Imagequant 5

Twenty μg from each sample were diluted in Laemmli loading buffer, denatured by boiling for 5 minutes, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Biorad, Hercules, CA). Membranes were blocked for 2 hrs in TBS containing 0.1% Tween 20 (Sigma-Aldrich) and 5% nonfat dry milk (Bio-Rad). After an overnight incubation at 4°C with the primary antibodies in TBS, 0.1% Tween 20, and 5% nonfat dry milk or bovine serum albumine (BSA), blots were washed three times with TBS containing 0.1% Tween (TBS-T) and incubated with a HRP-linked secondary antibodies for one hour. After three washes with TBS-T, the membranes were treated with ECL detection system (GE Healthcare, Piscataway, NJ), exposed to x-ray film (Denville Scientific, Metuchen, NJ) and developed to visualize the labeled protein bands. Molecular mass was estimated by comparison of sample bands with prestained molecular mass marker (Bio-Rad). For quantitative studies, the bands on x-ray films were scanned using a photodocumentation system (Alpha Innotech) and analyzed with ImageQuant 5.2 software (GE Healthcare). Where indicated, blots were stripped and reblotted with the corresponding antibody. Values were normalized respect to b-actin or total antibody as indicated.

Yeast U4 and U6 snRNA production (commonly used also in unwinding assays with human BRR2), purification, labeling and assembly were carried out as described before^{65 (link)}. All U4/U6 unwinding assays were performed at 30 °C. Unwinding of 0.6–1.5 nM radioactive U4/U6 di-snRNA was compared for 100 nM BRR2 (FL or HR) alone with 100 nM BRR2 (FL or HR) in complex with 150 nM GST-C9ORF78 in the presence or absence of 250 nM PRPF8^Jab1 or PRPF8^Jab1ΔC. All reactions were pre-incubated for 3 min in unwinding buffer (40 mM Tris-HCl, pH 7.5, 50 mM NaCl, 8% (v/v) glycerol, 0.5 mM MgCl₂, 15 ng/μl acetylated BSA, 1 U/μl RNase inhibitor, 1.5 mM DTT) in a total volume of 120 μl. Unwinding was initiated by addition of 1.7 mM ATP/MgCl₂, and 10 μl samples were withdrawn at the indicated time points and mixed with 10 μl of stop buffer (40 mM Tris-HCl, pH 7.4, 50 mM NaCl, 25 mM EDTA, 1% (w/v) SDS, 10% (v/v) glycerol, 0.05% (w/v) xylene cyanol, 0.05% (w/v) bromophenol blue). The samples were run on a 6% non-denaturing PAGE gel for 1 h at 200 V and 4 °C. RNA bands were visualized by autoradiography using a phosphoimager and quantified using the Image Quant 5.2 software (GE Healthcare). Data were fit to a single exponential equation (fraction unwound = A (1–exp[- k_ut])); A, amplitude of the reaction; k_u, apparent first-order rate constant of unwinding; t, time) using GraphPad Prism.

The DNA primer extension assay was performed as described previously (57 (link)). Briefly, TET-labeled primer (1 μM) was annealed to 1.25 μM oligonucleotides containing G4 or non-G4 (S. pombe ade6⁺) sequences. Extension of the annealed primer was performed at 37°C for 1 min in the presence of either 1% (v/v) DMSO and 100 mM KCl or 0.1 μM PhenDC3 and 100 mM KCl using 0.063 U of an exonuclease-deficient Klenow fragment of Escherichia coli DNA polymerase (Thermo Fisher Scientific). Reaction products were separated on 10% polyacrylamide gels containing 8 M urea, 25% formamide, and 1 × Tris/borate/ethylenediaminetetraacetic acid (TBE). Bands were visualized and quantified using a Typhoon Scanner 9400 (GE Healthcare) and the ImageQuant 5.2 software (GE Healthcare).

Western blot was performed according to regular protocol. Briefly, cell lysates of treated cells were extracted and transferred onto PVDF membranes. Membranes were blocked in PBS supplemented with 0.5% (v/v) Tween 20 (PBST) plus 10% (w/v) nonfat milk for 1 h followed by incubation with primary (1:100 to 2000) antibodies at 4 °C overnight. The membrane was then scanned by a Typhoon9400 Variable Mode Imager (GE Health Life Sciences, Uppsala, Sweden) to detect the fluorescent signals released from catalyzed ECF substrate (GE Healthcare, Little Chalfont, UK). Primary antibodies used were as follows: ZEB1 (Catalog #3396, Cell Signaling Technology), β-actin (Catalog #A5441, Sigma-Aldrich), β-catenin (Catalog #05-665, Sigma-Aldrich), histone (Catalog #MAB1276, Sigma-Aldrich), and GAPDH (Catalog #GTX100118, GeneTex). The results of Western blots were quantified by Image Quant 5.2 software (GE Healthcare, Little Chalfont, UK).