Peroxidase conjugated donkey anti goat secondary antibody

HEK293T cells were grown in 60 mm culture dishes containing 3 mL DMEM supplemented with 10% FCS, L-Glutamine and Pen-Strep, and transfected using linear polyethylenimine (PEI) (PolysciencesInc, Eppelheim, Germany) as described earlier [29 ]. Cell lysates were prepared in buffer D (20 mM HEPES, 125 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, and 1% Triton X-100, pH 7.6, supplemented with 1 mM PMSF and 10 μg/mL aprotinin). 20 μg protein lysate was separated by 10% SDS-PAGE and blotted onto nitrocellulose membrane. Ponceau staining was used to reveal equal protein loading and subsequent quantification. Blots were blocked with 5% (v/v) chicken egg yolk in TBST (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.05% (v/v) Tween-20) for 1 h at room temperature. K_IR2.1 WT and mutants were detected by N-terminal K_IR2.1 antibody (Santa Cruz Biotechnology, Dallas Tx, USA, cat. No. sc-18708) and peroxidase-conjugated donkey anti-goat secondary antibody (Jackson ImmunoResearch, West Grove, PA, cat. No. 705-035-003) followed by ECL detection procedure (GE Healthcare, Hoevelaken, The Netherlands). For quantification purposes, untransfected HEK293T cells were used as blank and protein levels were normalized to ponceau staining levels. Differences between group averages were tested by using a one-way ANOVA with a Bonferroni’s post-hoc test.

Western blotting was performed as described in our previously published study (Huang et al. 2020 ). The protein concentration of each sample was measured using a BCA Protein Assay Kit, and then the samples were heated for 5 min at 99 °C. Next, 30 µg of protein was loaded in each well of a 10% polyacrylamide gel, and then the proteins on the gel were transferred to a polyvinylidene fluoride membrane. The PVDF membrane was incubated in 5% skimmed milk for 1 h, washed and incubated overnight at 4 °C with the following primary antibodies: rabbit anti-TLR4 (Cell Signaling Technology (CST); 1:1000), rabbit anti-phospho-p44/42 MAPK (Erk1/2) (CST; 1:2000), rabbit anti-p44/42 MAPK (Erk1/2) (CST; 1:2000), rabbit anti-phospho-NF-κB p65 (CST; 1:1000), rabbit anti-NF-κB p65 (CST; 1:1000), rabbit anti-Iba1 (FUJIFILM Wako Chemicals; 1:500), mouse anti-GFAP (CST; 1:2000) and rabbit anti-GAPDH (CST; 1:5000) followed by peroxidase-conjugated goat anti-mouse or anti-rabbit secondary antibodies (1:3000; Jackson ImmunoResearch) and a peroxidase-conjugated donkey anti-goat secondary antibody (1:2000; Jackson ImmunoResearch). Finally, the proteins were visualized using Western peroxide reagent and Clarity Western ECL Substrate (Bio-Rad) and the ChemiDoc XRS system (Bio-Rad) with Image Lab software. NIH ImageJ software was used to quantify the intensity of the bands by measuring the optical density.