CD-NTase in-cell expression levels were verified by Western blot of lysed cells. Confluent HEK293T cells were seeded 24 h prior to transfection at a dilution of 1:4 in a 6-well dish. Cells were transfected with 2 μg of plasmid using Lipofectamine2000. At 24 h post transfection cells were harvested by washing cells from the dish using Hanks Buffered Saline Solution, pelleted at low speed, and flash frozen. Pelleted cells were lysed by re-suspending the pellet in 400 μL 1x LDS buffer (ThermoFisher Scientific) + 5% β-mercaptoethanol, boiling for 5 min, and vigorously vortexing. Samples were separated by SDS-PAGE, transferred to nitrocellulose membrane, and probed with primary antibodies 1:5,000 Rabbit anti-MBP (Millipore Cat# AB3596, RRID:AB_91531) and 1:10,000 Mouse anti-Tubulin (Millipore Cat# MABT205, RRID:AB_11204167), followed by secondary antibodies at 1:10,000 IRDye 680RD Goat anti-Rabbit IgG (LI-COR Biosciences Cat# 925–68071, RRID:AB_2721181) and IRDye 800CW Goat anti-Mouse IgG (LI-COR Biosciences Cat# 925–32210, RRID:AB_2687825). Stained membrane was imaged using a LI-COR Odyssey CLx imager.
Mabt205
Manufactured by Merck Group
MABT205 is a piece of lab equipment manufactured by Merck Group. It is designed for use in scientific research and analysis. The core function of MABT205 is to facilitate the measurement and analysis of various samples and materials.
Automatically generated - may contain errors
Lab products found in correlation
Irdye 800cw goat anti mouse igg, by LI COR (2 mentions)
Odyssey clx imager, by LI COR (2 mentions)
Irdye 680rd goat anti rabbit igg, by LI COR (2 mentions)
Lds buffer, by Thermo Fisher Scientific (2 mentions)
Ab3596, by Merck Group (2 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti iba1, by Fujifilm (1 mentions)
Anti neun, by Cell Signaling Technology (1 mentions)
Anti fmrp, by Cell Signaling Technology (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Abways (1 mentions)
Anti gfap, by Cell Signaling Technology (1 mentions)
4 protocols using mabt205
1
Western Blot Analysis of CD-NTase Expression
2
Smooth Muscle α-Actin Expression
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The HAoVSMCs were seeded in the pre-coated 96-well plates at a density of 2000–2500 cells per well in 150 µL of culture medium per well and incubated for 72 h. To identify the expression of SM α-actin, the lysed samples were separated on polyacrylamide gels and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). Then the PVDF membranes were incubated with the primary antibodies SM α-actin (1:1000, Abcam) and α-Tubulin (1:2000, MABT205, Millipore) overnight at 4 °C. Afterwards, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at RT for 1 h. The signal was acquired by using Azure 400 biosystem (Azure, USA). Then, ImageJ software was used to calculate the band intensities, and α-Tubulin antibodies were used as an internal control for signal normalization.
3
Western Blot Analysis of CD-NTase Expression
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CD-NTase in-cell expression levels were verified by Western blot of lysed cells. Confluent HEK293T cells were seeded 24 h prior to transfection at a dilution of 1:4 in a 6-well dish. Cells were transfected with 2 μg of plasmid using Lipofectamine2000. At 24 h post transfection cells were harvested by washing cells from the dish using Hanks Buffered Saline Solution, pelleted at low speed, and flash frozen. Pelleted cells were lysed by re-suspending the pellet in 400 μL 1x LDS buffer (ThermoFisher Scientific) + 5% β-mercaptoethanol, boiling for 5 min, and vigorously vortexing. Samples were separated by SDS-PAGE, transferred to nitrocellulose membrane, and probed with primary antibodies 1:5,000 Rabbit anti-MBP (Millipore Cat# AB3596, RRID:AB_91531) and 1:10,000 Mouse anti-Tubulin (Millipore Cat# MABT205, RRID:AB_11204167), followed by secondary antibodies at 1:10,000 IRDye 680RD Goat anti-Rabbit IgG (LI-COR Biosciences Cat# 925–68071, RRID:AB_2721181) and IRDye 800CW Goat anti-Mouse IgG (LI-COR Biosciences Cat# 925–32210, RRID:AB_2687825). Stained membrane was imaged using a LI-COR Odyssey CLx imager.
4
Western Blot Analysis of Neural Markers
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Treated cells and brain tissues were lysed in the TNEN lysis buffer (25mM Tris-HCl [pH 7.6], 150 mM NaCl, 1% sodium deoxycholate, 1% NP-40, and 0.1% SDS) supplemented with the protease inhibitor. Equal amounts of protein lysates were subjected to SDS-polyacrylamide gel electrophoresis and proteins were detected by the indicated antibodies. Primary antibodies used were as follows: anti-FMRP (Cell Signaling Technology, 4317S, 1:1,000), anti-NeuN (Cell Signaling Technology, 94403S, 1:1,000), anti-GFAP (Cell Signaling Technology, 3670S, 1:1,000), anti-Iba1 (Wako, 016–20001, 1:1,000), anti-GAPDH (Abways, AB0038, 1:5,000), and anti-α-tubulin (Millipore, MABT205, 1:10,000). HRP-conjugated secondary antibodies used were goat anti-rabbit IgG (H + L), HRP (Thermo Fisher Scientific, 31460, 1:5,000) and goat anti-mouse IgG (H + L), HRP (Thermo Fisher Scientific, 31430, 1:5,000).
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