Insig-2, a second endoplasmic reticulum protein that binds SCAP and blocks export of sterol regulatory element-binding proteins -- Data Supplement - Supporting Materials & Methods -- Proceedings of the National Academy of Sciences

Supporting information for Yabe et al. (2002) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.162488899

Supporting Materials and Methods
Materials.
We obtained Mouse Multiple Tissue Northern (MTN) Blot and SMART RACE cDNA Amplification Kit from CLONTECH; RNA LA PCR Kit (AMV) Version 1.1 from Takara; and Protein A/G agarose beads from Santa Cruz Biotechnology. Mouse monoclonal antibodies IgG-9D5 against hamster SCAP (1), IgG-2A4 against human SREBP-1 (2), and IgG-9E10 against c-Myc (3) have been described. Monoclonal antibody IgG-1D2, directed against amino acids 48-403 of human SREBP-2, was produced by immunizing a mouse (4) with a GST-SREBP(48-403) fusion protein expressed in Escherichia coli. IgG1 fractions of antibody were purified by protein G-Sepharose. All other reagents were obtained from previously reported sources (3, 5–7). cDNA Cloning of Insig-1 and -2 from Multiple Species.
Human insig-2 (GenBank accession no. XM_043953), encoded by a gene on chromosome 2q21.2, was identified as a protein homologous to human insig-1 (GenBank accession no. AY112745; ref. 3) by database search using blast program (www.ncbi.nlm.nih.gov/BLAST/). Subsequent blast searches in expression sequence tag (EST) databases using amino acids 1-50 of human insig-1 and human insig-2 identified multiple EST clones containing entire open reading frames (ORFs) for human, mouse, and zebrafish insig-2 and zebrafish insig-1. EST clones were purchased from Invitrogen and sequenced. Mouse insig-1 has an internal NotI site within its ORF, which prevented obtaining EST clones with the entire ORF for mouse insig-1. Based on sequences available from partial EST fragments, mouse insig-1 cDNA was amplified by PCR using first-strand cDNA made from mouse liver poly(A)⁺ RNA. The following pair of forward and reverse primers was used for PCR: 5'-GTGTCGTGGCGTGTACCTTCCATGCTG-3' and 5'-CCCCCTTACCCGACTCTCACATAC-3'. Partial ORF sequences of hamster insig-1 and -2 were amplified by PCR using first-strand cDNA from CHO-K1 cells. Pairs of forward and reverse primers whose sequences were highly conserved between human and mouse insigs are, respectively, as follows: hamster insig-1, 5'-CTGGTGCTCAACCTGCTGCAGATCCAG-3' and 5'-TCAATCACTATGGGGCTTTTCAGGAACACC-3'; and hamster insig-2, 5'-CATAAATTTAAAAGAGAGTGGTCCAGTG-3' and 5'-ATAACTTTGCATTCATACATTGCCAGTTG-3'. To obtain their entire ORF sequences, 5'- and 3'-rapid amplification of cDNA ends (RACE) were performed using SMART RACE cDNA Amplification Kit (CLONTECH) with poly(A)⁺ RNA of CHO-K1 cells. Pairs of outer and inner nested primers used for RACE are, respectively, as follows: 5'-RACE for hamster insig-1, 5'-ACAGGGGTACAGTAGGCCGACAACAGC-3' and 5'-GGGCACCCACCAGGCGGAGGAGAAGATGG-3'; 3'-RACE for hamster insig-1, 5'-TATTTTTCTCAGGAGGCGTCACAGTGGG-3' and 5'-TCAGGAGGCGTCACAGTGGGAAACATAG-3'; 5'-RACE for hamster insig-2, 5'-GATTCCAACCCCAAGGCCAAAACCAC-3' and 5'-TGATTGATACCTACGAACACCGCCACAC-3'; 3'-RACE for hamster insig-2, 5'-GTGTGGCGGTGTTCGTAGGTATCAATCAC-3' and 5'-GTGGTTTTGGCCTTGGGGTTGGAATC-3'. All PCR products were subcloned into pCRII vector (Invitrogen) and sequenced. Construction of Expression Plasmids.
pCMV-Insig-2-Myc encodes amino acids 1–225 of human insig-2, followed by six tandem copies of the c-Myc epitope tag (EQKLISEEDL) under control of the CMV promoter. To generate this plasmid, the coding region of human insig-2 (amino acids 1-225) was amplified by PCR using one EST clone encoding human insig-2 as a template. Forward and reverse primers used for PCR were as follows:

5'-TCGGATCCATGGCAGAAGGAGAGACAGAGTC-3' and 5'-TTGCTCAGAGCGGCCGCCTTCCTGATGAGATTTTTCTGC-3' (underlines denote BamHI and NotI sites). The PCR product was digested with BamHI and NotI and ligated into pcDNA3 vector between the BamHI and NotI sites. Then, a fragment encoding a NotI site, six tandem copies of the c-Myc epitope tag, a stop codon (TGA), and a XbaI site were introduced between the NotI and XbaI sites of the newly created plasmid. pCMV-Insig-1-Myc used in the current studies was created as follows: a BamHI-NotI fragment containing human insig-1 (amino acids 1–277) was released from the previously reported plasmid encoding a fusion protein of human insig-1(1-277) and six tandem copies of the c-Myc epitope tag (3). The resulting BamHI-NotI fragment was replaced with the BamHI-NotI fragment from pCMV-Insig-2-Myc. Both plasmids were sequenced in their entirety.

Northern Blot Analysis.
cDNA probes were prepared by PCR using first-strand cDNA from following RNAs: mouse liver poly(A)⁺RNA for mouse insig-1 (306 nt) and mouse insig-2 (400 nt) and total RNA of CHO-K1 cells for hamster insig-1 (405 nt) and hamster insig-2 (504 nt). Forward and reverse PCR primers used to generate these probes were, respectively, as follows: mouse insig-1, 5'-AGGACGACAGTTAGCTATGGGTG-3' and 5'-CCCCCTTACCCGACTCTCACATAC-3'; mouse insig-2, 5'-CTGGTGTGCCTTTTCCCGTTTCTA-3' and 5'-ACCTTGATCTGCCTGTGTTCCTGT-3'; hamster insig-1, 5'-AGGACGACAGTTAGCTATGGGTG-3' and 5'-CACAAAACTGCAGGTTGGTGATTC-3'; hamster insig-2, 5'-GTTTCTGCAGTCTGTGATTGCTTCTG-3' and 5'-CATACACCTCTGGTAATATCTGTC-3'. The PCR products were subcloned into pCRII vector (Invitrogen) and sequenced. Fragments were released by EcoRI digestion, purified on agarose gels, and radiolabeled with [α-³²P]dCTP using the Megaprime DNA Labeling System (Amersham Pharmacia Biotech). Total RNA was prepared using an RNA STAT-60 Kit, subjected to electrophoresis (10 μg per lane), and transferred to Hybond N⁺ membranes (Amersham Pharmacia Biotech). Filters were hybridized at 68° C for 1 h with radiolabeled probe (1.5 ´ 10⁶ cpm/ml for each probe) using the ExpressHyb Hybridization Solution (CLONTECH) according to the manufacturer’s instructions. Filters were exposed to Kodak X-Omat films with intensifying screens for the indicated time at –80° C.

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