Anti psmad

Liver tissue (100 mg) was homogenized using Precelllys Evolution tissue homogenizer (Bertin Instruments, Montigny-le-Bretonneux, France) in a buffer with phosphatase inhibitors [29 (link)]. Protein lysate (25 µg) was electrophoresed on a Bolt™ 4–12% Bis-Tris Plus gels (Thermo Fisher Scientific, Massachusetts, United States). After transferring to a nitrocellulose membrane, the proteins were blocked with 10% non-fat milk in Tris buffer with 0.1% Tween-20 (TBST) for 2 h at room temperature, and incubated with primary antibodies: anti-Tfr2 [30 (link)] 1:10000, anti-pSmad (Cell Signaling Technology, Danvers, MA) 1:1000, anti-prohepcidin [31 (link)] 1:1000, anti-ferritin-H (Cell Signaling Technology) 1:1000, anti-actin (Sigma–Aldrich, St. Louis, Missouri) 1:20000 and anti-GAPDH (Merck Millipore, Kilsyth, Victoria, Australia) 1:200000 overnight at 4°C. After washing with TBST and incubated with anti-rabbit IgG-horseradish peroxidase (Invitrogen, Life Technologies) 1:10000, diluted in 10% non-fat milk in TBST for 1 h at room temperature. Excess secondary antibodies were washed off and the blots were incubated with chemiluminescent substrate (Lumina Forte; Merck Millipore) for 5 min. Blots were exposed to X-ray film (Fujifilm, Brookvale, NSW, Australia) and developed using the Minolta film processor (Konica Minolta Medical and Graphic, Tokyo, Japan).

anti-jagged1 (R&D Systems, Minneapolis, MN), anti-E-cadherin (BD Transduction Laboratories, UK), anti-wt1 (Santa Cruz, Dallas, TX), anti-LEF1 (Cell Signaling, The Netherlands), anti-Pax8 (Proteintech Europe, UK), anti-Pax2 (Covance, Princeton, NJ), anti-phospho-β-cat (S33, S37, T41) (Cell Signaling), anti-phospho-β-cat (S45) (Cell Signaling), pan β-cat (SIGMA), anti-dephospho-β-cat (AG Scientific, San Diego, CA), anti-podocalyxin, anti-laminin (SIGMA), LTL (Vector Labs, Burlingame, CA), anti-ZO1 (DSHB, Iowa City, IA), anti-pSMAD (Cell Signaling), anti-pAKT (Cell Signaling), 6-CF (SIGMA), PNA (Vector Labs), Annexin V (Biovision, San Francisco, CA), TUNEL (ROCHE, Switzerland), anti-phospho-β-cat (S552) (Cell Signaling).

IP assays of larval brains and ventral ganglia were carried out according to a previous report [61 (link)]. Immunoprecipitation from S2 or HEK293 cell lysates was performed following a previously described protocol [43 (link)]. Antibodies used for western blotting were: anti-myc (1:1000; 9E10 from Clontech), anti-flag (1:1,000; M2 from Sigma), anti-K48 (1:1,000; D9D5 from Cell Signaling Technologies), anti-GFP (1:1,000), anti-Ube3a (1:300), anti-HA (1:1,000; 3F10 from Roche), anti-ubiquitin (1:1000; P4D1 from Cell Signaling Technologies), anti-Wit (1:50; DSHB), anti-pSmad (1:1000; Cell Signaling Technologies), and anti-α-tubulin (1:25,000; mAb B-5-1-2 from Sigma).
To analyze protein levels in different genotypes, larval brains and ventral ganglia were homogenized in ice-cold lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% (v/v) NP-40, 0.1% (w/v) SDS, 1% (v/v) proteinase inhibitor). Western blots were probed with primary antibodies anti-Ube3a, anti-pMad (1:500, Cell Signaling Technologies), anti-Wit, anti-GFP, and anti-α-tubulin, followed by HRP-conjugated secondary antibody (1:25,000; Sigma).