Anti ph20

Cells were harvested and washed once with cold PBS. Cells were then lysed in lysis buffer (50 mM Tris-HCl, 250 mM NaCl, 5 mM EDTA, 50 mM NaF, 0.1% NP40, and 1% protease inhibitor cocktail). For each immunoprecipitation assay, proteins were incubated with anti-HA antibody at 4 °C overnight, followed by incubation with protein A/G agarose beads (Santa Cruz Biotechnology). Beads were washed three times with NP40 buffer, the bound proteins were eluted with 2×SDS loading buffer and then boiled at 100 °C for 5 min. Precipitated proteins were detected using western blot. Antibodies against anti-DNMT1 (Abcam), anti-GAPDH (ZSGB-BIO), anti-SUV39H1 (Active Motif), anti-vigilin (Abcam), and anti-PH20 (Abcam) were used.

Dynabeads^® M-270 Carboxylic Acid (Invitrogen, Carlsbad, USA) were activated by treatment with 3-(ethyliminomethyleneamino)-N,N-dimethyl-propan-1-amine (EDC) and N-hydroxysuccinimide (NHS) according to the manufacturer’s recommendations. A 60 μg sample of antibody (anti-PH-20, anti-SP-10, anti-ADAM2 or anti-JLP; Abcam, Cambridge, UK) was added to 3 mg of activated beads dissolved in 25 mmol/L 2-(N-morpholino)ethanesulfonic acid (MES), pH 5.0. The magnetic beads and antibody mixture was vortex mixed thoroughly and then incubated for 60 min at room temperature with slow tilt rotation. After incubation, the mixture was transferred to a magnet (Promega, Madison, WI). The beads were sufficiently collected in 2 min before the supernatant was removed. The beads were then incubated in 50 mmol/L Tris (pH 7.4) for 15 min to block the unreacted carboxylic acid groups, washed three times with reaction buffer (10 mmol/L PBS, pH 7.4, 0.1% (w/v) bovine serum albumin) containing 0.1% (v/v) Tween-20 and suspended in 100 μL of reaction buffer.