Mouse anti ha

First, RNA interference in S2 cells was performed with 20µg of dsRNA targeting GFP, BRWD3, or Cul4 for two days in six-well plates as described above. Cells were then transfected with 200μg/mL HA-KDM5-expressing plasmid DNA using the Effectene II (Invitrogen) kit according to the manufacturers protocol. After two days, cells were treated with the translation inhibitor Cycloheximide (Sigma) dissolved in DMSO at 1mg/mL. Cells were harvested at different time points and centrifuged and lysed in 50μL 2 x Laemilli sample buffer (with 50 mM DTT) and boiled for 5 minutes. To inhibit Cullin E3 ubiquitin ligase activity, cells were treated with 50μM of the Nedd8 inhibitor MLN4924 (Selleckchem). To measure KDM5-HA protein levels, 5μL of each sample was used for Western blot analysis with anti-mouse HA (Cell signaling, 1:1000).

First, RNA interference in S2 cells was performed with 20 µg of double-stranded RNA (dsRNA) targeting GFP, BRWD3, or Cul4 for 2 d in six-well plates as described above. Cells were then transfected with 200 μg/mL HA-KDM5-expressing plasmid DNA using the Effectene II (Invitrogen) kit according to the manufacturer’s protocol. After 2 d, cells were treated with the translation inhibitor CHX (Sigma) dissolved in dimethyl sulfoxide (DMSO) at 1 mg/mL. Cells were harvested at different time points, centrifuged, and lysed in 50 μL 2× Laemmli sample buffer (with 50 mM DTT) and boiled for 5 min. To inhibit Cullin E3 ubiquitin ligase activity, cells were treated with 50 μM of the Nedd8 inhibitor MLN4924 (Selleckchem). To measure KDM5-HA protein levels, 5 μL of each sample was used for western blot analysis with anti-mouse HA (Cell Signaling, 1:1,000).

Gonads were dissected from 1-day adults of him-17::3xHA worms in 1X sperm salts (50 mM PIPES pH 7.0, 25 mM KCl, 1 mM MgSO4, 45 mM NaCl, and 2 mM CaCl2) with 1 mM levamisole and fixed in 1% paraformaldehyde diluted in 1X sperm salts for 5 min in a humid chamber. Slides were then frozen on a metal block on dry ice for at least 10 min prior to flicking off the coverslip and immersing in 100% ethanol at −20° C for 2 min. Slides were then washed in PBSTB [1XPBS with 0.1% Tween and 0.1% bovine serum albumin (BSA)], and incubated overnight at 4°C with primary antibody: mouse anti-HA (Cell Signaling), guinea pig anti-XND-1(Wagner et al., 2010 (link)), and rabbit anti-HTP-3 (Das et al., 2022 (link)) (all diluted 1:2000 in PBST). The next day, slides were washed 3X in PBSTB for 10 min and incubated with secondary antibodies: anti-mouse Alexa 488 (Molecular Probes), anti-guinea pig 633, and goat anti-rabbit Alexa 568 (Invitrogen, all diluted 1:2000 in PBSTB) for 2 h at room temperature in the dark. Slides were then washed 2 X 10 min in PBSTB, mounted in Prolong Gold without DAPI (Invitrogen) and put in the dark to cure overnight before imaging. The images were taken using a Stellaris8 TauSTED equipped with three depletion lines.