The protein levels of megalin, angiotensin-converting enzyme 2 (ACE2), and tumor necrosis factor α (TNFα) were measured using western blotting. The hippocampal tissue was lysed using lysis buffer (Pre-prep, Intron). The protein was purified and quantified using a microplate reader. The quantified protein samples were loaded to 8-10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gels were transferred to polyvinylidene difluoride membranes (Merck Millipore, Burlington, MA, USA) and incubated in blocking buffer (5% nonfat dry milk in Tris-buffered saline containing Tween-20) for 1 hour. The membranes were incubated with 1 : 1000 of anti-megalin (Santa Cruz, Dallas, Texas, U.S.A., Sc515772), anti-ACE2 (Santa Cruz, Dallas, Texas, U.S.A., Sc390851), anti-TNFα (Abcam, Cambridge, England, Ab6671), and anti-rabbit monoclonal β-actin (Cell Signaling Technology, Danvers, MA, USA, D6A8). Then, membranes were incubated with horseradish peroxidase- (HRP-) conjugated secondary antibodies (anti-rabbit IgG, HRP-linked (Cell Signaling Technology, #7074S) and goat anti-mouse IgG H&L (HRP) (Abcam, Cambridge, England, #ab97023)). Protein bands were evaluated using an enhanced chemiluminescence kit (Bio-Rad, Hercules, CA, USA) and quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Anti megalin
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-megalin is a laboratory product used in scientific research. It is an antibody that recognizes and binds to the megalin protein, which is a receptor involved in the uptake of various molecules. The primary function of Anti-megalin is to facilitate the study of megalin and its role in cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescence kit, by Bio-Rad (1 mentions)
Anti ace2, by Santa Cruz Biotechnology (1 mentions)
Anti tnf α, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Goat anti mouse igg h l hrp, by Abcam (1 mentions)
Lsm 510 meta confocal scanning microscope, by Zeiss (1 mentions)
Total gsk3β, by Cell Signaling Technology (1 mentions)
Anti phospho akt ser473, by Cell Signaling Technology (1 mentions)
Anti phospho gsk3β ser9, by Cell Signaling Technology (1 mentions)
Anti nephrin, by Santa Cruz Biotechnology (1 mentions)
Anti podocin, by Santa Cruz Biotechnology (1 mentions)
Total akt, by Cell Signaling Technology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Eclipse e200, by Nikon (1 mentions)
4 protocols using anti megalin
1
Quantification of Protein Levels in Hippocampus
2
Fluorescent Labeling and Localization of LCN2 in Mice
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LCN2 was fluorescently labelled with Alexa568-Succinimidyl Ester (Molecular Probes) by following previous protocols58 (link) and instructions from the manufacturer (https://tools.thermofisher.com/content/sfs/brochures/TR0031-Calc-FP-ratios.pdf ) including clean up by gel filtration followed by microcon (10 K) washes. The molar ratios of Alexa dye to native, K3, K3Cys proteins were 0.98, 1.20 and 0.73. The proteins were introduced (80 μg for LCN2, 8-week old mice) and after 1 h, kidney, liver, heart and spleen were fixed in 4% PFA. Immunohistochemical staining of the kidney proximal tubules (Anti-Megalin, Santa Cruz; LTL, Vector Labs) collecting ducts (Anti-Tromal Ab, Developmental Studies Hybridoma; DSHB; Anti-AE1 Ab), Principal cells (Anti-AQP2), Intercalated cells (Anti-V-ATPase Ab) were visualized using Zeiss LSM 510 Meta Confocal Scanning Microscope. Immunoblots were assayed with anti-mouse LCN2 (R&D Systems) and anti-mouse Tf (Bethyl Laboratories, A90-129A).
3
Podocyte Protein Expression Analysis
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Anti-podocin (Santa Cruz, catalog# sc-518088), anti-nephrin (Santa Cruz, catalog# sc-377246), anti-phospho-Akt (Ser-473) (Cell Signaling, catalog# 9271), anti-phospho-GSK3β (Ser9) (Cell Signaling, catalog# 9336), β-actin (Santa Cruz, catalog# sc-47778), total Akt (Cell Signaling, catalog# 9272), total GSK3β (Cell Signaling, catalog# 9315), anti-megalin (Santa Cruz, catalog# sc-515750), anti-cubilin antibody (Santa Cruz, catalog# sc-518059), anti-LAMP1 (Development Studies Hybridoma Bank, catalog# 1D4B), Alexa fluor 488 anti-mouse for megalin, Alexa fluor 488 anti-rabbit for cubilin, Alexa fluor 568 anti-rat for LAMP1.
4
Immunofluorescent Localization of Megalin
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Kidneys, liver and distal colon were fixed in 10% neutral formalin in PBS 0.1 M (pH: 7.4).
The tissue sections were dehydrated and embedded in paraffin. Sections of 5 μm were For immunofluorescence studies, the slides were preincubated with non immune rabbit serum in PBS (1:50) at room temperature for 30 min, followed by incubation with a polyclonal anti-megalin (1:50, Santa Cruz Biotechnology, Santa Cruz, CA) antibody overnight in a humidified chamber at 4°C. After several washes in PBS, the slides were incubated with an anti-rabbit IgG antibody conjugated with fluorescein (1:200, Santa Cruz Biotechnology) for 1 h at room temperature in a humidified chamber. Finally, all the slides were mounted on a mixture containing PBS:glycerol (1:3), and observed in an epifluorescent microscope (Nikon Eclipse E200, Nikon, Tokyo, Japan). Negative control was performed without primary antibody.
The tissue sections were dehydrated and embedded in paraffin. Sections of 5 μm were For immunofluorescence studies, the slides were preincubated with non immune rabbit serum in PBS (1:50) at room temperature for 30 min, followed by incubation with a polyclonal anti-megalin (1:50, Santa Cruz Biotechnology, Santa Cruz, CA) antibody overnight in a humidified chamber at 4°C. After several washes in PBS, the slides were incubated with an anti-rabbit IgG antibody conjugated with fluorescein (1:200, Santa Cruz Biotechnology) for 1 h at room temperature in a humidified chamber. Finally, all the slides were mounted on a mixture containing PBS:glycerol (1:3), and observed in an epifluorescent microscope (Nikon Eclipse E200, Nikon, Tokyo, Japan). Negative control was performed without primary antibody.
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