Anti p75ntr, by Promega - Product details

1

Proximity Ligation Assay for p75NTR-TRAF6 Interaction

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Four DIV WT CGNs were treated with 500 μM AraC for 10 min. After treatment, CGNs were fixed for 15 min in 4% paraformaldehyde (PFA)/4% sucrose, permeabilized, and blocked in 10% normal donkey serum and 0.3% Triton X-100 in PBS for 1 h. Neurons were then incubated overnight at 4 °C with anti-p75^NTR (Promega; G323A; 1:500) and anti-TRAF6 (Santa Cruz; sc-8490; 1:100) antibodies in PBS supplemented with 3% BSA. The Duolink In Situ Proximity Ligation kit (Sigma; DUO92007) was used as per the manufacturer’s instructions. Cells were imaged with an LSM Imager Z2 confocal microscope (Zeiss) to detect PLA signals. PLA puncta were quantified using NIH ImageJ software with the plugin particle analyser.

Lopes-Rodrigues V., Boxy P., Sim E., Park D.I., Habeck M., Carbonell J., Andersson A., Fernández-Suárez D., Nissen P., Nykjær A, & Kisiswa L. (2023). AraC interacts with p75NTR transmembrane domain to induce cell death of mature neurons. Cell Death & Disease, 14(7), 440.

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2

Western Blot Analysis of Myelination Proteins

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Samples from cultures and mouse sciatic nerves were prepared for Western blot analysis as previously described (Lewallen et al., 2011). Briefly, samples were dounced in RIPA buffer containing: 50 mM Tris pH 8.0, 150 mM NaCl, 2 mM EDTA, 1 mM Na3V04, 2.5 mM NaF, Complete protease inhibitor (Roche), 1% NP-40, 0.5 % Na deoxycholic acid, 1 mM PMSF and 0.1% SDS for Western analysis. Electrophoretic separation of the samples and the extracts was performed on 10% SDS polyacrylamide gels. Proteins were transferred to nitrocellulose membranes and probed with specific antibodies. Antibodies for Western blot analysis: rat monoclonal anti-MBP (Millipore), mouse monoclonal anti-P0 (Astexx), mouse monoclonal anti-β-actin (Sigma), rabbit polyclonal anti-Oct-6 (provided by Dr. Dies Meijer), rabbit polyclonal anti-Oct-6 (provided by Dr. Dies Meijer), mouse monoclonal anti-LKB1 (Abcam), mouse monoclonal anti-p-LKB1 (Santa Cruz), rabbit polyclonal anti-p75^NTR (Promega). The Alexa Fluor goat antirabbit, anti-mouse, and anti-rat 680 IgG antibodies (Invitrogen) were used as secondary antibodies for near-infrared fluorescent detection performed on the Odyssey Infrared Imaging System (LI-COR). Full Western blots from all the figures are displayed in Supplementary Figure 3.

Shen Y.A., Chen Y., Dao D.Q., Mayoral S.R., Wu L., Meijer D., Ullian E.M., Chan J.R, & Lu Q.R. (2014). Phosphorylation of LKB1/Par-4 Establishes Schwann Cell Polarity to Initiate and Control Myelin Extent. Nature communications, 5, 4991.

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3

Immunocytochemistry of Neural Markers

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The following primary antibodies were used: anti-MBP (rat, 1:800, Chemicon),
anti-neurofilament (mouse, 1:800, SMI31, Covance), anti-Olig1 (rabbit, 1:1000,
Abcam), anti-p75^NTR (rabbit, 1:500, Promega), anti-S100 (rabbit,
1:500, Dako).
The following secondary antibodies were used: donkey-anti-rabbit AlexaFluor488,
donkey-anti-mouse DyLight549, donkey-anti-rat AlexaFluor647 (all 1:500, Jackson
ImmunoResearch Laboratories), DAPI (4′,6-diamidino-2-phenylindole)
(1.25 μg/ml; Molecular Probes) was used as cellular counter
stain.

Schmid D., Zeis T, & Schaeren-Wiemers N. (2014). Transcriptional regulation induced by cAMP elevation in mouse Schwann cells. ASN NEURO, 6(3), e00142.

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4

Protein expression and signaling assays

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Anti-p75^NTR (Cat. # G323A; 1:1000 dilution) was purchased from Promega (Madison, WI, USA). Anti-RhoA (Cat. # 05-778; 1:1000 dilution) and agarose conjugated rhotekin- Rho-binding domain (RBD) was purchased from Millipore (Billerica, MA, USA). Phospho-p38MAPK (Cat. # 9221; 1:1000 dilution) and non-phospho p38MAPK antibodies (Cat. # 9212; 1:1000 dilution), JNK (Cat. # 9252S; 1:1000 dilution) and phospho JNK (Cat. # 9251S; 1:1000 dilution), cleaved caspase-3 antibodies (Cat. # 9664; 1:1000 dilution) were purchased from Cell signaling (Danvers, MA, USA). Cleaved poly ADP-ribose polymerase (PARP) antibody (Cat. # 556494; 1:1000 dilution) was obtained from BD Bioscience Pharmingen (San Diego, CA, USA). ProNGF was obtained from Alomon (Israel), NGF (Cat. # 04-1142) from Bioproducts for science (Indianapolis, IN, USA), and Y-27632 (Cat. # 10005583) was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Enhanced chemiluminescence (Cat. # 32106) was from Thermo Scientific (Waltham, MA, USA), MTT Cell Proliferation Assay Kit (Cat. # ab211091) was procured from abcam (Cambridge, MA, USA), and all other reagents were obtained from Sigma-Aldrich.

Sycheva M., Sustarich J., Zhang Y., Selvaraju V., Geetha T., Gearing M, & Babu J.R. (2019). Pro-Nerve Growth Factor Induces Activation of RhoA Kinase and Neuronal Cell Death. Brain Sciences, 9(8), 204.

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5

p75NTR Thermal Stability Assay

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The protein lysate from 293 T HEK cells constitutively expressing p75^NTR protein was homogenised in the same buffer as described above for CETSA and incubated with different concentrations of AraC (0, 0.1, 1, 30, 100, 300, 500 and 1000 μM) for 3 min at RT. The protein extracts were heated at a constant temperature of 37 or 53 °C for 3 min. After subsequent cooling on ice for 3 min, the heat-treated lysate was centrifuged (20,000g, 4 °C, 20 min), followed by supernatant collection. Samples were then subjected to immunoblotting and probed for anti-p75^NTR (Promega, Cat: G323A, 1:300) and anti-GAPDH (Sigma, Cat: G8795, 1:1000). Densitometry data acquired from 37 °C ITDR-CETSA were used as non-denaturing controls to normalise those from ITDR-CETSA conducted at 53 °C. The densitometry was done on the immunoblot bands, and restricted cubic spline curve fitting was generated using GraphPad Prism 9 (GraphPad Software, Inc., La Jolla, CA, USA).

Lopes-Rodrigues V., Boxy P., Sim E., Park D.I., Habeck M., Carbonell J., Andersson A., Fernández-Suárez D., Nissen P., Nykjær A, & Kisiswa L. (2023). AraC interacts with p75NTR transmembrane domain to induce cell death of mature neurons. Cell Death & Disease, 14(7), 440.

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6

Identifying p75NTR-Interacting Proteins

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For the detection of p75^NTR-interacting proteins, wild-type and mutant p75^NTR peptides, or a random peptide (control), were labeled on the amino terminus with biotin (synthesized by CanPeptide, Montréal, QC, Canada). U87 glioma cells were lysed as described above and incubated with biotin-labeled peptides for 1 h at 4 °C. NeutrAvidin agarose (Thermo Scientific, Rockford, lL, USA) was added and incubated for 1 h. Peptide bound to the agarose beads was centrifuged, washed with PBS and boiled with 2 × Laemmli buffer. Proteins that bound to each peptide were resolved on a 10% sodium dodecyl sufate–polyacrylamide gel electrophoresis gel and silver-stained as described previously^{95 (link)} or transferred to nitrocellulose for western blot analysis. Proteins that bound to the wild-type p75^NTR peptide and not to the mutant peptides were excised from the gel, and processed LC-MS/MS analysis as described.^{95 (link)} A similar procedure was followed for the detection of serine phosphorylation on the p75^NTR mutant and wild-type receptors following immunoprecipitation with anti-p75^NTR (Promega).

Ahn B.Y., Saldanha-Gama R.F., Rahn J.J., Hao X., Zhang J., Dang N.H., Alshehri M., Robbins S.M, & Senger D.L. (2015). Glioma invasion mediated by the p75 neurotrophin receptor (p75NTR/CD271) requires regulated interaction with PDLIM1. Oncogene, 35(11), 1411-1422.

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7

Western Blot and Immunoprecipitation Analysis

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Cells were lysed with RIPA buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% (v/v) NP-40, protease inhibitor cocktail (Roche, Indianapolis, IN, USA), phosphatase inhibitor (Sigma)), protein quantification was determined, and proteins were resolved by sodium dodecyl sufate–polyacrylamide gel electrophoresis. Western blot analysis was performed using rabbit polyclonal anti-human p75^NTR intracellular domain (Promega), mouse monoclonal anti-β-actin (Millipore, Billerica, MA, USA) or mouse monoclonal PDLIM1 (Abcam, Cambridge, MA, USA) with the appropriate HRP-conjugated secondary antibodies (SantaCruz Biotechnology, Santa Cruz, CA, USA). For immunoprecipitation experiments, whole-cell lysates were incubated with the indicated antibody in the presence of Sepharose-Protein A/G (Sigma). Immunoprecipitates were boiled with 2 × Laemmli buffer, resolved by sodium dodecyl sufate–polyacrylamide gel electrophoresis and western blotting were performed using anti-p75^NTR (Promega), anti-PDLIM1 (Abcam) or anti-HA (Abcam) antibodies.

Ahn B.Y., Saldanha-Gama R.F., Rahn J.J., Hao X., Zhang J., Dang N.H., Alshehri M., Robbins S.M, & Senger D.L. (2015). Glioma invasion mediated by the p75 neurotrophin receptor (p75NTR/CD271) requires regulated interaction with PDLIM1. Oncogene, 35(11), 1411-1422.

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8

Western Blot Analysis of Myelination Proteins

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Samples from cultures and mouse sciatic nerves were prepared for Western blot analysis as previously described (Lewallen et al., 2011). Briefly, samples were dounced in RIPA buffer containing: 50 mM Tris pH 8.0, 150 mM NaCl, 2 mM EDTA, 1 mM Na3V04, 2.5 mM NaF, Complete protease inhibitor (Roche), 1% NP-40, 0.5 % Na deoxycholic acid, 1 mM PMSF and 0.1% SDS for Western analysis. Electrophoretic separation of the samples and the extracts was performed on 10% SDS polyacrylamide gels. Proteins were transferred to nitrocellulose membranes and probed with specific antibodies. Antibodies for Western blot analysis: rat monoclonal anti-MBP (Millipore), mouse monoclonal anti-P0 (Astexx), mouse monoclonal anti-β-actin (Sigma), rabbit polyclonal anti-Oct-6 (provided by Dr. Dies Meijer), rabbit polyclonal anti-Oct-6 (provided by Dr. Dies Meijer), mouse monoclonal anti-LKB1 (Abcam), mouse monoclonal anti-p-LKB1 (Santa Cruz), rabbit polyclonal anti-p75^NTR (Promega). The Alexa Fluor goat antirabbit, anti-mouse, and anti-rat 680 IgG antibodies (Invitrogen) were used as secondary antibodies for near-infrared fluorescent detection performed on the Odyssey Infrared Imaging System (LI-COR). Full Western blots from all the figures are displayed in Supplementary Figure 3.

Shen Y.A., Chen Y., Dao D.Q., Mayoral S.R., Wu L., Meijer D., Ullian E.M., Chan J.R, & Lu Q.R. (2014). Phosphorylation of LKB1/Par-4 Establishes Schwann Cell Polarity to Initiate and Control Myelin Extent. Nature communications, 5, 4991.

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Anti p75ntr

Lab products found in correlation

8 protocols using anti p75ntr

Proximity Ligation Assay for p75NTR-TRAF6 Interaction

Western Blot Analysis of Myelination Proteins

Immunocytochemistry of Neural Markers

Protein expression and signaling assays

p75NTR Thermal Stability Assay

Identifying p75NTR-Interacting Proteins

Western Blot and Immunoprecipitation Analysis

Western Blot Analysis of Myelination Proteins

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