Ab167453

Equal number of wild-type and RNAi-treated adult worms were lysed in 75 ml M9/0.1% Triton X-100 (3 g KH 2 PO 4 , 6 g Na 2 HPO 4 , 5 g NaCl, 1 ml 1 M MgSO 4 , 1 ml Triton X-100, and H 2 O to 1l) and 25 ml 43 sample buffer (8% SDS, 0.04% bromophenol blue, 240 mM Tris/HCl [pH 6.8], 40% glycerol, and 5% b-mercaptoethanol) using water bath sonication at 4 C for 20 min. Ten worms were loaded in each lane for SDS-PAGE. Proteins were transferred to nitrocellulose membranes and probed using antibodies against a-tubulin (1:8,000; Abcam ab7291 DM1A), RbAp46/48 LIN-53 (1:400; abm SAB494-1), CENP-A HCP-3 (1:1,000; Novus 29540002 SDQ0806 or SDQ0804), mCherry (1:500; Abcam ab167453), H3K9ac (1:1,000; Millipore ABE18), H4ac (1:1,000; Millipore 06-598; recognizing acetylated lysines 5, 8, 12, and 16 of Tetrahymena histone H4), H3 (1:5,000; Abcam ab1791), H4 (1:1,000; Abcam ab10158), or mCherry (1:500; Abcam ab167453) at 4 C overnight. After washes with TBST, immunoblots were subjected to HRP-conjugated antibodies incubation (Abcam ab97051 or ab97023) at room temperature for 1 hr. Blot signals were detected using Amersham ECL Select western blotting detection reagent (GE Healthcare Life Sciences).