Anti trkc

We used the following antibodies: anti-GFP (A11122, Life technologies), anti-GAPDH (sc-25778, Santa Cruz), anti-Histone (07–449, Millipore), anti-Flag (F3165, Sigma), anti-Actin (MAB1501R, Chemicon), anti-Hey1 (anti-HRT1, sc-16424, Santa Cruz), anti-p53 (SAPU, [53 (link)]), anti-Ptc (sc-6149, Santa Cruz), anti-TrkC (AF1404, R&D), anti-MDM2 (VMA00406, BioRad), anti-COBRA1 (F7E4, GeneTex), anti-BAX (sc-526, Tebu Bio), anti-BAK (G-23, Santa Cruz), anti-NT-3 (sc-547, Santa Cruz), and anticleaved PARP (9541T, Cell Signaling). The following antibodies were used for proximity ligation assays (DuoLink): anti-GFP (TP401, Biolabs), anti-Flag (F3165, Sigma), anti-Hey1 (anti-HRT1, sc-16424, Santa Cruz), anti-p53 (sc-126, Santa Cruz), and anti-importins (I1784, Sigma Aldrich).

Western blotting was performed as previously described [9 (link)] with rabbit anti-NGF, -BDNF, -NT-3, -TrkA, -TrkB, -p75^NTR and goat anti-sortilin Ab (Santa Cruz) or anti-TrkC (R&D Systems, Minneapolis, MN, USA) at 1/200 dilution. Signalling activation was studied by using rabbit anti-protein kinase B (Akt) and -phospho-Akt Ab and -phospho-extracellular signal-regulated kinase (Erk)1/2 and mouse anti-Erk1/2 Ab (at 1/1000 dilution; R&D Systems). Proteins (50 μg/lane) obtained from whole-cell lysates of TASMCs using lysis buffer [9 (link)] were separated on 10 to 12% SDS-polyacrylamide gels (Invitrogen, Carlsbad, CA, USA) and transferred onto PVDF membranes (Merck Millipore, Billerica, MA, USA). After saturation (5% nonfat dry milk, 0.1% Tween in PBS), Ab were incubated overnight at 4°C, and revealed after 1 h incubation at room temperature (RT) with anti-rabbit or -goat Ig-HRP-conjugated Ab (Santa Cruz, dilution 1/1000) by chemiluminescence (ECL reagent; Amersham, Little Chalfont, UK). Protein-loading control was performed with anti-actin Ab (Santa Cruz, 1/10,000). Western blots were scanned using a bioimaging system (Genesnap; Syngene, Cambridge, UK).

After performing antigen retrieval (10 mM citrate buffer with 0.05% Tween-20, pH 6.0, at 95 °C for 20 min) and blocking (5% goat serum plus 0.3% Triton X-100). Following that, the tissue was incubated with antibodies under different conditions. The resulting images were captured using a Zeiss LSM510 confocal microscope. To estimate the cell profile, every third section was stained with DAPI to visualize the nucleoli. The validity of the method was confirmed through several control experiments. The following primary antibodies were used: Anti-α-tubulin (CST, 3873, 1:1000), Anti-acetyl-α-tubulin (CST, 5335, 1:800), anti-TrkA (Millipore,06-574, 1:200), anti-TrkB (Santa Cruz Biotechnology, sc-377218, 1:200), anti-TrkC (R&D systems, AF1404,1:200), anti-Tuj1 (Abcam, ab78078, 1:1000), anti-S100β (Sigma, Sab4200671,1:100), anti-DOK6 (Oasisbiofarm, epitope:1624, 1:250), anti-NF200 (Abcam, ab4680,1:1000), anti-MBP (RayBiotech, Aa82-87,1: 1000), anti-MPZ (Sigma, ABN363,1:2000). Secondary antibodies were Alexa Fluor 488 (goat anti-mouse, A-11001; goat anti-rabbit, A-11008); and 594 (goat anti-rabbit, A-11012; goat anti-mouse, A-11005) (Invitrogen) with dilution: 1:1000.