After treatment, cells were harvested and lysed in the cell extraction buffer (Invitrogen, FNN0011) supplemented with proteasome inhibitor (P2714, Sigma) and 1 mM phenylmethanesulfonyl fluoride (PMSF). Equal amounts (20 µg) of protein were loaded onto SDS-PAGE for separation and then transferred onto a PVDF membrane. The membrane was blocked with 5% w/v nonfat dry milk (in TBS with 0.05% Tween-20). After incubation with the specific primary antibody (1:1000,5% nonfat dry milk or BSA in TBS with 0.05% Tween-20) at 4 °C overnight, the membrane was washed three times with TBST (TBS with 0.05% Tween-20) solution. The membrane was then incubated with secondary antibody (1:5000, in 5% nonfat dry milk, TBS with 0.05% Tween-20) at room temperature for 1 h followed by another three washes with TBST. The protein bands were developed by incubation with a luminescent reagent. ImageJ was used to quantify the band intensity.
Fnn0011
Manufactured by Merck Group
Sourced in Germany
FNN0011 is a laboratory equipment product manufactured by Merck Group. It is designed to perform a core function, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
Lab products found in correlation
P2714, by Merck Group (1 mentions)
Polyvinylidene difluoride membranes pvdf, by Merck Group (1 mentions)
Supersignal west pico substrate, by Thermo Fisher Scientific (1 mentions)
Semi dry transfer system, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Endothelial cell growth medium 2, by PromoCell (1 mentions)
4 protocols using fnn0011
1
Western Blot Analysis of Protein Expression
2
Quantitative Western Blotting of CDH19
3
Isolation of Brain Endothelial Cells
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Primary brain endothelial cells (BECs) were isolated from BACE1 knockout mice and littermate wildtype controls. The brains were minced and incubated in 0.2 mg/mL collagenase dispase dissolved in DMEM (1 h, 37 °C, 5% CO2). The samples were homogenised using a cannula attached to a stripette, strained in a 70 uL cell strainer, centrifuged (1000× g, 10 min at room temperature), and resuspended in 5 mL 20% BSA solution. The samples were centrifuged (1000× g, 20 min, room temperature) the myelin layer discarded and resuspended in 5 ml PBS before a final centrifuge (400× g, 5 min, room temperature). The pellet was resuspended in MV2 media (Promocell, Heidelberg, Germany; C-22022), supplemented with antibiotic antimycotic solution (Merck, Darmstadt, Germany; A5955-100ML), and cells incubated (37 degrees, 5% CO2) until confluent with the media changed every 2–3 days. We expect >95% of remaining cells to be BECs [76 (link)]. Cells were grown on 0.2% gelatine-coated plates. Once confluent, BECs were washed with ice cold PBS and lysed with a cell extraction buffer (Fisher Scientific, FNN0011) supplemented with protease inhibitor (Merck, Darmstadt, Germany).
4
hCMEC/D3 Cells Transfection and BACE1 Inhibition
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hCMEC/D3 cells were grown on 0.2% gelatine-coated flasks/plates, and grown in ECGM2 (Endothelial Cell Growth Medium 2, Promocell) supplemented with antibiotic antimycotic solution (Merck, Darmstadt, Germany, A5955-100ML) at 37 degrees, and 5% CO2. For transfection, cells were plated on to 6-well plates at 100,000 cells per well and grown for 3 days. Cells were then transfected with 1ug of plasmid DNA of either empty vector (pcDNA3.1) or BACE1 per well, using Lipofectamine™ 2000 reagent (Invitrogen, Waltham, MA, USA), according to the manufacturer’s instructions. For BACE1-inhibitor treatments, cells were plated onto 6-well plates at 100,000 cells per well and grown for 3 days, and treated with Merck-3 (250 nM, Merck, Darmstadt, Germany) for 24 h. At 24 h post-transfection or inhibitor treatment, the cells were washed with ice cold PBS and lysed with a cell extraction buffer (Fisher Scientific, FNN0011) supplemented with protease inhibitor (Merck), or pelleted for RNA extraction.
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