Nupage lds 2x sample buffer

The cell extracts were centrifuged at 8000× g for 10 min at 4 °C. The supernatant was precleared by incubation for 20 min with 40 µL of 50% slurry of Protein G-conjugated to Sepharose (Invitrogen, Carlsbad, CA, USA) on ice before centrifugation at 15,300× g at 4 °C. The supernatant was incubated overnight in primary antibody (HIF-1α) at 4 °C. Protein G-conjugated to Sepharose (30 µL 50% slurry) was added with end-over-end mixing for 2 h at 4 °C. The immunoprecipitate on beads was washed once with 1 mL ice cold HEPES-buffered saline (150 mM NaCl; 10 mM HEPES, pH 7.4). Protein was eluted with 30 µL NuPAGE LDS 2x sample buffer (Invitrogen) including 10% reducing agent for 5 min at 90 °C. Cell extracts (1.5%) or immunoprecipitants were immunoblotted and developed with Bio-Rad Clarity ECL (Bio-Rad Laboratories, Hercules, CA, USA) and a ChemiDoc Imaging System (Bio-Rad Laboratories).

Brain tissue homogenates in TSB buffer (crude) were centrifuged at 14,000 rpm for 10 min (Centrifuge 5424R, Eppendorf). The supernatant (S1) was added with TSB to a final volume of 600 µl and sonicated (40 pulses of 0.5 s, amplitude 40%, 20 kHz). The brain tissue homogenates were then spun at 49, 000 rpm for 1 h (Optima TLX ultracentrifuge equipped with a TLA-110 rotor, Beckman). The supernatant was collected, and a pellet was resuspended in 600 µl of a Tris-Triton (2%) solution (Tris-HCl 10 mM pH 7.4, 2% Triton X-100) (S2). The S2 samples were sonicated and spun at 49, 000 rpm for 1 h. The resulting S3 supernatant was recovered, and the pellet (C3) was resuspended in one volume of NuPAGE™ LDS 2X Sample Buffer supplemented with NuPAGE™ Sample Reducing Agent (10x) (Invitrogen), following the manufacturer’s instructions. The NUPAGE™ Western blot protocol was applied, and 8 μL of crude, 10 μL of S1, 15 μL of S2 and S3, and 20 μL of C3 were loaded per well. Western blot signals were acquired using the LAS-3000 (Fuji), and protein expression levels were determined using ImageQuantTL software. Results (n = 4 per group of animals) were expressed as the ratio of the protein in the insoluble fraction divided by the protein signal detected in the soluble fraction plus that measured in the insoluble fraction.