Rabbit mAb to LC3I antibody (12741, dilution 1/800), rabbit mAb to p62 (23214, dilution 1/1000), rabbit anti-Beclin-1 (3495, diluted 1/1000), rabbit anti-Phospho-ULK1 (14202, diluted 1/800) and ULK1 (8054, diluted 1/1000) were all purchased from Cell Signaling Technology, Inc. Rabbit polyclonal anti-LC3II antibody (ab63817, dilution 1/1000) was purchased from Abcam. Anti-actin was obtained from Epitomics, an Abcam company (Cambridge, MA, USA). miR-34a inhibitors (5’-AGCCUUGCUGCAGGUGCGCAU-3’) and a nonsense control (NC) sequence (5’-UGCCUUACUGACGGUCGGAGA-3’) were obtained from Shanghai GeneChem, Inc. (Shanghai, China). According to the protocol, Huh7-R and HepG2-R cells (1 × 105) were transfected with 50 nM miR-34a inhibitors, 50 nM miR-34a mimics, 50 nM scramble control inhibitors or 50 nM scramble control mimics using Lipofectamine® 2000 (Thermo Fisher Scientific, Inc.) for ~ 30 min at room temperature. After 48 h of transfection, the transfected cells were used for subsequent experiments. 3-Methyladenine (3-MA), a class III phosphoinositol 3-kinase (PI3K) inhibitor, and choroquine (CQ), were used as selective inhibitors of autophagy, was purchased from Santa Cruz Biotechnology (Texas, U.S.A). Rapamycin (rapa), a potent and specific mTOR inhibitor used as an autophagy agonist, was obtained from Selleck.
Anti actin
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Anti-actin is a laboratory reagent used to detect and quantify the presence of actin, a ubiquitous and essential cytoskeletal protein in eukaryotic cells. It is commonly used in various biochemical and cell biology assays.
Automatically generated - may contain errors
Lab products found in correlation
Anti flag, by Merck Group (17 mentions)
Protease inhibitor cocktail, by Roche (11 mentions)
Pvdf membrane, by Merck Group (9 mentions)
Anti gapdh, by Abcam (7 mentions)
Anti histone h3, by Abcam (6 mentions)
Anti caspase 3, by Abcam (5 mentions)
Alkaline phosphatase, by Merck Group (5 mentions)
Anti gfp, by Abcam (5 mentions)
Anti lc3b, by Abcam (5 mentions)
Anti akt, by Cell Signaling Technology (5 mentions)
Scanjet 5300c, by Hewlett-Packard (4 mentions)
Anti ha, by BioLegend (4 mentions)
Anti β tubulin, by Abcam (4 mentions)
Ripa buffer, by Merck Group (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Quantity one software, by Bio-Rad (3 mentions)
Glial fibrillary acidic protein (gfap), by Merck Group (3 mentions)
Phosphatase inhibitor cocktail, by Thermo Fisher Scientific (3 mentions)
Proteinase inhibitor cocktail, by Roche (3 mentions)
210 protocols using anti actin
1
Autophagy Regulation by miR-34a
2
Protein Degradation Mechanisms Analysis
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MG132 (S2619, Selleck Chemicals), bortezomib (S1013, Selleck Chemicals), MLN4924 (M2189, Abmol), Cycloheximide (R750107, Sigma-Aldrich), Paxilline (P2928, Sigma-Aldrich) and BMS-204352 (SML1313, Sigma-Aldrich) were dissolved in DMSO. Anti-CRBN (SAB045910, Sigma-Aldrich), anti-DDB1 (37–6200, Invitrogen), anti-actin (1844–1, Epitomics), anti-β-actin (4967, cell Signaling Technology; 5779–1, Epitomics), anti-β-tubulin (32–2600, Invitrogen), anti-Pan-Cadherin (4068, Cell Signaling Technology), anti-ubiquitin (sc-271289, Santa Cruz), anti-Cul1 (AP16324b, Abgent), anti-Fbxo7 (ab84129, Abcam), anti-Cul4A (A300-739A, Bethyl Laboratories), anti-Cul4B (2527–1,Epitomics), anti-Slo1 (NBP1-48250, Novus Biologicals), anti-mSlo1 (Millipore, MABN70), anti-HA (H6908; H3663, Sigma-Aldrich), anti-Flag (F3165, Sigma-Aldrich), anti-Myc (2272, Cell Signaling Technology), anti-GFP (sc-8334, Santa Cruz), anti-HA agarose (E6779, Sigma-Aldrich), anti-Flag M2 agarose (A2220, Sigma-Aldrich), goat anti-mouse IgG-HRP (sc-2005; Santa Cruz) and goat anti-rabbit IgG-HRP (sc-2004; Santa Cruz) were used following the manufacturers’ protocol.
3
Quantitative Western Blot Analysis
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A total of 50 mg protein was separated by denaturing 8–15% SDS–polyacrylamide gel electrophoresis. Western analysis was conducted as previously described [12] (link). The films were analyzed by densitometry with a VersaDoc Imaging System; Model 3000 (BioRad) using Quantity One software. Analysis of variance with Bonferroni correction for multiple tests was used to determine significance. The primary antibodies used were anti-Ncadherin (1∶5000), anti-Ecadherin (1∶2000), anti-fibronectin (1∶1000), anti-vimentin (1∶1000) and anti-actin (1∶2000) as an endogenous control, all from Epitomics Biotechnology (Epitomics).
4
Protein Interaction Assay with Compounds
5
Western Blot Protein Analysis
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Cells were lysed using a RIPA buffer (Beyotime, Shanghai, China) with 0.5% sodium dodecyl sulfate in the presence of proteinase inhibitor cocktail (Complete mini; Roche). Then the protein concentration was measured using a BCA Protein Assay Kit (Beyotime). Twenty-five micrograms of protein were separated by 10% SDS-PAGE mini gels and then transferred onto a PVDF membrane (Immobilon P -SQ ;Millipore, Billerica, MA). After blocking with 5% nonfat milk, the membranes were incubated with primary anti-RhoA (1:1000, Abcam), anti-ROCK1 (1:1000,Abcam) or anti-actin (1:5000, Epitomics, Burlingame, CA) at 4°C overnight, followed by incubated with HRP-labeled secondary antibodies (anti-rabbit IgG, 1:10000, KPL, Gaithersburg, MA) for 1 h at room temperature. Finally, the band signals were developed with Super Signal West Pico chemoluminescent substrate (Pierce, Rockford, IL), visualized by the GeneGnome HR Image Capture System (Syngene, Frederick, MD) and analyzed by Gene tools (Syngene).
6
Protein Expression Analysis in Rat Brain
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The primary antibodies, including anti-caspase-3, anti-caspase-9, anti-Nrf2, anti-NQO1, anti-HO-1, anti-NF-κB p65, and anti-actin, were purchased from Abcam (Cambridge, UK). After 24 h of reperfusion, the rat brain samples were homogenized, washed with phosphate buffered saline (PBS), and then lysed with radio immunoprecipitation assay (RIPA) lysis buffer. The total protein content was determined using a BCA kit (Solarbio, Beijing, China). After centrifugation at 12,000 rpm for 10 min, the supernatants were harvested for the measurement of protein concentration. The protein was isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Solarbio, Beijing, China) and then transferred to polyvinylidene difluoride (PVDF) membranes (Solarbio, Beijing, China). The membranes were blocked with 5% nonfat dry milk and incubated overnight at 4 °C with anti-caspase-3, anti-caspase-9, anti-Nrf2, anti-NQO1, anti-HO-1, anti-NF-κB p65, and anti-actin. Following that, the membranes were washed with tris-buffered saline and Tween 20 (TBST) buffer. The secondary antibody was added to the membranes. The blots were detected with an ECL detection kit (Solarbio, Beijing, China). Actin was served as a loading control.
7
Measuring Secreted Collagen by Western Blot
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For measuring collagen secretion into the cellular medium, cells were washed with serum free medium and incubated in serum free medium for 3 h. The medium was collected and collagen was directly measured by western blot by loading 50–100 μL of the medium onto 7.5% SDS-PAGE gels27 (link),67 (link). Western blots were performed with 1:1000 dilutions of the antibodies. The antibodies used were: anti-collagen α1(I) from Rockland (600-401-103), anti-collagen α2(I) from Santa Cruz Biotechnology (sc-8786), anti-fibronectin from BD Transduction Laboratories (610523 and 610077). When cell extracts were analyzed anti-actin from Abcam (ab8227), anti-LARP6 from Novus Biologicals (H00055323) antibodies were used.
8
Azurocidin and Cofilin Signaling in Cells
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Proteins from 3 × 105 RLT cells were isolated with 150 µL RIPA-buffer with protease and phosphatase inhibitors according to the manufacturer's recommendation (Santa Cruz). Protein samples were heated for 10 min at 95°C and separated by SDS-PAGE (7%). After blotting to a PVDF (Merck/Millipore, Darmstadt, Germany) the following antibodies were used: mouse anti-azurocidin (Abcam, 1:1000), mouse anti-α-SMA (Sigma, 1:500), rabbit anti-p-cofilin (Cell Signaling, Danvars, MA, USA, 1:1000), mouse anti-MMP9 (1:500), rabbit anti-pp38 (Biovision, Milpita, CA, USA, 1:500), and rabbit anti-p-MAPKAP-2 (Santa Cruz; 1:300). After washing, membranes were incubated using a goat anti rabbit IgG POX or, goat anti mouse IgG POX, respectively (Becton and Dickinson) as secondary antibodies (room temperature for 30 min). To control for equal loading, β-actin was determined using anti-actin (Abcam). For detection, Amersham ECL plus Western Blotting Detection System (GE Healthcare, Munich, Germany) was used. Densitometrical analysis was performed using Quantity one software (Biorad, Hercules, CA, USA).
9
Oxidative Stress Protein Expression
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Anti caspase 3, anti HO1, anti Nrf2, anti SOD2, anti actin, anti lamin B1 and anti GCLC antibodies were purchased from Abcam (Cambridge, MA, USA). K2Cr2O7 and all other reagents were bought from Sisco Research Laboratory (Mumbai, India).
10
Analyzing TRPC3 Protein Localization in Cerebellar Slices
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Cerebellar
organotypic slice cultures were prepared as described previously,5 (link) and biotinylation experiments were conducted
as described previously15 (link) using Sulfo-NHS-SS-Biotin
(Thermo Scientific) and StrepTactin sepharose (GE Healthcare). Protein
lysates prepared with RIPA buffer (Thermo Scientific) were subjected
to sodium dodecyl sulfate–polyacrylamide gel electrophoresis
(SDS–PAGE) followed by immunoblotting with anti-TRPC3 (1:200,
Alomone) and anti-Actin (1:1000, Abcam) antibodies. Antibody binding
was detected by enhanced chemoluminescence (ECL; GE Healthcare).
organotypic slice cultures were prepared as described previously,5 (link) and biotinylation experiments were conducted
as described previously15 (link) using Sulfo-NHS-SS-Biotin
(Thermo Scientific) and StrepTactin sepharose (GE Healthcare). Protein
lysates prepared with RIPA buffer (Thermo Scientific) were subjected
to sodium dodecyl sulfate–polyacrylamide gel electrophoresis
(SDS–PAGE) followed by immunoblotting with anti-TRPC3 (1:200,
Alomone) and anti-Actin (1:1000, Abcam) antibodies. Antibody binding
was detected by enhanced chemoluminescence (ECL; GE Healthcare).
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