Rpn1231

Amersham ECL mouse IgG HRP-linked whole Ab; sheep polyclonal at 1:10 000 dilution (NA931V, Cytiva)
Streptavidin–horseradish peroxidase conjugate at 1:10 000 dilution (RPN1231, Cytiva)

Total cell lysates were boiled for 10 min at 99°C in 1× Laemmli buffer supplemented with 4.2% (vol/vol) β-mercaptoethanol. Proteins were separated by electrophoresis on precast 4 to 20% SDS-PAGE gels (Bio-Rad Laboratories) and semidry blotted on nitrocellulose membranes. Membranes were blocked with a 5% (wt/vol) lowfat milk solution in PBS. Proteins were detected with the following primary antibodies: rabbit anti-Flag (F7425; Sigma-Aldrich), rabbit anti-HA (ab9110; Abcam), and rabbit anti-MED25 (NBP2-55868; Novus Biologicals). As a control for equal loading, we used mouse anti-α-tubulin (T5168; Merck) or rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) (ab9485; Abcam). We used the following secondary antibodies: streptavidin-horseradish peroxidase (HRP) (RPN1231; Amersham Life Science), ECL donkey anti-rabbit IgG–HRP (NA934V, GE Healthcare), and ECL sheep anti-mouse IgG–HRP (NA931V; GE Healthcare). Protein bands were visualized by using Pierce ECL Western blotting substrate (32106; Thermo Fisher Scientific) and a chemiluminescence imager (Amersham Imager 600; GE Healthcare).

The Neurobiotin-injected tissues were fixed overnight at 4°C with 4% PFA and 1% glutaraldehyde (GA) in 0.067 M phosphate buffer (PB, pH 7.4) containing 1% saturated picric acid. After fixation, the brain was dissected and embedded in a gelatin/albumin mixture (4.8% gelatin and 28% albumin in distilled water) and then post-fixed overnight at 4°C in 7–8% PFA in PB. The post-fixed tissues were sectioned in a frontal plane at a thickness of 40 μm with a vibrating blade microtome, then washed in PB, and incubated overnight at 4°C with streptavidin-horseradish peroxidase (HRP, 1:200; RPN1231, Amersham Biosciences, Bucks, UK) in 0.01 M phosphate-buffered saline (PBS, pH 7.4) containing 0.5% Triton X (PBST). The sections were developed with a solution of 0.032% 3,3’-diaminobenzidine tetrahydrochloride (DAB) in 0.1M Tris-HCl (pH 7.4) containing 0.0145% H₂O₂ and 0.3% nickel ammonium sulfate. After washing in PB, the sections were mounted on a gelatin-coated glass slide, dehydrated in a graded ethanol series, cleared in xylene, and mounted in Mount-Quick (Daido Sangyo, Tokyo, Japan) beneath a cover slip.