo, 32209) was used to develop the signal in a dark room.
Ab32572
Manufactured by Abcam
Sourced in United States, United Kingdom, China, Germany, Canada
Ab32572 is a laboratory equipment product. It is a centrifuge designed for high-speed separation of biological samples. The core function of this product is to separate components of a liquid mixture based on their relative density differences.
Automatically generated - may contain errors
Lab products found in correlation
Ab32072, by Abcam (49 mentions)
Pvdf membrane, by Merck Group (42 mentions)
Ab181602, by Abcam (32 mentions)
Ab8245, by Abcam (28 mentions)
Ab16663, by Abcam (27 mentions)
Ab8226, by Abcam (27 mentions)
Ab205718, by Abcam (25 mentions)
Ab40772, by Abcam (23 mentions)
Ab6721, by Abcam (22 mentions)
Ab92547, by Abcam (21 mentions)
Ab15251, by Abcam (19 mentions)
Ripa lysis buffer, by Beyotime (19 mentions)
Ab32503, by Abcam (18 mentions)
Ab8227, by Abcam (18 mentions)
Ab32391, by Abcam (17 mentions)
Ripa buffer, by Beyotime (14 mentions)
Ab9485, by Abcam (14 mentions)
Ab134175, by Abcam (13 mentions)
Ab76011, by Abcam (13 mentions)
Bca kit, by Beyotime (13 mentions)
419 protocols using ab32572
1
Western Blot Analysis of Apoptosis Markers
2
Protein Extraction and Western Blot Analysis
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Total protein was prepared from GC tissues and cells using RIPA buffer, and protein quantification was performed by a spectrophotometer (Thermo Fisher Scientific, Carlsbad, USA). The extracts of protein were subjected to SDS-PAGE, followed by move to PVDF (Millipore, Bradford, MA, USA). After being blocked with nonfat milk, membranes were probed with primary antibodies at 4 °C overnight, and then incubated with secondary antibody for 1 h at dark room. Primary antibodies were as follows: anti-ß-catenin (ab32572, Abcam, Cambridge, USA), anti-GLI1 (ab49314, Abcam), anti-P13K (ab53610, Abcam), anti-AKT (ab81283, Abcam), anti-CTNNB1 (ab32572, Abcam) and anti-GAPDH (ab181602, Abcam), antibody against E-cadherin (ab40772), N-cadherin (ab76057), Vimentin (ab8978, Abcam), Twist (ab187008). The quantification of protein was analyzed by chemiluminescence system (GE Healthcare, Chicago, USA).
3
Protein Expression Profiling in WiT49 Cells
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The levels of HOXA11, β-catenin, receptor–related protein 6 (LRP6), phosphorylation-LRP6 (p-LRP6), E-cadherin (E-cad), N-cadherin (N-cad), Vimentin, HIF-1α, C/EBPβ, and β-actin protein expression were assessed in WiT49 cells. The total cellular proteins were isolated with RIPA lysis buffer (#R0278, Sigma), and a standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method was used to separate the proteins [19 ]. Next, the separated protein bands were transferred onto membranes that were subsequently blocked, and then cultured with primary antibodies against HOXA11 (#ab54365; 1:1000), β-catenin (#ab32572; 1:800), LRP6 (#ab134146; 1:1000), p-LRP6 (#ab76417; 1:800), E-cad (#ab6528; 1:800), N-cad (#ab6258; 1:800), Vimentin (#ab92547; 1:800), HIF-1α (#ab51608; 1:1000), C/EBPβ (#ab32358; 1:1000), and β-actin (#ab32572; 1:5000) (Abcam, Cambridge, MA, USA). Next, the membranes were then washed and incubated with HRP conjugated Goat Anti-Rabbit IgG H&L (1: 5000, #ab6721, Abcam). Finally, the immunostained bands were observed using a Tanon 6600 Luminescence imaging workstation (Tanon, China).
4
Western Blot Analysis of β-catenin
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Cells were cultured for 24 h and treated with BBR or DMSO (control), respectively for 24 h. Cells were washed and lysed in lysis buffer containing protease inhibitor (1% PMSF, Sigma Aldrich, St. Louis, MO, USA). The concentrations of proteins were determined by BCA assay (Beyotime, Shanghai, China). Proteins were separated by SDS/PAGE and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk for 1 h, and incubated overnight at 4 °C using primary antibodies directed against β-actin, 1:2 000 (sc-47778, ab32572; Abcam, Cambridge, UK); β-catenin, 1:1 000 (ab32572; Abcam, Cambridge, UK), following corresponding secondary antibodies (m-IgGBP-HRP, 1:5 000, sc-516102; mouse anti-rabbit IgG-HRP, 1:5 000, sc-2357, Santa Cruz Biotech, Delaware Avenue, CA, USA) incubated for 2 h at room temperature. β-actin was used as the internal control. Immunocomplexes were visualized using Super Signal reagents (Pierce, Rockford, IL, USA), and ImageJ software (NIH, Bethesda, MD, USA) was used for densitometric analyses of the membranes.
5
Protein Expression Analysis in WiT49 Cells
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The protein expression levels of HOXA11, β-catenin, receptor-related protein 6 (LRP6), Phosphorylation-LRP6 (p-LRP6), E-cadherin (E-cad), N-cadherin (N-cad), Vimentin, HIF-1α, C/EBPβ, and β-actin were assessed in WiT49 cells. The proteins were isolated by RIPA lysis buffer (#R0278, Sigma). Standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method was used to separate the proteins [20] . Then, the gels were transferred, blocked, and cultured with primary antibodies against HOXA11 (#ab54365; 1:1000), β-catenin (#ab32572; 1:800), LRP6 (#ab134146; 1:1000), p-LRP6 (#ab76417; 1:800), E-cad (#ab6528; 1:800), N-cad (#ab6258; 1:800), Vimentin (#ab92547; 1:800), HIF-1α (#ab51608; 1:1000), C/EBPβ (#ab32358; 1:1000), and β-actin (#ab32572; 1:5000) bought from Abcam (Cambridge, MA, USA). The membranes were washed and incubated with the HRP conjugated Goat Anti-Rabbit IgG H&L (1: 5000, #ab6721, Abcam). Finally, the bands were observed under a Tanon 6600 Luminescence imaging workstation (Tanon, China). Bioinformatics analysis PROMO (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) and JASPAR(http://jaspar.genereg.net/) databases were used for the transcription factor prediction of HOXA11-AS with the threshold.
6
Immunohistochemical Analysis of RPN2 and β-Catenin
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Sections underwent dewaxing, re‐hydration, antigen retrieval, and blocking, and then were incubated with antibodies against RPN2 (1:200; ab244399; Abcam) and β‐catenin (1:500; ab32572; Abcam) overnight at 4°C and then washed three times with PBST. Sections were incubated with HRP‐conjugated secondary antibody for 15 min at room temperature and then stained with diaminobenzidene (DAB) and hematoxylin.
7
Western Blot Analysis of Protein Signaling Pathways
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Cell total protein was extracted using RIPA lysis buffer containing protease inhibitor cocktail (Beyotime, China). The protein concentration was detected using a BCA Protein Assay Kit (Pierce Biotechnology). Thirty micrograms of protein were separated using 6-10% SDS-PAGE (v/v) and transferred to nitrocellulose membranes (Millipore, MA, USA). Then, the nitrocellulose membranes were blocked with 5% nonfat milk, followed by probing corresponding primary antibodies: anti-mTOR (#2983, 1 : 1,000; Cell Signaling Technology), anti-p-mTOR (#2971, 1 : 1,000; Cell Signaling Technology), anti-Akt (#4685, 1 : 1000; Cell Signaling Technology), anti-p-Akt (#4060, 1 : 2,000; Cell Signaling Technology), anti-β-catenin (ab32572, 1 : 2000; Abcam), anti-O-GlcNAc (ab2739, 1 : 1000; Abcam), and anti-β-actin antibody (ab8226, 1 : 2000; Abcam). After incubation with primary antibodies overnight at 4°C, the membranes were incubated the horseradish peroxidase-conjugated secondary antibodies in the next day. Finally, the antigen-antibody complexes were detected by enhanced chemiluminescence (Pierce, USA). Protein quantification was performed by ImageJ software (NIH Image), and the β-actin was used as an internal control.
8
Immunohistochemical Analysis of Cell Proliferation Markers
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Formalin-fixed, paraffin-embedded sections were cut into 4 μm sections. The sections were deparaffinized and rehydrated using xylene and graded alcohol solutions. They were blocked with 2% bovine serum albumin and incubated with specific primary antibodies for 12 hours at 4°C, followed by incubation with a biotinylated secondary antibody. Subsequently, diaminobenzidine (DAB) was added dropwise over a 3- to 5-minute period and sections were then counterstained with hematoxylin. The primary antibodies included rabbit polyclonal antibodies of CyclinD1 (dilution 1 : 200, cat. no.: ab134175, Abcam), β-catenin (dilution 1 : 500, cat. no.: ab32572, Abcam), Ki-67 (dilution 1 : 500, cat. no.: ab92742, Abcam), and C-Myc (cat. no.: GT220607, Gene Tech).
9
Protein Isolation and Western Blot Analysis of RCC Cells
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The total protein from RCC cells was isolated using RIPA lysis buffer (Beyotime), and the protein concentration was measured by the BCA Kit (Pierce). A total of 30 μg protein was separated by SDS‐PAGE electrophoresis. Then, the membrane was incubated with LHPP (ab116175, Abcam), N‐cadherin (ab76011, Abcam), E‐cadherin (ab40772, Abcam), β‐catenin (ab32572, Abcam), Vimentin (ab92547, Abcam), Caspase‐3 (ab32351, Abcam), Bcl‐2 (ab32124, Abcam), Bax (ab32503, Abcam), and β‐actin (ab8227, Abcam) primary antibodies and goat derived IgG H&L secondary antibodies, respectively. The immunoreactive bands were visualized by an enhanced chemiluminescence ECL kit and then scanned by a LAS‐4000 imaging system (Fujifilm).
10
Retinal Protein Expression Analysis
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Total protein was isolated from the retinal samples and run on 10% polyacrylamide gels following a standard protocol. Equal amounts of protein resolved by polyacrylamide gels were immunoblotted using antibodies against β-tubulin (1:10000, RM2003; Beijing Ray Antibody Biotech, Beijing, China), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:10,000, RM2002; Beijing Ray Antibody Biotech), caspase-3 (1:1000, 9662; Cell Signaling Technology, Danvers, MA, USA), Bax (1:1000, 2772; Cell Signaling Technology), B-cell lymphoma-2 (Bcl-2; (1:1000, ab182858; Abcam, Cambridge, UK), Akt (1:1000, YT0177; ImmunoWay, Plano, TX, USA), phosphorylated Akt (p-Akt; 1:1000, YP0006; ImmunoWay), glycogen synthase kinase-3 beta (GSK3β; 1:1000, 12456; Cell Signaling Technology), phosphorylated GSK3β (p-GSK3β; 1:1000, 9323; Cell Signaling Technology), and β-catenin (1:1000, ab32572; Abcam). They were then detected with a chemiluminescence kit. Gray value analysis was conducted using Image J software.
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