Using the operating microscope (Leica A60), metastatic tumors in the brain were isolated from the tumor-bearing mice. Brain tissues isolated from normal mice were used as controls. Activity of NE in tumor and normal brain tissues were measured by using a SensoLyte® Green Elastase Assay Kit (#AS-72178, AnaSpec, CA). For characterization of CXCR4, Western Blot analysis was performed according to the standard procedures as previously described [25 (link)]. Antibodies used in this study included anti-CXCR4 (#NB100–74396, Novus), HRP anti-rabbit IgG (#656120, invitrogen), beta-actin (c643802, BioLegend), and HRP anti-mouse IgG (AP307P, Sigma-Aldrich).
Beta actin
Manufactured by BioLegend
Sourced in United States
Beta-actin is a cytoskeletal protein that is involved in cell motility, structure, and integrity. It is a highly conserved and ubiquitously expressed protein that is essential for various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Anti mouse igg hrp, by Merck Group (2 mentions)
Nb100 74396, by Novus Biologicals (2 mentions)
Anti rabbit igg hrp, by Thermo Fisher Scientific (2 mentions)
Sensolyte green elastase assay kit, by AnaSpec (1 mentions)
Ab216880, by Abcam (1 mentions)
Anti ho 1, by Novus Biologicals (1 mentions)
Anti nrf2, by Novus Biologicals (1 mentions)
Anti claudin 5, by Thermo Fisher Scientific (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
Glut1, by Santa Cruz Biotechnology (1 mentions)
Hif 1α, by R&D Systems (1 mentions)
Foxp1, by Cell Signaling Technology (1 mentions)
Gapdh, by LI COR (1 mentions)
α tubulin, by Synaptic Systems (1 mentions)
Intercept tbs blocking solution, by LI COR (1 mentions)
Vegfa, by Proteintech (1 mentions)
Pparγ h 100, by Santa Cruz Biotechnology (1 mentions)
Hrp conjugated anti mouse igg h l, by Promega (1 mentions)
Hrp conjugated anti rabbit igg, by Promega (1 mentions)
8 protocols using beta actin
1
Characterization of Metastatic Brain Tumors
2
Characterization of CXCR4 and BBB Repair
3
Subcellular Fractionation and STAT3 Analysis
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For whole lung protein fractions, mice were euthanized 24 h after inoculation as described above, lungs were homogenized, and protein was extracted using RIPA buffer (50 mM Tris-HCl pH 7.5, 2 mM EDTA, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% Na-deoxycholate, 50 mM NaF). Primary alveolar epithelial cells were grown to ~80–90% confluence on T-75 flasks and were treated with IAV (MOI 1) or PBS. At 24 h after treatment cells were washed with ice-cold PBS and cytoplasmic protein fraction was extracted with Buffer A (10 mM HEPES pH 8.0, 0.5% NP-40, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, and 200 mM sucrose). The tubes were incubated for 30 min on ice, and then centrifuged at 14,000 rpm at 4°C and the supernatant was collected. The pellets were resuspended in 30 μl of RIPA buffer and incubated on ice for 30 min. Then they were centrifuged at 14,000 rpm at 4°C and the supernatant was collected as the nuclear protein fraction. Proteins were quantified using BCA (Pierce). Proteins were run on a 10% SDS-Page gel, transferred to nitrocellulose, and stained for either STAT3 (Cell Signaling; D1B2), P-STAT3 (Cell Signaling; Tyr705, D3A7), or beta-actin (BioLegend; Poly6221). Protein levels were normalized to beta-actin and fold change was calculated over WT PBS.
4
Antioxidant Effects of BA Nanoparticles
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To determine the anti-oxidant effect of BA NPs on cells, NHA were randomly divided into 4 groups, which were treated with PBS, 2 μg/ml BA NPs, 10 μg/ml BA NPs and 30 μg/ml BA NPs. After 24 h, cells were lysed in RIPA lysis buffer containing protease for 30 min on ice. The protein concentration of each cell lysate sample was determined using the BCA and adjusted to equivalent amounts. Western blot analysis was performed according to the standard procedures as described in our previous report 29 (link), using antibodies targeting Nrf2 (Novus Biologicals), HO-1 (Novus Biologicals), and beta-actin (#643802, BioLegend). To determine the anti-oxidant effect in vivo, mice with successful MCAO surgery were randomly divided into 2 groups (n = 3 for each group), which received treatment of PBS or 2mg BA NPs, respectively. After 24 h, the brains were harvested, and the right hemispheres containing the ischemic area were excised. The brains from normal mice without surgery were used as controls. Western blot analysis was performed as described above.
5
Protein Extraction and Western Blot Analysis of Mouse Embryonic Stem Cells
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Whole-cell extracts from mESCs cultured in feeder-free 2i media were prepared in a modified RIPA lysis buffer: 50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, and 1x SigmaFast protease inhibitor cocktail. Spheroids were lysed in RIPA buffer and protease inhibitors. Protein concentration was determined with a BCA kit (Thermo Fisher Scientific) and normalized to 1.0–2.0 μg/μl. 20–25 μl of protein was mixed with sample buffer and 50–100 mM DTT. Protein was transferred to a PVDF membrane, blocked with either 1% BSA or Intercept (TBS) Blocking Solution (LI-COR), and blotted with primary antibodies at 4 °C overnight. Primary antibodies used: Foxp1 (1:1000, D35D10, #4402, Cell Signaling) (Glut1 (2 μg/ml, #sc-377228, Santa Cruz Biotechnology), HIF-1α (1 μg/ml, #AF1935, R&D Systems), Ldha (1:1000, #2012 Cell Signaling), Vegfa (1:500, #19003, Proteintech), Beta-Actin (1 μg/ml, 664802, Biolegend), Gapdh (1:1000, #926-422116, LI-COR), α-Tubulin (Synaptic Systems, 1:2000, #302 204). Western blots were imaged on an Odyssey DLx Imager system.
6
Western Blot Analysis of Cell Signaling
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Total protein lysates were prepared using RIPA buffer (Santa Cruz) according to manufacturer’s instructions. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by blotting on polyvinylidene fluoride (PVDF) membranes (Thermo Fisher Scientific) using Trans-Blot SD semi-dry transfer cell (Bio-Rad Laboratories). Antibodies against AMPK (Abcam, 1:2500), PPARγ (H-100) (Santa Cruz, 1:1000), phospho-(Ser112)-PPARγ (Merck Millipore, 1:500), p44/42 mitogen-activated protein kinase (MAPK, a.k.a. extracellular signal-regulated kinases 1 and 2, ERK1/2) (Cell Signaling, 1:1000), phospho-p44/42 MAPK [(ERK1) (Tyr204)/(ERK2) (Tyr187) (D1H6G)] (Cell Signaling, 1:1000), and cleaved poly(ADP-ribose) polymerase 1 (PARP1) (Abcam, 1:2000) were used overnight at 4 °C. Beta-actin (ACTB) was used as a loading control (Biolegend, 1:2500). HRP-conjugated anti-rabbit IgG (Promega, 1:5000), HRP-conjugated anti-mouse IgG (H+L) (Promega, 1:5000), and HRP-conjugated anti-rat IgG (Biolegend, 1:5000) were used as secondary antibodies for 1 h at room temperature (RT). Subsequently, membranes were covered with AceGlow chemiluminescence substrate (Peqlab) and imaged immediately using ChemiDoc XRS + (Bio-Rad Laboratories).
7
Western Blot Analysis of HSV-1 Glycoprotein E
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Vero cells were infected with either KOS or either isolate of vUs7-8mCherry at a multiplicity of infection (MOI) of 5. At 18 hours post-infection (hpi), cells were lysed in RIPA buffer (50 mM Tris pH 7.5, 1% Triton X-100, 0.5% DOC, 0.1% SDS, 500 mM NaCl). Proteins were resolved by SDS polyacrylamide gel electrophoresis, and transferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore). Proteins were detected by Western blotting using a mouse monoclonal antibody directed against HSV-1 gE (Fitzgerald) or a mouse monoclonal antibody directed against beta-Actin (Biolegend), followed by appropriate secondary antibodies conjugated to horseradish peroxidise (Bethyl and Sigma respectively). Detection was done by enhanced chemiluminescence (GE) according to the manufacturer's instructions.
8
Prostate Protein Extraction and Western Blot
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Proteins were extracted from prostate tissues and separated through SDS-PAGE, Proteins were transferred to nitrocellulose membranes (Advantec, Japan). The membranes were then blocked with the 5% Bovine serum albumin, followed by overnight incubation at 4°C with appropriate primary antibodies (Rabbit polyclonal anti-AR, anti-ER-α (Abclonal, Woburn, United states) and Beta actin (Biolegend, San Diego, USA). After that, secondary antibody incubation at 25°C for one hour. Blots were projected with a chemiluminescence blotting substrate (Roche, Germany) kit under Chemidoc system.
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