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Files in this Data Supplement:
Fig. S1. Developmental change of mig-6 isoforms. RNase protection assay of embryonic, larval (L1-L4), and adult stage pools of synchronized worms. Each preparation was hybridized to a mixture of probes specific to mig-6S, mig-6L and fem-1 (internal control). For staged populations, eggs were harvested by bleaching a mixed stage culture in 500 ml S medium containing micrococcus (Sigma) or OP50 bacteria. By culturing 0.5 ml of packed eggs in 500 ml S medium without food for 12 to 18 hours in room temperature, worms hatch and starve at the L1 stage. Synchronized cultures were started by adding 0.1 g of micrococcus and 1 g of frozen dry OP50. L1, L2, L3, L4 and young adults were collected by harvesting worms at 8-10 hours, 18-20 hours, 31-32 hours, 40 hours and 49 hours after adding food, respectively. Total RNAs were isolated from each worm preparation using TRIZOL (Gibco BRL). Radio-labelled probes for RNase protection assays were produced using T7 polymerase. These probes contained the NsiI-HincII fragments (exon 13−14) from mig-6L cDNA, SspI-AvaI fragments from the 3′ UTR of the mig-6S cDNA and BstYI-PstI fragments (exon 6−7) from the fem-1 cDNA (a kind gift from Dr A. Spence). After hybridization to the probe mixture at 43°C, for 16 hours, the RNA samples (10 µg) were digested by a mixture of ribonucleases A and T1 at 30°C, for 60 minutes, and then analyzed on a urea gel. The sizes protected by these probes are 333, 210 and 150 bp, respectively.
Fig. S2. Accumulation of MIG-6 proteins in mig-6 class-s background. Anti-MIG-6 antibody staining of the class-s mig-6(ev701) mutant at L2 stage shown in Nomarski (A) and fluorescent microscopy (B-D). The mutant animals showed abnormal accumulation of MIG-6 proteins in the CAN neurons (C) in addition to apparently normal distribution of the proteins in pharynx, intestine and gonad basement membranes (B). (D) A higher magnification of the CAN neuron in C. The CAN neurons were not visible in control animals. The reason for this accumulation is unknown and is unique to the CAN neuron. Scale bar: 20 µm.
Fig. S3. Expression of MIG-17::GFP. Two independently established MIG-17::GFP transgenic lines (evIs209 and evIs212) and their counterparts in a mig-6(ev700) background were analyzed on SDS-PAGE and western blotted with anti-GFP antibody. Each lane was loaded with a worm lysate prepared from ∼300 larvae. L3 to L4 worms were harvested from several large plates and washed three times with M9 buffer. Approximately 12,000 worms were treated with 800 µl SDS-PAGE sample buffer by boiling for 5 minutes and then cooled on ice. Supernatants were collected after centrifugation at 13,000 rpm for 5 minutes at 4°C and 20 µl of the supernatants were loaded onto a 10% acrylamide Precise precast mini-gel (Pierce) and run in HEPES-SDS-Tris running buffer. Proteins were transferred to PVDF membrane using a BioRad mini trans-blot cell. The membrane was soaked in Tris buffered saline, including 5% skim milk and 0.1% Tween 20 overnight at 4°C and then incubated with anti-GFP mouse monoclonal antibody (Roche clones 7.1+13.1) diluted 1000 times in the same buffer. After extensive washing, antigens were visualized using horse radish peroxidase conjugated anti-mouse IgG (1:2500 dilution) and Super signal West Dura extended duration substrate (Pierce) according to the manufacturer’s instructions.
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