Anti flag m2 horse radish peroxidase antibody

Western blot analyses were performed as described previously 15. Primary antibodies used for western blot and dilution are as follows: anti‐FLAG M2–horse radish peroxidase (HRP) antibody (Sigma‐Aldrich, 1 : 1000), anti‐myc–HRP antibody (Thermo Fisher Scientific, 1 : 5000) and anti‐V5–HRP antibody (Thermo Fisher Scientific, 1 : 5000), anti‐Lamin A/C antibody (Cell Signaling Technology, Danvers, MA, USA, 1 : 1000), anti‐glyceraldehyde‐3‐phosphate dehydrogenase (Cell Signaling Technology, 1 : 500), anti‐p27^kip1 antibody (MBL, 1 : 1000), anti‐JAB1 antibody (Santa Cruz Biotechnology, Dallas, TX, USA, 1 : 200), anti‐Cdk2 antibody (Santa Cruz Biotechnology, 1 : 200), and anti‐cyclin E antibody (MBL, 1 : 1000). Anti‐mouse IgG HRP‐linked and anti‐rabbit IgG HRP‐linked (Cell Signaling Technology, 1 : 5000) secondary antibodies were used. Data were analyzed using lumivision analyzer 400 software (Aisin Seiki, Kariya, Aichi, Japan).

Spores of the parental strain and of S. albus Δantall and the ΔclpXclpP1clpP2 mutant harboring pPDA or pPDD were grown on MS agar (buffered with 50 mM TES, pH 7.2) covered with cellophane discs. Protein was isolated from mycelium collected during growth at the following regular intervals: 14 h, 17 h, 24 h, and 30 h for the Δantall and ΔclpXclpP1clpP2 mutants harboring 3xFLAG-AntA constructs and 17 h, 20 h, 23 h, and 30 h for the Δantall and ΔclpXclpP1clpP2 mutants harboring the 3xFLAG-FscRI construct. Protein samples were generated as follows: 100-mg volumes of cells were resuspended in 200 μl lysis buffer (50 mM sodium phosphate buffer [pH 7.0]; 150 mM sodium chloride; 10 mg ml⁻¹ lysozyme; cOmplete, Mini, EDTA-free protease inhibitors [Roche]; 100 mg of 0.1-mm-diameter glass beads [PowerLyzer]) and lysed by vortex mixing for 30 min at 2,000 rpm and 37°C, with a subsequent incubation for another 30 min at 37°C. The obtained suspension was centrifuged for 20 min at 20,000 × g at 18°C. Each clarified protein sample (30 μg) was subjected to SDS-PAGE and then transferred to a nitrocellulose membrane (pore size, 0.2 μm) for Western blot analysis. The membrane was probed with mouse monoclonal anti-FLAG M2-horseradish peroxidase (HRP) antibody (Sigma) (1:10,000, and the signals were detected using Pierce 1-Step Ultra TMB blotting solution (Thermo Scientific).

A PAWS1 antibody was generated by injecting GST-PAWS1 (amino acids 715–815) into sheep. The P-PAWS1 S610 antibody was generated by injecting peptide GPGPRRPS*VAS (* denotes phospho-Ser) into rabbit. The antibodies were subsequently affinity purified. Anti-FLAG-M2-horseradish peroxidase (HRP) antibody was from Sigma. HA-HRP antibody was from Roche. Antibodies recognizing phospho-SMAD1/5/8, phospho-SMAD2, phospho-SMAD3, SMAD2/3, GAPDH and lamin A/C were from Cell Signalling Technology. HRP-conjugated secondary antibodies and light-chain-specific HRP-conjugated antibodies were from Jackson Laboratories. BMP-2 and BMP-4/7 were from R&D Systems. The nuclear cytoplasmic extraction kit was from Thermo Scientific. The first strand cDNA synthesis kit was from Invitrogen. 2X SYBR green master mix was from BioRad. pBABE-Puro, pCMV-Gag-Pol and pCMV-VSVG constructs were from Cell Biolabs. All plasmids for mammalian cell expression were cloned into pCMV5, pBABE-Puro or pcDNA-FRT-TO vectors with N-terminal FLAG, HA or GFP tags as indicated. For bacterial expression of proteins, SMAD1, SMAD2 and PAWS1 (523-end; other mutants) were cloned into pGEX6T vectors. All DNA constructs were verified by DNA sequencing (by the DNA Sequencing Service at University of Dundee; www.dnaseq.co.uk). Bacterial protein expression in BL21 cells and purification were performed as described previously [31 (link)].