Infrared secondary antibody

Protein was isolated from ipsilateral hippocampal tissue using TRIzol (Life Technologies, Carlsbad, CA, USA) according to manufacturer’s instructions. Equal amounts of protein samples were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked in Superblock (Pierce, Waltham, MA, USA) and incubated overnight at 4°C with primary antibodies. After washing, membrane was incubated with infrared secondary antibodies (LI-COR, Lincoln, NE, USA) for 1 h at room temperature. Imaging and densitometry was performed with an Odyssey Imaging System (LI-COR, Lincoln, NE, USA). The following antibodies and concentrations were used for Western blotting: suppressor of cytokine signaling 1 (SOCS1; 1:1000, 38-5200, Thermo, Waltham, MA, USA), caspase 3 (1:1000, 9662, Cell Signaling, Danvers, MA, USA), beta-actin (AM1829B, 1:20,000, Abgent, San Diego, CA, USA). For statistical analysis, each sample was normalized to beta-actin.

After lysis, SDS-PAGE, and transfer to polyvinylidene difluoride (PVDF) or nitrocellulose membranes, membranes were blocked with 7% milk in Tris-buffered saline (TBS) containing 0.1% Tween 20 or TBS blocking buffer (Li-COR, Cambridge, UK). Primary antibodies (Table 2) were diluted in blocking buffer (or Li-COR TBS blocking buffer + Tween 0.2%), incubated overnight at 4°C, and detected with either horseradish peroxidase (HRP)–conjugated secondary antibodies (Dako, California, USA) or infrared secondary antibodies (Li-COR) and incubated for 1 hour at room temperature. HRP-labeled secondary antibodies were detected by enhanced chemiluminescence (ECL; Pierce, ThermoFisher, Massachusetts, USA) and either exposure of membranes to x-ray film (GE Healthcare, Illinois, USA) and development in an SRX 101A processor (Konica Minolta, Basildon, UK) or detection with a ChemiDoc system (BioRad, California, USA). infrared secondary antibodies were detected using a Licor imager (Li-COR). Densitometric quantification of bands was performed in ImageJ.

Cells were lysed in radioimmunoprecipitation (RIPA) buffer (R0278, Sigma Aldrich) and total protein quantified by Bicinchoninic acid (BCA) assay. For the fractionation of soluble and polymerized tubulin, extraction buffer A (137 mM NaCl, 20 mM Tris–HCl, 1% Triton X-100, and 10% glycerol) was added to cells at 4 °C for 3 min, plates were gently swirled and the buffer removed and saved as the soluble tubulin fraction. Immediately after, extraction buffer B (buffer A + 1% sodium dodecyl sulphate (SDS)) was added for 1 min, the remaining sample was scraped, incubated on ice for 30 min and saved as the polymerized tubulin fraction.
SDS–PAGE was performed under reducing conditions and proteins transferred to nitrocellulose membranes. Membranes were blocked in odyssey blocking buffer (Li-Cor Cambridge, UK) prior to overnight incubation with primary antibodies and infrared secondary antibodies (Li-Cor). Proteins were visualized using the Li-Cor Odyssey and quantified using Image Studio Lite software (Li-Cor).