BMM were treated for 24 h with 100 ng/ml LPS, after which the cells were washed twice with PBS and lysed in ice-cold Nonidet P-40 (NP-40) lysis buffer (50 mM Tris–HCl pH7.4, 100 mM NaCl, 10 mM NaF, 1 mM Na3PO4, 10 % glycerol, 1 % NP-40). After a 10 min incubation on ice the lysates were collected and boiled in sample buffer containing DTT. Samples were thereafter analyzed by SDS-PAGE. All proteins of interest were separated on 12 % polyacrylamide SDS gels. Proteins were transferred to 0.2 μm nitrocellulose membranes using the Trans-blot Turbo transfer system (Biorad). Nitrocellulose membranes were subsequently blocked in 5 % (w/v) non-fat milk in Tris-buffered saline (TBS) and incubated with CX3CR1 (Santa Cruz) and anti-α-tubulin (Cedarlane) primary antibodies overnight at 4 °C, followed by horse radish peroxidase-labelled secondary antibodies (Bio-Rad) for 1 h. Proteins were visualized with an enhanced chemiluminescence (ECL) detection system (Thermoscientific) and quantification of signal was performed using intensity measurements in ImageJ software.
Anti α tubulin
Manufactured by Cedarlane
Sourced in United States, United Kingdom
Anti-α-tubulin is a primary antibody used in various laboratory techniques to detect and quantify the presence of α-tubulin, a major component of the cytoskeleton in eukaryotic cells. It is a useful tool for studying cellular structure and function.
Automatically generated - may contain errors
Lab products found in correlation
Ecl detection system, by Thermo Fisher Scientific (1 mentions)
Cx3cr1, by Santa Cruz Biotechnology (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Anti mettl1, by Proteintech (1 mentions)
Hrp conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Supersignal west pico plus chemiluminescent substrate kit, by Thermo Fisher Scientific (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Anti cd40, by Abcam (1 mentions)
Ibrightfl1000 system, by Thermo Fisher Scientific (1 mentions)
Ibright analysis software, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Pngase f, by New England Biolabs (1 mentions)
4 protocols using anti α tubulin
1
Activation of CX3CR1 protein by LPS
2
Detecting tRNA Modifying Enzymes
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Rabbit polyclonal anti-NSUN2 [17] (link) and anti-METTL1 (ProteinTech Group, Chicago, IL, USA) antibodies were used to detect endogenous tRNA modifying enzymes. Monoclonal anti-Xpress (Invitrogen, Carlsbad, CA, USA) and anti-α-tubulin (Cedarlane Laboratories, Burlington, Ontario, Canada) antibodies were also used.
3
CD40 Signaling in Macrophages
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CD40flflLysMcre and CD40flfl BMDMs were cultured as described above, treated for 10 min or 24 h with FGK45 (30 µg/ml), IFN‐γ (5 ng/ml) and LPS (100 ng/ml) and subsequently lysed in RIPA lysis buffer. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Equal amounts of protein samples were loaded on to a SDS polyacrylamide gel and transferred to a nitrocellulose membrane (Bio‐Rad). After blocking with 5% BSA in PBS containing 0.05% Tween‐20, blots were incubated overnight with anti‐CD40 (Abcam, Cambridge, UK) and anti‐α‐tubulin (Cedarlane, Burlington, ON, Canada). Blots were then washed and incubated with the appropriate HRP‐conjugated secondary antibody (1:800; Thermo Scientific) and visualized using the SuperSignal West Pico Plus Chemiluminescent Substrate kit (Thermo Scientific).
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CD40flflLysMcre and CD40flfl BMDMs were cultured as described above, treated for 10 min or 24 h with FGK45 (30 µg/ml), IFN‐γ (5 ng/ml) and LPS (100 ng/ml) and subsequently lysed in RIPA lysis buffer. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Equal amounts of protein samples were loaded on to a SDS polyacrylamide gel and transferred to a nitrocellulose membrane (Bio‐Rad). After blocking with 5% BSA in PBS containing 0.05% Tween‐20, blots were incubated overnight with anti‐CD40 (Abcam, Cambridge, UK) and anti‐α‐tubulin (Cedarlane, Burlington, ON, Canada). Blots were then washed and incubated with the appropriate HRP‐conjugated secondary antibody (1:800; Thermo Scientific) and visualized using the SuperSignal West Pico Plus Chemiluminescent Substrate kit (Thermo Scientific).
4
Membrane Protein Deglycosylation and Western Blotting
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Brain membrane proteins were prepared from P60 to 90 mice as described.41 (link) Complete protease inhibitors (Roche Diagnostics) were added to all solutions at twice the recommended concentration to minimize protein degradation. Deglycosylation of membrane protein samples, where indicated, was performed using PNGaseF (New England BioLabs Cat. #P0704S) as previously described.17 (link) For western blotting, 50–80 µg aliquots of membrane protein were separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and processed for western blotting with anti-β1intra antibody (1:1000) (Cell Signaling Technologies 13950) or anti-NaV1.1 Na+ channel antibody (K74/71) (1:200) (NeuroMab 75-023) as previously described.22 (link) Anti-α-tubulin (1:1000) (Cedar Lane) or anti-transferrin receptor (TfR) (1:200) (Invitrogen H68.4 13-6800) antibodies were used as loading control, as indicated. Immunoreactive bands were detected using Supersignal West Dura Extended Duration Substrate (Therma Scientific #34076) and imaged using an iBrightFL1000 system (Invitrogen). Immunoreactive signals for the deglycosylated bands were quantified using iBright analysis software (Invitrogen) and normalized to the level of α-tubulin for each sample.
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