Na931

Polyclonal anti-uL11K3me3 antibodies were generated in rabbit using a peptide corresponding to the first 16 amino acids of uL11 with methylated lysine 3 [PPK(me3)FDPTEVKLVYLRC] (Eurogentec). The serum was first loaded on a uL11K3me3 peptide affinity column which allowed to retain anti-uL11K3me3 and anti-uL11 antibodies.
After elution, they were separated by passage through an unmethylated uL11 peptide affinity column. Specificity of the antibodies was checked by dot blot (Supplementary Figure 1). Proteins were extracted from third instar larvae in RIPA buffer (150 mM sodium chloride, 1 % NP40, 0.5 % sodium deoxycholate, 0.1 % SDS, 50 mM Tris-HCl pH 8,0) supplemented with phosphatase and protease inhibitors (Roche). 30 μg of proteins were separated by SDS-PAGE electrophoresis on 15 % acrylamide gels. Western blots were performed according to standard protocols using either goat anti-uL11 (SantaCruz sc82359, 1/1000), rabbit anti-uL11K3me3 (1/6000), or mouse anti-α-tubulin (DSHB E7c, 1/2500) as primary antibodies. Anti-goat (Jackson ImmunoResearch; 705035147; 1/10000), anti-rabbit (1/20000) or anti-mouse (Sigma NA931; 1/20000) were used as secondary antibodies.

S2 cells transiently transfected with either pA-uL11 K3A -Myc or pA-uL11 K3Y -Myc and pA-FLAG-CortoCD (17) were harvested after 48 h and washed in Schneider medium at room temperature. Co-immunoprecipitations were performed as described in (30) without fixation. 30 μl of Protein G coated Bio-Adembeads (Ademtech) were incubated with either 1 μg of mouse monoclonal anti-FLAG antibody (F3165, Sigma), or goat anti-HA antibody as mock (sc-805, Santa Cruz Biotechnology).
Western blot analyses were performed according to standard protocols with mouse anti-Myc (Abcam ab9132; 1/10000) or anti-FLAG (Sigma F3165; 1/5000) as primary antibodies and anti-mouse (Sigma NA931; 1/20000) as secondary antibodies.

Puromycin assays were adapted from (28) with the following modifications: 20 third instar larvae were turned inside-out and incubated for 1 h at 25 °C under gentle rotation in Schneider's medium supplemented or not with 10 mg/mL cycloheximide (Sigma). Puromycin (antpr1, InvivoGen) was then added at a final concentration of 0.28 mg/mL and incubation was continued for 2 h. Total proteins were extracted in a buffer containing 30 mM Hepes pH 7.4, 0.1 % NP40, 150 mM NaCl, 2 mM Mg(OAc)2 supplemented with phosphatase and protease inhibitors (Roche) (adapted from (29) (link)). 60 μg of protein extracts were deposited on a 12 % acrylamide gel. Western blot analyses were performed according to standard protocols with mouse anti-Puromycin (Kerafast, 3RH11; 1/500) or mouse anti-H3 (Diagenode; C15200011; 1/1000) as primary antibodies, and anti-mouse (Sigma; NA931; 1/20000) as secondary antibodies.
Puromycin and H3 signals were measured using ImageJ. The Puromycin signal (signal in the samples treated with CHX and Puromycin minus signal in the untreated sample) was normalized towards the H3 signal. Statistical significance was assessed by Student's t-tests.