Xcell 2

Protein expression of KDM4 family members and cell cycle proteins was carried out by Western blotting. Protein from transfected cells was extracted using cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA). Proteins were resolved on 3–8% Bis-Tris acrylamide gels and transferred onto PVDF membranes using the X-Cell II and iBlot systems according to manufacturer’s instructions (Invitrogen). Antibodies can be found in Table S7.

20 fly heads were homogenized in 50 μl LDS sample buffer (Invitrogen) including 5% beta-mercaptoethanol (Sigma-Aldrich). Samples were boiled at 95 °C for 5 min and centrifuged at 15,000 g for 5 min at 4 °C. The supernatant was collected and stored at − 20 °C until loaded on 4–12% Bis-Tris NuPAGE gels (Invitrogen) using 5 μl of sample per well. Gel electrophoresis was performed at 140 V for 70 min and the gels were blotted on a PVDF membrane using XCell II (Invitrogen) at 30 V for 1 h. Membranes were blocked in 3% bovine serum albumin in tris buffered saline with 0.1% Tween20 (TBST) for 30 min and incubated with primary antibodies in blocking buffer over-night at 4 °C. Following washes in TBST, membranes were incubated with HRP-conjugated secondary antibodies (Jackson Immunoresearch) at 1:10,000 for 2 h, washed and the luminescent signal was developed using ECL prime (Amersham) and detected with Amersham Imager 600. Primary antibodies: anti β-tubulin (CAT#E7, DSHB, 1:500), anti β-Galactosidase (CAT#Z378A, Promega, 1:2000), anti-TDP-43 (CAT#10782, Proteintech, 1:1000), anti Tau (CAT#A0024, Dako, 1:1000).

The purified PAP enzyme preparation was also analyzed by native gel for in-gel PAP activity assay using Xcell II, Mini-Cell and 3–12% Novex NuPage Bis-Tris gels with NuPAGE native running buffer (Life Technologies). The native gel was loaded with 0.5–12 µg of proteins purified from the Affigel Blue column. The protein bands on the gel were visualized with Coomassie Blue staining (Simple Blue SafeStain, Life Technologies) using native mark unstained protein standards (20 to 1236 kDa) as size standards (Thermo Scientific). PAP activity on the unstained native gel was performed using 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as the substrate and B-phycoerythrin as the positive control (Life Technologies). The activity assay was performed by incubating the native gel into 3-mL reaction mixture containing 0.5 mM DiFMUP, 50 mM imidazole, pH 6.5, 3 mM MnCl₂ and 100 mM DTT at 37°C for 5 min.