Horseradish peroxidase hrp conjugated anti rabbit igg antibody

Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane and probed with rabbit anti-mouse Wnt3a (1:6000 dilution, Abcam). Positive bands were detected with a horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody (1:5000 dilution, Jackson Immuno Research). The amount of Wnt3a present in samples was calculated from a standard curve generated with isolated Wnt3a [12 (link)].

To check PJA1 protein expression, brains were sampled from 9‐ to 10‐week‐old mice (N = 4 WT and 4 Pja1^KI/Y and N = 3 WT and 3 Pja1^KO/Y). One hemisphere per sample was homogenized in ice‐cold 1X phosphate‐buffered saline (PBS) supplemented with protease inhibitors (Complete, Roche). Homogenates were centrifuged at 20,000g for 15 min. 20 μg of proteins was separated on 5–20% gradient SDS‐polyacrylamide gel (Super Sep Ace, Wako pure reagents) and performed Western blotting. The membrane was processed through sequential incubations with primary anti‐PJA1 rabbit polyclonal antibody (1:200 dilution, 17687‐AP, Proteintech) overnight at 4°C and then with secondary horseradish peroxidase (HRP) conjugated anti‐rabbit IgG antibody (1:10,000 Jackson Immuno Research). Labeled proteins were revealed using enhanced chemiluminescence (ECL) detection (Perkin‐Elmer). Membranes were re‐probed with anti‐GAPDH rabbit antibody (1:2,000; Santa Cruz Biotechnology) and revealed as described earlier. Band intensities were quantified using NIH ImageJ software (National Institute of Health).

Neutrophil lysates heated under reducing conditions were applied to 7.5% sodium dodecyl sulfate–polyacrylamide gels and electrophoresed. After transfer onto a polyvinylidene difluoride membrane, the membrane was soaked in PBS-T solution containing 3% bovine albumin serum to avoid nonspecific antibody binding. Immunoblotting was conducted using anti-Btk antibody (1:1000 dilution; Cell Signaling), anti-phosphorylated Btk antibody (1:1000 dilution; Cell Signaling), anti-Vav antibody (1:3000 dilution; GeneTex, Irvine, CA, USA), and anti-phosphorylated Vav antibody (1:1000 dilution; GeneTex) as primary antibodies. These rabbit polyclonal antibodies were reacted at 4 °C overnight. After washing with PBS-T, the membrane was allowed to react with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody (1:10,000 dilution; Jackson ImmunoResearch, West Grove, PA, USA) as a secondary antibody for 1 h at RT. After washing with PBS-T, antibody binding was detected using a chemiluminescent substrate and ImageQuant LAS4000 (Cytiva, Tokyo, Japan).