Immun star

SEMA3A-GFP was purified from conditioned media using magnetic GFP-Trap beads (Chromotek, Germany). FLAG tagged proteins (SEMA3C-FLAG and SEMA3E-FLAG), and Myc tagged proteins (SEMA3F-AP) were immunoprecipitated from conditioned media using Dynabeads Protein A/G (Invitrogen, Australia) and 5 μg α-FLAG antibody (Sigma-Aldrich, Australia), or 5 μg α-Myc antibody (CellSignaling, USA). Proteins were eluted in 2X Laemmili Buffer (250 mM Tris, 10% (v/v) glycerol, 4% (w/v) SDS, 2% (v/v) β-mercaptoethanol, 0.005% (w/v) bromophenol blue, in DDW pH 6.8), separated by SDS-PAGE (Mini-PROTEAN TGX Stain-Free Precast Gels; Bio-Rad, Australia) and transferred to nitrocellulose membrane (Trans-Blot Turbo Mini Nitrocellulose Transfer Pack; Bio-Rad, Australia). Recombinant proteins were detected by western blot using 5% (w/v) skim milk powder in Tris buffered saline containing Tween (TBS-T; 100 mM Tris, 154 mM NaCl, 0.1% (v/v) Tween 20, in DDW pH 7.5) blocking buffer, α-GFP antibody (mouse monoclonal clones 7.1 and 13.1, 0.4 μg/mL; Roche, Australia), α-FLAG antibody (mouse monoclonal, 10 μg/mL; Sigma-Aldrich, Australia), α-Myc antibody (mouse monoclonal, 1:1,000 dilution; Cell Signaling, USA), and α-mouse-HRP secondary antibody (1:10,000 dilution; Peirce Scientific, Australia). Proteins were visualised by chemiluminescence (Immun-Star; Bio-Rad, USA) using the Chemi-Doc system (Bio-Rad, USA).

Protein bands were detected by incubation of the membranes in Immun-Star chemiluminescent reagent (BioRad, Hercules, CA, USA) for 5 min, followed by exposure to autoradiographic film (Thermo Scientific, Waltham, MA) for 2 min. Signal intensity was analyzed by densitometry using LabWorks software (UVP Laboratory Products, Upland, CA, USA). In some experiments, the chemiluminescent signal was directly imaged and quantitated using the iBright FL1500 imaging system (Thermo Scientific, Waltham, MA, USA).

Total cellular proteins were isolated from all six human thyroid cancer cell lines as previously described [47 (link)]. Protein concentrations were quantified by BCA Protein Assay Kit (Thermo Scientific, Waltham, MA). Denatured proteins (20μg) from each sample were resolved by gel electrophoresis (Bio-Rad Laboratories) and transferred to nitrocellulose membranes (Bio-Rad Laboratories). Protein-bound membranes were blocked in 5% nonfat milk solution and incubated with dysadherinmAb NCC-M53 antibodies, diluted 1:1000, overnight at 4°C. The next day, the nitrocellulose membranes were washed and incubated for 1 hour at room temperature with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, Beverly, MA). Immunoreactive protein bands were detected by Immunstar (Bio-Rad Laboratories).

For aldose reductase and fructokinase expression (Fig. 2), protein lysates were obtained after homogenization of renal cortical tissues (50 mg) in MAPK lysis buffer67 (link) containing 0.5% triton X-100, 50 mM β-glycerophosphate, 2 mM MgCl₂, 1 mM EGTA, 1 mM DTT and a cocktail of protease inhibitors (Roche). Homogenates were centrifuged in cold at 13,000 r.p.m. for 10 min and supernatants collected for protein quantification using the BCA assay (Pierce). Nuclear and cytosolic extracts were extracted from the kidney cortex using the nuclear/cytosol fractionation kit from Biovision. Pure nuclear and cytosolic fraction were determined by analysing the expression of specific markers (Lamin A/C for nucleus and tubulin for cytosol). Western blot was performed using specific primary antibodies at a concentration of 1:1,000 in TTBS as follows: aldose reductase (kind gift from Dr Mark Petrash at University of Colorado), fructokinase (Sigma, HPA007040), p65 and IκBα (Cell signaling, 8242 and 4814) and detected with HRP-conjugated antibodies (Cell Signaling) and Immun-star (Bio-Rad).

To confirm that both the BDNF antibodies bound specifically to BDNF we performed an absorption control test using recombinant BDNF protein (Human BDNF; PeproTech). First, BDNF protein was eluted in 2× Laemmli buffer [250 mm Tris, 10% (v/v) glycerol, 4% (w/v) SDS, 2% (v/v) β-mercaptoethanol, 0.005% (w/v) bromophenol blue (Sigma-Aldrich)] in double-deionized water (DDW; pH 6.8), separated by SDS-PAGE (Mini-PROTEAN TGX Stain-Free Precast Gels; Bio-Rad) and transferred to nitrocellulose membrane (Trans-Blot Turbo Mini Nitrocellulose Transfer Pack; Bio-Rad). Recombinant proteins were detected by Western blot using 3% (w/v) skim milk powder in TBS-T [100 mm Tris, 154 mm NaCl, 0.1% (v/v) Tween 20, in DDW, pH 7.5] blocking buffer, antibodies to BDNF (rabbit; Biosensis R-172-20, 1:500 or Santa Cruz Biotechnology, SC-546; 1:500) and α-rabbit-HRP secondary antibody (1:10,000 dilution; Pierce Scientific). Proteins were visualized by chemiluminescence (Immun-Star; Bio-Rad) using the ChemiDoc system (Bio-Rad). In the absorption control, the anti-BDNF antibody (1 µg, rabbit; Biosensis R-172-20) was incubated with 10 μg of BDNF diluted in 100 µl blocking agent and incubated overnight at 4°C. The pre-absorbed primary antibody was then used to immunostain purified RGC cultures using the protocol described above.