Odyssey gel documentation system

To examine the uptake of ODNs, A375 cells were placed on glass coverslips in the presence of 4 µM 5′-Cy3-labelled CpG-1-PTO and nCpG-6-PTO for 24 h. Consecutively, cells were examined using a BZ-X800 fluorescence microscope (Keyence, Neu-Isenburg, Germany). Nuclei were stained with DAPI. Formation of G4 was tested as described [22 (link),23 (link)]. Briefly, 0.2 µg 5′-Cy5-labelled ODNs were mixed with 200 and 400 ng of the G4-specific antibody BG4 (Biozol ABA-AB00174-1.1, Eching, Germany) for 15 min at room temperature. After separation by 10% non-denaturing PAGE (100 V, corresponding to 14.7 V/cm) with 0.5× TBE, fluorescence was captured using the LI-COR Odyssey gel documentation system (Bad Homburg, Germany). Likewise, specific interaction with the IFNGR was tested by incubating the Cy3-labelled ODNs with either 200 ng or 400 ng IFNGR1 (R&D Systems) or IFNGR2 (Novus Biologicals, Centennial, CO, USA) before separation by PAGE.

For the isolation of cellular proteins, cells were lysed in RIPA lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.5% NP-40, 15% Glycerol, supplemented with 10 µg/ml of each aprotinin, leupeptin and pepstatin as well as 0.8 µM Pefabloc (Roche, Mannheim, Germany), 1 mM NaF, and 1 mM Na₃VO₄). The protein concentration of the lysates was determined using Bradford Assay according to manufacturer´s instructions (Carl Roth, Karlsruhe, Germany). Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Antigens were detected by incubation with specific primary antibodies (1:1,000) followed by incubation with DyLight-coupled secondary antibodies (1:10,000) (Licor, Lincoln, NE, USA). List of primary antibodies: (p)Y SHP2 (#3751), (p)Y STAT3 (#9145), STAT3 (#9139), (p)Y/T ERK1/2 (#4370), ERK1/2 (#4695) (Cell Signaling Technology); SOCS3 (#18391) (Immuno-Biological Laboratories, Fujioka, Japan); HSC70 (#7009) (Stress Marq, Victoria, Canada); SHP2 (#K0810) (Santa Cruz Biotechnology, Dallas, TEX, USA); Tubulin (#T5168) (Sigma-Aldrich Chemie, Munich, Germany). Detection was performed using an Odyssey gel documentation system (Licor). Analysis of Western blots was performed using Image Studio Lite (version 5.2, Licor).

Brains from mice at 61–65 days were dissected on the sagittal plane and flash frozen. Following weighing, samples were ribolysed in Dulbecco’s phosphate-buffered saline (DPBS) with ceramic homogenisation beads (Bertin Technologies) at max speed for 105 s to produce 10 and 20% homogenates which were stored at − 80 °C until use.
Homogenates were diluted in DPBS with 4× SDS sample buffer (250 mM Tris base, 40% glycerol, 8% SDS, 0.04% bromophenol blue) and boiled at 95 °C for 5 min. 35 μg of total protein was loaded onto a 4–12% (w/v) Bis-Tris polyacrylamide gel (Invitrogen) and electrophoresed before being electroblotted to a nitrocellulose membrane. Membranes were blocked in Odyssey Blocking Buffer for 1 h at room temperature (RT), before incubation with anti-syntaxin-6 (1:500 clone C34B2 (Cell Signalling; 2869S) or clone 3D10 (Abcam; Ab12370)) overnight at 4 °C. Membranes were washed with 0.05% Tween-20 in phosphate buffered saline (PBST) for 3 × 5 min with agitation and incubated with anti-β actin (1:1000 rabbit polyclonal (Sigma Aldrich; A2066) or 1:5000 mouse monoclonal (Sigma Aldrich; A5441)). After washing, membranes were probed with fluorophore-conjugated secondary antibodies (IRDye 800CW Donkey anti-rabbit IgG and IRDye 680RD goat anti-mouse IgG both at 1:4000) for 1 h at RT. Membranes were washed again and imaged with Odyssey Gel Documentation System (LI-COR; Model 9120).