Western blots were performed using standard protocols. Cell lysates were prepared using RIPA buffer. Protein samples were subjected to SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a PVDF membrane (Millipore). The membrane was blocked with 5% non-fat milk. The membrane was then incubated with anti-E-Cadherin antibody (1:1000, bs-10009R, Bioss, Beijing, China), anti-Vimentin antibody (1:1000, bs-0756R, Bioss, Beijing, China), anti-TRAF6 antibody (1:1000, bs-1184R, Bioss, Beijing, China), anti-N-Cadherin antibody (1:1000, AF5237, Beyotime, Shanghai, China), anti-RGS2 antibody (1:1000, sc-100761, Santa Cruz), anti-Flag antibody (1:1000, M185-3, MBL, Beijing, China) or anti-αTubulin antibody (1:5000, AF5012, Beyotime, Shanghai, China) at 4 °C overnight, followed by incubation with HRP-anti-rabbit or HRP-anti-mouse secondary antibody at room temperature for 1 h. Signals were detected using an ECL Kit (P0018AS, Beyotime, Shanghai, China) according to the manufacturer’s protocol.
Af5012
Manufactured by Beyotime
Sourced in United Kingdom
The AF5012 is a laboratory equipment designed for general purpose use. It features a compact and durable construction. The core function of the AF5012 is to provide a controlled environment for various scientific applications.
Automatically generated - may contain errors
Lab products found in correlation
Ecl kit, by Beyotime (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Sc 100761, by Santa Cruz Biotechnology (1 mentions)
Bs 10009r, by Bioss Antibodies (1 mentions)
Bs 0756r, by Bioss Antibodies (1 mentions)
Ab28340, by Abcam (1 mentions)
Bradford kit, by Beyotime (1 mentions)
Ab5103, by Abcam (1 mentions)
Af1002, by R&D Systems (1 mentions)
Df7011, by Affinity Biosciences (1 mentions)
Af743, by R&D Systems (1 mentions)
Ab10516, by Abcam (1 mentions)
Pico17, by Thermo Fisher Scientific (1 mentions)
Ab8245, by Abcam (1 mentions)
Ab52495, by Abcam (1 mentions)
Iseq00005, by Merck Group (1 mentions)
Zb 2305, by ZSGB-BIO (1 mentions)
Mab3726, by Cell Signaling Technology (1 mentions)
Supersignal chemiluminescent detection system, by Thermo Fisher Scientific (1 mentions)
Ab177178, by Abcam (1 mentions)
8 protocols using af5012
1
Western Blot Analysis of EMT Markers
2
Protein Extraction and Western Blot Analysis
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Liver tissue (100 to 500 mg) was homogenized and used for protein extraction. The protein extracts from the cultured cells were also used for the assays that are described below. The protein sample concentrations were determined using a Bradford kit (Beyotime Co., Jiangsu, China). The same amount of protein samples were separated in sodium dodecyl sulfate-polyacrylamide gels and transferred onto polyvinyl difluoride (PVDF) membranes. The membranes were blocked with 5% bovine serum albumin (BSA) and incubated with anti-PKCε (1:1000) or anti-IRS-1 tyr (1:1000), anti-DPP4 (1:1000, ab28340, Abcam, Cambridge, UK), or anti-HSP90 (1:2000, AH732, Beyotime Co.) antibodies. After incubation with the corresponding secondary antibodies, the membranes were detected using an electrochemiluminescence (ECL) kit (Beyotime Co.). The corresponding bands were quantified using grayscale analyses, and the relative expression was calculated by normalizing to β-tubulin (1:2000, AF5012, Beyotime Co.).
3
Western Blot Analysis of AAV-Induced Meningeal Protein Expression
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WB analysis was performed as described previously. For plasma WB analysis, 0.5 μL of plasma was collected 7 days after AAV injection. For WB analysis of the meninges, meningeal tissue homogenate was obtained 24 h after IVH. RIPA lysis buffer and 1% PMSF were used to extract total proteins. The following primary antibodies were added and incubated overnight at 4 °C with gentle agitation: anti-VEGFR3 (goat, 0.1 μg/mL, AF743, R&D), anti-CitH3 (rabbit, 1:2000, ab5103, Abcam), anti-VEGF-C (rabbit, 1:1000, DF7011, Affinity), anti-VE-cadherin (goat, 0.2 μg/mL, AF1002, R&D), anti-PROX1 (rabbit, 1:2000, 11067-2-AP, Proteintech), anti-LYVE-1 (rabbit, 1:1000, 67538S, Cell Signaling Technology), anti-FOXC-2 (rabbit, 1:500, DF3252, Affinity), and anti-CX3CR1 (rabbit, 1:1000, 13885-1-AP, Proteintech). Subsequently, the membrane was incubated at room temperature for 2 h with horseradish peroxidase-conjugated secondary antibodies. The membrane was probed for GAPDH (1:10000, 200306-7E4, ZEN BIO) or ɑ-Tubulin (1:2000, AF5012, Beyotime) as a loading control.
4
Quantifying Protein Expression in Nanoparticle-Treated Cells
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Cells were treated with TM-NPs, CQ/NPs or TM-CQ/NPs nanoparticles for 24 h at the concentration mentioned in crystal violet assay. Cells were lysed and the resulting lysates were centrifuged at 10,000 rpm for 10 min at 4 °C (Pico17, Thermo Fisher). The supernatants were collected and the protein concentration was quantified and normalized by BCA protein assay. Proteins were separated on 10% SDS-PAGE (P0455M, Beyotime, Shanghai, China), and transferred to PVDF membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% BSA for 1 h and immunoblotted with antibodies against TRAIL (ab10516, 1: 1000 dilution, v/v, Abcam, Cambridge, UK), β-tubulin (AF5012, 1: 5000 dilution, v/v, Beyotime, Shanghai, China), Na+/K+ ATPase (A01483, 1: 1000 dilution, v/v, GenScript, Nanjing, China), PARP (#9532, 1:1000 dilution, v/v, Cell signaling Danvers, MA, USA), cleaved-PARP (#5625, 1:1000 dilution, v/v, Cell signaling Danvers) or GAPDH (ab8245, 1:5000 dilution, v/v, Abcam). Following incubation with the secondary antibodies, proteins were visualized with enhanced chemiluminescence reagent (P0018S, Beyotime, Shanghai, China).
5
Western Blot Analysis of Protein Expression
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To detect protein expression in mouse materials, total proteins were extracted using WIP Tissue and Cell lysis solution (BioChip, 110000) according to the manufacturer’s protocol. Electrophoresis was performed with 50 μg total proteins separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, ISEQ00005). The membranes were incubated overnight at 4 °C with the appropriate primary antibodies listed below: anti-RDX (78 kDa, 1:500, ab52495, Abcam), anti-p-ERM (78 kDa, 1:500, mAb#3726, Cell Signaling Technologies). The appropriate secondary antibody (ZB-2301, ZB-2305 from ZSGB-BIO) was diluted 1:5000 in TBST (TBS plus 0.5% Tween 20). Tubulin (1:5000, Beyotime, AF5012) was used as an internal control. The membranes were visualized using the SuperSignal chemiluminescent detection system (Thermo Scientific, 32109). Original blots can be found in the Source data file.
6
Immunoblotting for Protein Expression Analysis
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For immunoblotting, protein extracts were prepared by lysing BmE cells in RIPA buffer (20 mM Tris-HCI pH7.5, 150 mM NaCl, 10 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS) and boiling in SDS sample buffer for 5 min. Protein samples were resolved on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, transferred onto activated polyvinylidene difluoride membranes, and blocked for 1 h in 5% skim milk. Membranes were incubated with primary antibodies against the EcR (DDA2.7, 1:1000; Developmental Studies Hybridoma Bank (DSHB), created by the NICHD of the NIH, maintained at The University of Iowa, Department of Biology, Iowa City, IA, USA), CBP (aa1269-1580, 1:5000; homemade), FLAG (F7425, 1:5000; Sigma-Aldrich, St Louis, MO, USA), HA (AH158, 1:1000; Beyotime, Shanghai, China), H3K27ac (ab177178, 1:10,000; Abcam, Cambridge, UK), and α-tubulin (AF5012, 1:5000; Beyotime, Shanghai, China) overnight at 4 °C, then further incubated with HRP conjugated secondary antibody for 1 h. Signals were detected using the SuperSignal West Pico Chemiluminescent Substrate (34580; Thermo Fisher, Waltham, MA, USA).
7
Protein Expression Analysis of CeO2 NP-Treated Cells
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Western blotting analysis was used to determine the protein expression levels. HTR-8/SVneo Cells were seeded and treated with CeO2 NPs for another 24 h, and when incubation finished, the cells were lysed using RIPA, containing phenylmethyl sulfonyl fluoride (PMSF), for subsequent protein extraction. The BCA Protein Assay Kit (Beyotime, China) was used to examine the protein concentrations in each sample. 80 µg of the total proteins were loaded to 10% SDS-PAGE for electrophoresis. The antibodies of α-tubulin (Beyotime, AF5012), β-tubulin (Beyotime, AF1216), actin (Beyotime, AA128), protein kinases B (AKT) (CST, #9272), mammalian target of rapamycin (mTOR) (CST, #2972), matrix metalloproteinase 2 (MMP2) (Abcam, ab92536), MMP9 (Abcam, ab76003), and LC3A/B (CST, #4108) were used to quantify the protein levels, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Beyotime, AF5009) was used as the loading control. Immunoblots were visualized with an enhanced chemiluminescence (ECL) western blot detection kit (Millipore) combined with the Bio-Rad Imaging System. All experiments were repeated at least three times.
8
Isolation and Analysis of ER Proteins
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ER fraction was collected from pancreatic cancer cells by ER enrichment kit (NBP2-29482, Novus Biologicals). After pretreatment with or without 1%, Triton X-100 for 3 min, the solution (0.625 g/L trypsin + 0.05 g/L EDTA in PBS) was added to ER fraction. Samples were incubated for the indicated time. After trypsinization, samples were analyzed by Western blotting with primary antibodies against IRE1α (3294, Cell Signaling Technology), HSP90B1 (NBP2-42379, Novus Biologicals), EGFR (4267T, Cell Signaling Technology), NEK2 (24171-1-AP, Proteintech Group), and a-Tubulin (AF5012, Beyotime).
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