Mouse monoclonal anti ha antibody

RiboTag mice were injected intravitreally with AAV-Cre for labeling of RGCs. Subsequently, AAV-F-iTrkB was injected intravitreally. Purification of RGC ribosomes was performed as described previously,⁴⁴ (link)^,45 (link) with minor modifications. The mice were perfused transcardially with ice-cold phosphate-buffered saline (PBS), and retinas were dissected. Six retinas were pooled as one sample and were homogenized with a Dounce homogenizer in a homogenization buffer (50 mM Tris-HCl [pH 7.5], 100 mM KCl, 12 mM MgCl₂, 1% Nonidet P-40, 1 mM DTT, 200 U/mL RNasin, 100 μg/mL cycloheximide, 1 mg/mL heparin). The homogenates were centrifuged at 15,000 × g at 4°C for 15 min. The supernatant was incubated with a mouse monoclonal anti-HA antibody (1:50; Biolegend) at 4°C for 16 h. Ribosomes bound to an anti-HA antibody were purified using protein G magnetic beads (GE Healthcare). The RNA bound to ribosomes was purified with an RNeasy Protect Mini Kit (QIAGEN) according to the manufacturer’s instructions.

The anti-Rpd3 and anti-CBP antibodies were raised against the C-terminal 124 amino acids of Rpd3 and the N-terminal 1000 amino acids of CBP, respectively, by Japan lamb (Hukuyama, Hiroshima, Japan). Sera were collected and affinity-purified using a resin conjugated with the antigens. The anti-CoRest antibody raised against the amino acids 634–820 was provided by G. Mandel^{58 (link)}. The antibodies used in western blot analysis were rabbit anti-CoRest antibody at a 2000-fold dilution; mouse monoclonal anti-myc antibody (626802; BioLegend, San Diego, CA, USA) at a 500-fold dilution; rabbit monoclonal anti-HA antibody (3724; Cell Signaling Technology, Beverly, MA, USA) at a 1000-fold dilution; mouse monoclonal anti-HA antibody (901515; BioLegend, San Diego, CA, USA) at a 1000-fold dilution; rabbit anti-histone H3 antibody (ab1791; Abcam, Cambridge, MA, USA) at a 4000-fold dilution, and mouse anti-α-tubulin antibody (T6199; Sigma, St. Louis, MO, USA) at a 20,000-fold dilution.