P0161

ELISA plates were coated with 50 μL/well of VLP at 5 mg/mL in carbonate coating buffer and incubated at 4°C overnight in a humidified chamber. The coating antigen (VLP) was then discarded and wells were blocked with 100 μL of 1% BSA/PBS for 1 h at 37°C in a humidified chamber. Immunised mice serum was initially diluted 1 in 10 in blocking buffer and serially diluted. Fifty microlitres of each serum dilution was added to coated wells. Plates were incubated for 1 h at room temperature and wells were then washed four times with PBS with 0.05%Tween20. Fifty microlitres of anti-mouse antibody conjugated to HRP (P0161, Dako) diluted 1 in 400 was then added to wells and incubated at room temperature for 1 h. Plates were then washed 4 times with PBS with 0.05%Tween20, and developed by adding tetramethylbenzidine (TMB) substrate (002023, Thermofisher Scientific) and stop solution as described above. Absorbance values were determined on a Labsystems Multiskan Multisoft plate reader (Thermo Scientific) at 450 nm.

Testicular protein extract and WB were performed as previously described [46 (link)]. Fifteen pairs of testes or seminal vesicles from 1–2 days-old males were dissected in each genotype. Mouse monoclonal anti-α-tubulin (DM1A; Sigma #T9026) was used at a 1/10 000 dilution, rabbit polyclonal anti-Mst77F [FL]; 1/5000 dilution, rabbit polyclonal anti-Mst77F [171–184]; 1/2500 dilution, rabbit polyclonal anti-Mst35Bb; 1/1000 dilution. HRP-conjugated anti-rabbit or anti-mouse (Dako #P0448 or #P0161) secondary antibodies were used at a 1/5000 dilution.

Western blot analysis was performed as previously described [62 (link)]. Antibodies were anti-ACTB mAb (017-24551, Wako Pure Chemical, Osaka, Japan), anti-GFP (GF-200, Nacalai Tesque), and anti-TLS/FUS (ab154141, Abcam, MA, USA) for primary antibodies at the concentration of 1:2000, anti-rabbit IgG HRP-linked antibody (7074S, Cell Signaling Technology, Danvers, MA, USA) and anti-mouse immunoglobulins/HRP (P0161, DAKO, Glostrup, Denmark) for secondary antibodies at the concentration of 1:5000.

The relative expression levels of some BlaP-polyQ chimeras and of Q82::YFP animals were confirmed by dot blot. For this purpose, worm extracts were prepared and collected as described before (Western blot analysis).
2 μl of each sample was spotted on a nitrocellulose membrane and the samples were allowed to dry for 1 h. The membrane was then incubated at room temperature (1) for 2 h in 5% blocking agent (GE Healthcare) and (2) with primary mouse anti-polyQ antibody (1/1000 dilution in Tris-Saline pH 7.6, 5TF1-1C2, Millipore) or primary rabbit anti-Histone H3 antibody (1/20000 dilution in Tris-Saline pH 7.6, ab8580, Abcam). Horse Radish Peroxidase-conjugated rabbit anti-mouse (1/50000 dilution in Tris-Saline pH 7.6, P0161, Dako) or goat anti-rabbit (1/50000 dilution in Tris Saline, pH 7.6, P0160, Dako) were used as secondary antibodies for visualization with Supersignal West Dura (Thermo Scientific).
Dot blot signals were analysed using ImageJ. Upon removal of background signals, the polyQ signal was normalized to the Histone H3 signal, which functioned as an endogenous loading control.

Cells were lysed in 50 mM Tris pH 8.0, 150 mM NaCl, 0.5% NP-40, 50 mM NaF, 1 mM Na₃VO₄, 1 mM PMSF, one tablet protease inhibitors (EDTA free, Roche) per 10 ml and 30 μg/ml DNaseI (Sigma-Aldrich) and analyzed by immunoblotting. For detection of proteins by chemoluminescence (Advansta, K-12049-D50) a mouse anti-CHK1 (CS 2360, 2G1D5) 1:750, mouse anti-CHK1Ser345 (CS 2348) 1:500, mouse anti-MYC cMYC (Y69 Abcam 32072) 1:1000, rabbit anti-GAPDH (CS 2118, 14C10) 1:5000, rabbit anti-γH2AX (CS 2577) 1:1000, rabbit anti-p53 (Santa Cruz sc-6243) 1:200, mouse anti-p21 (BD clone SX118) 1:500 or rabbit anti-PARP1 (CS 9542) 1:1000 were used (5 ml/membrane). Goat anti-rabbit Ig/HRP (Dako, P0448) or rabbit anti-mouse Ig/HRP (Dako, P0161) were used as secondary reagents (1:10,000). See Supplementary Fig. 8 for uncropped scans of western blots.

Cells were lysed in 50 mM Tris pH 8.0, 150 mM NaCl, 0.5% NP‐40, 50 mM NaF, 1 mM Na₃VO₄, 1 mM PMSF, one tablet protease inhibitors (EDTA free, Roche) per 10 ml, and 30 μg/ml DNaseI (Sigma‐Aldrich) and analyzed by Western blot analysis. For detection of proteins by chemoluminescence (Advansta, K‐12049‐D50), a mouse anti‐CHK1 (CS 2360, 2G1D5), rabbit anti‐ɣH2A.X (CS 2577), rabbit anti‐p53 (Santa Cruz sc‐6243), rabbit anti‐PARP1 (CS 9542), mouse anti‐HSP90 (Santa Cruz sc‐13119), mouse anti‐CycD3 (BD 554195), mouse anti‐hBCL2 (clone S100), rabbit anti‐ATR‐pSer428 (CS 2853), or rabbit anti‐actin (CS 4967) was used. Goat anti‐rabbit Ig/HRP (Dako, P0448) or rabbit anti‐mouse Ig/HRP (Dako, P0161) was used as secondary reagent.

Cells were lysed on ice in buffer containing 10 mM HEPES (pH 7.9), 0.1 mM ethylenediaminetetraacetic acid, 0.2 mM ethylene glycol-bis (β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid, 10 mM potassium chloride, and 0.5% Nonidet P40, supplemented with 10 mg/mL aprotinin, 10 mg/mL leupeptin, 1 mM dithiothreitol, 1 mM phenylmethylsulphonyl fluoride, 1 mM sodium vanadate, and 1 mM sodium fluoride. The lysates were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The membranes were then incubated with an antibody against Smad7 (1:1000, R&D Systems), p-Stat3 Tyr705 (1:1000, catalog number 9145, Cell Signaling Technology), total Stat3 (1:1500, catalog number 10913, Santa Cruz), BCL-xL (1:500, catalog number sc-8392, Santa Cruz), survivin (1:500, catalog number sc-17779, Santa Cruz), p-Stat1 (1:500, catalog number sc-8394, Santa Cruz), or β-actin (1:5000, catalog number A544, Sigma) and, subsequently, with a secondary antibody conjugated to horseradish peroxidase (1:20,000, rabbit anti-mouse, catalog number P0161, goat anti-rabbit, catalog number P0448, Dako, Santa Clara, CA, USA). Computer-assisted scanning densitometry was used to analyze the intensity of the immunoreactive bands.