Antibodies and reagents
Anti-Myc antibody-conjugated agarose was purchased from Bethyl Laboratories (Montgomery, TX). Anti Myc monoclonal antibody was purchased from BD Bioscience (Palo Alto, CA). Anti-FLAG-M2 antibody, antibody-conjugated agarose and anti β-actin antibody were from Sigma-Aldrich (St. Louis, MO). Rabbit anti IRS4 polyclonal antibody was from Santa Cruz (Santa Cruz, CA) and mouse anti human IRS4 monoclonal antibody (M01) was from Abgent (San Diego, CA). Anti Asb-4 antibody was from Abcam Inc. (Cambridge MA). Anti phospho-AKT at Thr308 and total AKT was from Cell Signaling Technology (Beverly, MA).
In situ hybridization
All studies were approved by the University of Michigan Committee on Use and Care of Animals.
A 568 bp fragment of the rat Asb-4 cDNA (codon 681 to 1248) was generated by PCR and subcloned into pBluescript SK (Stratagene, La Jolla, CA) at BamH I and EcoR I sites. The sense primer was: 5' CGG GAT CCG GAG CAG GAG TAC AGC AGG GAA CA 3'. The antisense primer was 5' CGG AAT TCT GAC AGT GGG AGG GAC AGC ATA GG 3'. A 566 bp fragment of the rat IRS4 cDNA (codon 3112 to 3677 according to gene bank number XM_001056753 was generated by PCR and subcloned into pBluescript SK at BamH I and Hind III sites. The sense primer was: 5' CCCCCAATGAACCAGGCTAAG 3'. The antisense primer was 5' ACAGGTGGTGGTTCGGAGTATCG 3'. The NPY cDNA (AI045437) in pT7T3D-PAC was purchased from Invitrogen (Carlsbad, CA). The rat POMC plasmid construct consisted of an 833 bp insert that included the full coding region of the POMC gene inserted into pGEM4Z (Promega, Madison, WI). The POMC-pGEM4Z plasmid was constructed by R. C. Thompson. The [35S]-labeled antisense and sense IRS4 and Asb-4 RNA probes, Dig-labeled antisense and sense Asb-4, NPY and POMC RNA probes were generated using standard in vitro transcription methodology .
Seven week old male Sprague-Dawley rats (280-300 g) were anesthetized with ketamine/xylazine and perfused via the ascending aorta with 200 ml of phosphate buffered saline (PBS), followed by 200 ml of 4% paraformaldehyde in PBS. The brain was postfixed for 16 h in 4% paraformaldehyde and then transferred to 20% sucrose (20% sucrose in PBS with 0.02% sodium azide) for 5 days at 4°C. The brain was embedded with 20% sucrose and Tissue-Tek O.C.T. (2:1) and coronal sections of 14 μ were cut on a cryostat. The sections were dried overnight at room temperature and were stored at -80°C until further processing. Sections studied covered the telencephalon and diencephalon from coordinates of bregma -0.80 mm to bregma -4.16 mm according to Paxinos and Watson .
In situ hybridization was conducted using a modification of a previously described technique [34, 35]. The brain sections were washed 3 times with 2 × SSC and then digested with 0.45 μg/ml proteinase K (Invitrogen, Carlsbad, CA) in 100 mM Tris, pH 8.0, 50 mM EDTA) for 15 minutes at 37°C. After brief washing with distilled water, the sections were acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine, pH 8.0, for 10 min. The sections were subsequently dehydrated through a graded series of ethanols.
Antisense or sense probes were diluted in hybridization buffer (50% formamide, 3 × SSC, 1 × Denhart's, 200 μg/ml yeast tRNA, 50 mM phosphate buffer, pH 7.4, 10% dextran sulfate and 10 mM DTT) to yield ≈ 30,000 cpm/μl. 40 μl diluted probes were applied to each slide and the sections were coverslipped. Slides were then placed in sealed plastic boxes lined with filter paper moistened with 50% formamide. The boxes were wrapped with plastic wrap and incubated at 55°C for 16 h. For dual label in situ hybridization, the sections were hybridized with antisense Dig-labeled Asb-4, NPY or POMC and [35S]-labeled IRS-4 riboprobes.
Following overnight incubation, coverslips were removed by dipping in 2 × SSC and the slides were washed with 2 × SSC. Slides were then incubated with 40 μg/ml RNase A (Roche; Indianapolis, IN) in 10 mM Tris-HCl (pH 8.0, 0.5 M NaCl) at 37°C for 1 h. The slides were washed with 2 x, 1 x, 0.5 × and 0.1 × SSC at room temperature for five minutes each time and then incubated in 0.1 × SSC at 67°C for 1 h. The sections were washed briefly with distilled water. For single label in situ hybridization the slides were dehydrated in graded alcohols and air-dried. Dried slides were exposed to Kodak BioMax film. For dual label in situ hybridization, after washing with distilled water, the sections were incubated with anti-Dig antibody (Roche, Indianapolis, IN) conjugated with alkaline phosphatase (1:20,000) overnight at room temperature. The slides were next incubated in nitroblue terazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate solution for 2-4 h for color reactions. Then the slides were dipped in ILFORD K.5D nuclear emulsion and exposed in dark for 14 days at 4°C. Sections were developed with D-19 developer.
The full length coding region of mouse Asb-4 cDNA was cloned by RT-PCR from the mouse hypothalamic neuronal cell line, GT1-1, and subcloned into the pCMV-Myc vector (BD Clontech, Palo Alto, CA) using EcoR1 and Not I sites. The Myc tag was fused to the N-terminus of Asb-4. The correct frame of the construct was confirmed by DNA sequencing. Mouse IRS4 cDNA with full length coding region was amplified with the Expand Long Template PCR System (Roche, 68298 Mannheim, Germany) from C57BL genomic DNA since the full length coding region is encoded by a single exon. The cDNA was subcloned into 3xFlag CMV-10 vector (Sigma-Aldrich) at Hind III and Xba I sites. The 3xFlag was fused to the N-terminus of mouse IRS4. The correct frame of the construct was confirmed by DNA sequencing.
HEK293 cells were grown in Dulbecco's modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum and 100 units/ml of penicillin and 100 μg/ml of streptomycin in a 37°C incubator supplemented with 5% CO2. Cells were transfected with 2.5 μg (100 mm dish) of pCMV-Myc-Asb-4 plasmid DNA along with 2.5 μg of p3xFlag-IRS4-CMV-10 plasmid DNA using lipofectamine 2000 as described by the manufacturer (Invitrogen). Forty hours after transfection, cells were disrupted by sonication in lysis buffer [150 mM NaCl, 20 mM Na2HPO4, pH 7.4, 1% Triton X-100 supplemented with protease inhibitor cocktail (Sigma)] and Flag-IRS4 or Myc-Asb-4 were immunoprecipitated using anti Flag-M2 or anti-Myc antibody conjugated agarose. After extensive washing the beads were resuspended in SDS-PAGE loading buffer and heated at 80°C for 5 min. The protein was resolved with SDS-PAGE gel and transferred to polyvinylidene membranes. The membranes were blotted with either anti-Myc, or anti Flag antibody.
For the co-immunoprecipitation of endogenous IRS4 from HEK cells, the cells were transfected with 5 μg Myc-Asb-4 vector (100 mm plate). Myc-Asb-4 was precipitated with anti-Myc antibody conjugated agarose and IRS4 was detected with anti human IRS4 monoclonal antibody (Abgent).
In order to confirm the interaction between Asb-4 and IRS4 in vivo twenty hypothalami were collected from adult rats (250-300 g body weight), homogenized in lysis buffer, centrifuged at 14,000 × g for 10 min at 4°C and the supernatant was used for immnoprecipitation with anti IRS4 antibody (Santa Cruz). Asb-4 was detected with anti Asb-4 antibody.
For the insulin stimulation of HEK cells, the cells were cultured in serum free DMEM with 0.1% BSA overnight; insulin was added to the cells and incubated for 20 min. The cells were harvested in lysis buffer and sonicated. Proteins were subjected to SDS-PAGE and transferred to polyvinylidene membranes.
CHO cells co-transfected with pRK5 myc-ubiquitin, which is provided by Dr. Liangyou Rui (Department of Molecular and Integrative Physiology, University of Michigan), Flag-IRS4, and empty vector, or Myc-Asb-4, or Myc-Asb-4/Δsb. Forty hours after transfection, cells were disrupted by sonication in lysis buffer. The supernatants (cell lysates) were immunoprecipitated with anti-Flag conjugated agarose and blotted with anti-Myc antibody. Membranes were stripped and reblotted with anti-Flag antibody.
Equal amounts of protein were resolved on SDS-PAGE gel, transferred to polyvinylidene membranes and immunoblotted with primary antibodies. Horseradish peroxidase conjugated secondary antibodies were used to detect antigen-antibody complexes via the ECL detection system (Amersham Biosciences, Piscataway, NJ).
The density of bands was analyzed and normalized with internal controls (β-actin or total AKT) using the Kodak Gel Logic 440 Imaging System All. Values are expressed as mean ± SEM. Analysis of variation (ANOVA) followed by post Bonferroni test was used for statistical analysis. Significance was accepted as P < 0.05.