One hour western detection kit

A Qubit Protein Assay Kit was used for protein extraction; total protein amount was measured using a Qubit 3.0 Fluorometer (Thermo Fisher Scientific). Equal amounts of protein (15 μg) from each sample, or 2 ng of artificial truncated IL-33 (amino acids 112–270, 20 kDa) (Recombinant Human IL33 carrier-free, Biolegend, San Diego, CA, USA) used as positive control to confirm that the bands correspond to IL-33 full length (amino acids 1–270, 34–30 kDa), to the cleaved inactive forms (amino acids 1–178, 22–20 kDa; amino acids 179–270, 13–12 kDa) [2 (link), 3 (link)] or, finally, to the cleaved active forms (amino acids 95–270, amino acids 99–270, and amino acids 109–270; 19–15 kDa) [4 (link)], were separated by 10% SDS-PAGE and transferred to PVDF membranes (Genscript). A pre-stained marker, broad range 11–190 KDa (Cell signaling, Danvers, MA, USA), was also used, PVDF membrane were treated by ONE-HOUR Western detection kit (Genscript) and proteins were visualized using a Chromosensor TMB substrate (Genscript). After protein transfer, PVDF membranes were incubated with a pretreatment solution for 5 min RT and then with the WB solution and an α-human IL-33 Ab (Nessy-1) (Abcam, Cambridge, UK) for 40 min. After three washes, membranes were developed with a TMB substrate.

First, a HisTrap HP (GE Healthcare) 1 mL column was equilibrated with a buffer containing 20 mM phosphate buffer pH 7.5, 300 mM NaCl, 10 mM imidazole, 2 mM TCEP, and 0.4% NG. Purified PIP was bound to the column and all unbound PIP was washed off with the buffer. Next, full-length AQP5 without His-tag was looped over the column overnight after which any unbound AQP5 was washed. Proteins were eluted with 300 mM (sample E1) and 400 mM (sample E2) imidazole. The peak fractions were concentrated and analyzed on SDS-PAGE with SYPRO Ruby total protein staining and glycosylation specific staining. A WB was performed using antibodies against the 6× His-tag on PIP and the signal was detected using the ONE-HOUR western Detection Kit (GenScript, Piscataway, NJ, USA).

Mouse tissues (muscle and brain) were homogenized in lysis buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, halt proteinase inhibitor cocktail (ThermoFisher Scientific), and halt phosphatase inhibitor cocktail (ThermoFisher Scientific), and subjected to immunoprecipitation with anti-BCL2 monoclonal antibody conjugated agarose beads (Santa Cruz Biotechnology 7382 AC). Eluates were separated by SDS-PAGE and detected by anti-BCL2-HRP antibody (C2 Santa Cruz Biotechnology, 1:500) and anti-BECN1 antibody (Santa Cruz Biotechnology, 1:200) using the ONE-HOUR Western Detection Kit (GenScript Corporation) following the manufacturer’s instruction.