Rabbit anti trka

At the conclusion of experimentation, subjects were anesthetized (n = 8 subjects per group), and tissue collected as previously described.10 Free‐floating basal forebrain samples (every sixth section; approximately Bregma: 1.60‐0.20 mm based on the atlas of Paxinos and Watson28) were processed as previously described.10 Briefly, sections were incubated in a primary antibody solution containing goat anti‐ChAT (Millipore, Temecula, CA, Cat. #AB144P), rabbit anti‐TrkA (Millipore, Cat. #06‐574), mouse anti‐p75^NTR (Millipore, Cat. #MAB365), mouse anti‐NeuN (Millipore, Cat. #MAB377), rabbit antiphosphorylated NF‐κB p65 Ser 536 (pNF‐κB p65; Abcam, Cambridge, MA, Cat. #ab86299), or mouse anti‐BrdU (Millipore, Cat. #MAB3424) for 24 hours at 4°C. For BrdU immunohistochemistry, DNA was additionally denatured by incubation in 2 N HCl for 30 minutes at 37°C followed by incubation in 0.1 M boric acid for 10 minutes at room temperature (pH: 8.5). The chromogen, nickel‐enhanced diaminobenzidine (Sigma‐Aldrich) was used to visualize immunoreactivity.

Forty µg of hippocampal protein per sample was subjected to PAGE electrophoresis using 4–12% Bis-Tris Midi Gel (Invitrogen) and transferred to a blotting membrane with the iBlot system (Invitrogen). The membrane probed with Goat anti-CHAT (1∶1000, Millipore) was blocked with Western Blocker Solution (Sigma). All other membranes were blocked with 5% milk in TBS/1.5% Tween (TBS-T), washed with TBS-T, and probed overnight with either rabbit anti-p75NTR (1∶3000, Advanced Targeting Systems), mouse anti-GFAP (1∶1000, Cell Signaling Technology), rabbit anti-TrkA (1∶1000, Millipore), rabbit anti-DCX (1∶1000, Cell Signaling Technology), rat anti-ALK-1 (1∶1000, R & D Systems), rabbit anti-BMP9 (1∶1000, Abcam), or mouse anti-β-actin (1∶5000, Sigma). Following incubation with the primary antibody, blots were incubated in species-specific anti-IgG-HRP: anti-Rabbit-HRP (1∶4000, Bio-Rad), anti-Goat/Sheep-HRP (1∶2000; Sigma), or anti-mouse-HRP (1∶2000, Bio-Rad).
Reactive bands were detected with SuperSignal West Femto chemiluminescent substrate (Pierce, Rockford IL). Chemiluminescence was captured with a Kodak ImageStation 440CF and the band intensities were quantified with Kodak 1D Image Analysis software.

Western blots were performed as previously described28 (link). The primary antibody [anti-ILK 1:1000; anti-phospho-Akt (Ser⁴⁷³)1:1000; anti-phospho-Akt (Thr³⁰⁸) 1:1000; anti-Akt 1:1000] and peroxidase-labeled secondary antibody (1:2000 dilution) were purchased from Cell Signaling Technology (Berverly, MA). The rabbit anti-TrkA, anti-TrkB antibodies and anti-p75^NTR antibody were purchased from Millipore (1:1000 dilution, Temecula, CA). The blot was developed with chemiluminescent substrate (Santa Cruz Biotechnology, Santa Cruz, CA). To control for loading efficiency, the blots were stripped and re-probed with α-tubulin antibody (1:1000 dilution; Abcam, Cambridge, MA) and expression levels of total ILK and phospho-Akt proteins were normalized to that of α-tubulin.

Free-floating FSC sections were processed similar to previously reported methods (Vetreno and Crews, 2018 (link); Vetreno et al., 2019 (link)). To assess cholinergic neuron marker colocalization, sections were incubated for 48 h at 4°C in a primary antibody cocktail containing goat anti-ChAT (Millipore), rabbit anti-TrkA (Millipore, Cat. #06-574, RRID:AB_11213262), and mouse anti-nerve growth factor receptor (NGFR; Millipore, Cat. #MAB365, RRID:AB_2152788). To assess ChAT colocalization with phosphorylated (activated) NF-κB p65, FSC sections were incubated for 48 h at 4°C in a primary antibody cocktail containing goat anti-ChAT (Millipore) and rabbit anti-pNF-κB p65 (phospho S536; Abcam, Cat. #ab86299, RRID:AB_1925243). Sections were then washed in TBS and incubated for 2 h at room temperature in the secondary antibody cocktail (Invitrogen; rabbit Alexa Fluor 594 [Cat. #A21207, RRID:AB_141637], mouse Alexa Fluor 488 [Cat. #A21202, RRID:AB_141607], and goat Alexa Fluor 350 [Cat. #A21081, RRID:AB_2535738]). Tissue was mounted onto slides and cover slipped using Prolong Gold Anti-Fade mounting media (Life Technologies, Grand Island, NY, United States). Immunofluorescent images were obtained using a DS-RiZ scope (Nikon Inc., Melville, NY, United States), and colocalization and pNF-κB p65 + IR cells quantified using NIS Elements AR46 (Nikon Inc.).

The following antibodies were used in immunoblotting and immunofluorescence experiments: rabbit anti-human p75^NTR (1:1,000; G3231; Promega), rabbit anti-TrkA (1:1,000; Millipore), rabbit anti-phosphoTyr674/5 (1:1,000; Cell Signaling), mouse anti-HA (1:2,000; Sigma-Aldrich), mouse anti-FLAG M2 (1:1,000; Sigma-Aldrich), mouse anti β-actin (1:1,000; Sigma-Aldrich), rabbit MBP-probe (1:1,000; Santa Cruz), rabbit anti-Cleaved Caspase-3 (1:1,000, 9661S; Cell Signaling), rabbit anti phospho-p38 (1:1,000, 9211; Cell Signaling), rabbit anti p38 (1:1,000, 9212; Cell Signaling), rabbit anti JNK (1:1,000, 9252; Cell Signaling), rabbit anti phospho-JNK (1:1,000, 9251; Cell Signaling), goat anti-choline acetyltransferase (1:200, AB144P; Millipore), rabbit anti Cy3 (1:500; Jackson), goat anti mouse Ig/HRP (1:10,000; Jackson), goat anti rabbit Ig/HRP (1:10,000; Jackson), goat IRDye800 (1:15,000; Rockland), and goat anti-mouse antibodies coupled to either Alexa 555 or Alexa 488 (Invitrogen). The DNA was stained with DAPI (1:1,000).

Western blotting has been described previously [70 (link)]. Antibodies used included: rabbit anti-TRKA (#06-574; 1:1000) and mouse anti-pY (#05-1050; 1:2000) from Millipore; rabbit anti-TRK (#4609; 1:500) and rabbit anti-phospho-TRKA (#9141; 1:1000) from Cell Signaling; mouse anti-V5 (#R960-25; 1:5000) from Invitrogen; mouse anti-GAPDH (#G8795; 1:5000) from Sigma-Aldrich.
N-linked glycosylation was inhibited from 6 h after transfection by adding tunicamycin (2 µg/ml; AppliChem) and cells were lysed 9 h later in RIPA buffer.
Immunoprecipitation, immunofluorescence and confocal microscopy were done as described previously [70 (link)].

Cells were washed in PBS and lysed in cold lysis buffer (50 mM Tris–HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.1% Triton X-100, 1 mM PMSF, 10 mM NaF, 1 mM Na₂VO₃, 10 mM iodoacetamide, and protease inhibitor cocktail) at 4 °C. Cellular debris was removed by centrifugation at 13,000g for 15 min, and protein quantification was performed by Bradford assay. Proteins were resolved in reducing and nonreducing SDS-PAGE gels, and membranes were incubated overnight at 4 °C with the following antibodies: rabbit polyclonal antihuman p75 ICD (1:1000 dilution; Promega); mouse monoclonal anti-HA (1:2000 dilution; Sigma); rabbit polyclonal MBP probe (1:1000 dilution; Santa Cruz); rabbit anti-phosphoTyr674/5 (1:1000 dilution; Cell Signaling); and rabbit anti-TrkA (1:1000 dilution; Millipore). Following incubation with the appropriate secondary antibody, membranes were imaged using enhanced chemiluminescence and autoradiography.