UCHL1-targeted small interfering (si)RNA were designed using siDIRECT17 (link) and synthesized by Life Technologies-Invitrogen: sense = 5’-GAAGUUAGUCCUAAAGUGUAC-3’; and anti-sense = 5’-ACACUUUAGGACUAACUUCUU-3’. Control siRNA were designed by scrambling the constituent nucleotides to generate non-targeted sequence: sense = 5’-CCUUUAAAUUCCCAAAGGGUU-3’; and anti-sense = 5’-CCCUUUGGGAAUUUAAAGGAA-3’. Human choroidal or retinal endothelial cells were grown to at least 80% confluence in 6 or 10 cm diameter dishes, and subsequently incubated for 24 hours with UCHL1 or non-targeted siRNA, in complex with Targefect-siRNA Transfection Kit reagents (Targeting Systems, El Cajon, CA), according to the manufacturer’s instructions. After transfection, the endothelial cells were rested at 37 °C in modified MCDB-131 medium with 5% FBS for 24 hours, ahead of use in cell proliferation or cell migration assays. For every cell proliferation or cell migration assay, protein was extracted from parallel, identically transfected cultures of human choroidal or retinal endothelial cells, and interrogated by Western blot using rabbit polyclonal anti-human UCHL1 antibody (Cedarlane Laboratories) and mouse monoclonal anti-human β-actin antibody (clone AC-15, isotype IgG1; Sigma-Aldrich), to verify UCHL1 protein knock-down.
Anti human β actin antibody
Manufactured by Merck Group
Sourced in United States
The Anti-human β-actin antibody is a laboratory reagent used for the detection and quantification of the β-actin protein, a key structural component found in eukaryotic cells. This antibody can be used in various immunoassay techniques, such as Western blotting, immunohistochemistry, and ELISA, to aid in the study of cellular processes and protein expression.
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Lab products found in correlation
Anti human uchl1 antibody, by Cedarlane (1 mentions)
Lumi light plus kit, by Roche (1 mentions)
Anti src, by Cell Signaling Technology (1 mentions)
Anti phospho src, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase labelled secondary antibodies, by GE Healthcare (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Ecl prime reagent, by Cytiva (1 mentions)
Supersep ace 15 precast gel, by Fujifilm (1 mentions)
Imagequant las 500, by Cytiva (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Alliance 2, by Uvitec (1 mentions)
4 protocols using anti human β actin antibody
1
UCHL1 gene silencing in endothelial cells
2
Integrin α5 Protein Extraction and Western Blot
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Protein extraction and western blot were carried out as previously described (Ponz-Sarvise et al, 2011 (link)). The primary antibodies (diluted at 1 : 1000) were as follows: anti-human ITGα5 (Sigma), anti phospho-Src (Cell Signaling, Danvers, MA, USA) and anti-Src (Cell Signaling). An anti-human β-actin antibody (Sigma) at 1 : 10 000 dilution was used as a loading control. Horseradish peroxidase-labelled secondary antibodies (GE Healthcare, Waukesha, WI, USA) against the corresponding primary antibodies were used. Immunoreactive bands were visualised by a chemiluminescent method using the Lumi-lightPLUS kit (Roche, Palo Alto, CA, USA).
3
Harvesting and Quantifying Intracellular and Secreted Proteins
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Proteins were harvested from the intracellular and culture supernatants. Before collection, MH7A cells, FLS, monocytes, and macrophages were washed with phosphate-buffered saline. The cells were dissolved in radioimmunoprecipitation assay (RIPA) buffer. For the supernatant protein assay, the last 24 h of incubation was performed in a serum-free medium. Cell supernatants for FLS, MH7A, HEK293T, and macrophages were suspended in acetone and kept at –20 °C for 1 h. The samples were then centrifuged for 10 min at 15,000 × g at 20–25°C. Protein precipitates were dried for 30 min and dissolved in RIPA buffer. Proteins were processed using a SuperSep Ace 15 % precast gel (FUJIFILM Wako Pure Chemical Co.) and transferred onto a polyvinylidene fluoride membrane. The membranes were probed with anti-human β-actin antibody (0.1 μg/mL, mouse monoclonal, clone AC-15, Sigma-Aldrich), anti-human FXIII-A antibody (0.025 μg/mL, mouse monoclonal, Proteintech), anti-human FXIII-B antibody (0.1 μg/mL, rabbit polyclonal, Sigma-Aldrich), and anti-HA-tag antibody (0.2 μg/mL, goat polyclonal, Genscript). Horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch) were then added. Horseradish peroxidase activity was detected using ECL prime reagents (Cytiva, Tokyo, Japan), followed by imaging using the Image Quant LAS 500 (Cytiva) system.
4
Quantitative Western Blot Analysis
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For western blotting, total cellular lysates were separated by 10% SDS-PAGE electrophoresis under reducing conditions and transferred (wet transfer method for blotting, 120 Volts, 1 h) to PVDF membranes (Millipore). The membranes were blocked at room temperature for 1 h in 5% non-fat dry milk, and incubated overnight at +4 °C with a specific primary antibody (Human NALP-3 antibody monoclonal mouse IgG, 1:1000, Clone CL0210, Novus Biologicals; anti –ERβ, Human ERβ/NR3A2 Antibody Monoclonal Mouse IgG1, 1:1000, Clone 733930, R&D Systems, Bio-Techne Ltd., Abingdon, UK). The membranes were washed with PBST and incubated in specific horseradish peroxidase-conjugated IgG diluted 1:2000 in 5% non-fat dried milk in PBST. A bound secondary antibody was detected by chemiluminescence. Bands were analyzed using the enhanced chemiluminescence (ECL™, Amersham, UK) by chemiluminescence imaging system Alliance 2.7 (UVITEC, Cambridge, UK) and quantified by the software Alliance V1607.
As positive load control cytosolic, the β-actin band (42-kDa) was detected by a mouse monoclonal anti-human β-actin antibody, 1:5000 (Sigma-Aldrich, St. Louis, MO, USA).
As positive load control cytosolic, the β-actin band (42-kDa) was detected by a mouse monoclonal anti-human β-actin antibody, 1:5000 (Sigma-Aldrich, St. Louis, MO, USA).
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