Table S1. Primers used in the study

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Table S1. Primers used in the study Primer name Application Sequence I1F16 Genotyping GGCAAGTGAGTGAGTGCCTA I1R11 Genotyping CCCACTCGTATTGACGCTCT V19 Genotyping GGGTCTCAAAGTCAGGGTCA D18Mit184-F Genotyping CACACATGTGTAGGTAGGTAGGTAGG D18Mit184-R Genotyping CGCACAAGGACTACTGAAACA Probe-1 Probe for Southern TTGGTCATGTTCTGGTTTGG Probe-1a Probe for Southern TGTTATCCAGTCCTGCCACAA Lman1 F1 Standard PCR ACGTGGAATGGTGTTGGAAT Lman1 R1 Standard PCR GCTCGGTCAGTTGGAAAGTC Lman1 F2 Standard PCR CCAGGAGAGGAGCAGGAAC Lman1 R2 Standard PCR TCTGGGCATTTTGGTTTTTC Lman1 F3 Standard PCR ATATCGACAGCCTCGCACAG Lman1 R3 Standard PCR AGCTGCTTCTTGCTGAGTCC Gapdh-F Real-time PCR TGCACCACCAACTGCTTAG Gapdh-R Real-time PCR GATGCAGGGATGATGTTC Mcfd2 RT1s Real- time PCR CACGACCAAGAGCACATCAT Mcfd2 RT1as Real- time PCR CTCTAGGCCGTCAAGCAAAC Sec23a-F Standard PCR GGACTGACGACTGTGCAAGG Sec23a-R Standard PCR GGGACCACGCAGAACTACAT

The image cannot be displayed. Your computer may not have enough memory to open the image, or the image may have been corrupted. Restart your computer, and then open the file again. If the red x still appears, you may have to delete the image and then insert it again. A. Lman1 intron 1 around the vector insertion site AGCTGATTCTGCTTTCACAGGTTTTCCTAGAAACTGCCCAACTCATCCCATGTACCCTGTAACTGTAGGCAAGTGAGT I1F16 GAGTGCCTAGGAATCTATTAAGAGGCCAGTCCTTGTATACTAAAAATGTATTTCATAAGCAGGTCTGTACTTCCAAGAC GTAATAATGTTAAATGTGTGCTTATGAGAAAGTTGTACATGTTTTATATAAATTAAATTGTAATCCTTAATTGATGTTTTC AGGGGACAATTTAGATGCCCGTGGTGTTAAGATAAATTGCAATTGGTTTCTGACTGAGAAAGCCCCAGGTATTTTGTG GTTAGTGTTAAGTTTGTGCTGCTCATTCAAGTCAGATGGAACTCTTAAATTTAACTTAGTCTCTCTCTCTCTCTCACTCA CACACACACACACACTCACACTCACACTCACCCAGCATGACATAACTCTGACACTTTCTTTTCTTTTGTTTCATAGCTC Deleted in XST1 CTGCCTTTTACACTAAAATTCACACGGAGGAAGTGAAAGTTAAAACTTGATGGATTCATCTAAAAAGAGCCAGTATTGC TGATAGAAAGTCAAGATGGGAACCCTTCTCACAGAGCGTCAATACGAGTGGGAAAGTCTGCTGTTAGGGG I1R11 5 end of pgt2tmpfs vector sequence inserted into Lman1 intron 1 TAGCCCGGATGGCCTTTTCCTGCACGGCACCATATGAACCTTGTGACCCTGACTTTGAGACCCCTCTAACCCAAGGCC V19 B. Three-primer genotyping (I1F16, I1R11, V19) Two-primer genotyping (D18mit184) 5bp 4bp 2bp 1bp Figure S1. (A) Sequence around the insertion site in intron 1 of the Lman1 gene and the 5 end of the vector sequence inserted into intron 1. The locations of genotyping primer sequences are underlined. The insertion site is indicated by an arrow head and genomic sequence deleted in the gene-trap allele is underlined. (B) Representative gels of the two genotyping assays, performed on DNA samples prepared from mouse tail biopsies. Each lane represents an individual mouse.

A. Southern blot strategy EcoRV probe I1F16 I1R11 EcoRV 3149bp 1443bp Ex 9 Ex 1 Ex 11 4592 bp, WT allele EcoRV probe I1F16 3149bp Ex 9 Ex 1 V19 2bp EcoRV 5149 bp, KO allele B. Southern blot analysis C. RT-PCR efficiency cdna dilution factor: 1 1:1 1:5 1:1 1:5 1:1 1:5 1:1 1 1:1 1:5 1:1 1:5 1:1 1:5 1:1 6 kb 5 kb 4kb Lman1 (F2+R2) Sec23a 3kb Lman1 Lman1 Figure S2. (A) Southern blot strategy. Genomic DNA was digested with EcoRV and hybridized with a probe located to the 5 of the insertion site. The expected sizes of EcoRV fragments from the WT and the knockout alleles are shown. (B) Southern blot analysis of DNA prepared from mice heterozygous () or homozygous () for the Lman1 gene-trap allele. (C) RT-PCR reactions were performed on serial dilutions of cdnas from WT and Lman1 liver using primers flanking exons 1 and 11. Sec23a RT-PCR served as a positive control.

Relative mrna level 2 18 16 14 12 1 8 6 4 2 Lman1 Mcfd2 Figure S3. LMAN1 expression profile. Quantitative RT-PCR analysis of Lman1 and Mcfd2 RNA levels in different organs of WT pups. Total RNA was prepared from organs dissected from 2-5 E18.5 pups delivered by Cesarean section. The normalized mrna levels in both Lman1 and Mcfd2 are plotted as fold increases of the level from the heart, which is set as one. Error bars represent one standard deviation.

a b c d e f g h i j k l m n o p Fig. S4

Figure S4. X-gal staining of select organs of Lman1 mice (a) () Brain, positive staining in all five layers including molecular, granular, pyramidal cell, inner granular, ganglion g cell layer. (b) Salivary glands, positive staining in both serous and mucous acini. (c) Heart, showing cardiomyocytes of ventricular myocardium. (d) Stomach, highest expression in the epithelial cells of the pyloric glands. (e) Lung, highest expression in the bronchiolar and alveolar epithelium. (f) Liver, showing liver cell cord, with strong staining in hepatocytes. (g) Pancreas, positive staining in both islets and acini. (h) Eye, highest expression in the retinal layer. (i) Colon, highest expression in lamina propria and adjacent glands. (j) Small intestine (jejunum), highest expression in the epithelium of the intestinal villi. (k) Kidney, highest expression in proximal tubule and distal convoluted tubule. (l) Adrenal glands, strong expression in all cell layers of cortex. (m) Testis, highest h expression in spermatogonia, spermatocytes t and Leydig cells. n) Seminal vesicles, highest expression in the epithelium. o) Ovary, strong expression in corpus luteum. p) Uterus, positive staining in both endometrial and smooth muscles.

A 2 Rela ative mrna leve el 1.5 1.5 LMAN1 LMAN1 / B RP78 protein 3 25 2 P<.1 Re elative level of G 15 1 5 Lman1 Lman1 Figure S5. (A) Quantitative RT-PCR analysis of ER stress and UPR markers in liver RNA prepared from 3 WT and 3 Lman1 mice. Primer sequences were reported previously (49). Xbp1-s is the alternatively spliced transcript of Xbp1 mrna. (B) The relative levels of GRP78 shown in Fig. 4B were quantified by densitometry and normalized to -actin.

Th i t b di l d Y t t h h t th i th i h b t d R t t t d th th fil i If th d till h t d l t th i d th i t it i A B Lman1 Lman1 Lman1 Lman1 CatC AAT (mouse) CatZ LMAN1 LMAN1 LMAN1 Actin AAT (human) Ca atc Level (%) 14 14 12 1 8 6 4 2 C atz Level (%) 12 1 8 6 4 2 se AAT Level (%) Mou 15 15 1 5 Huma an AAT Level (%) 1 5 Figure S6. Liver cathepsin C (CatC), cathepsin Z (CatZ) and plasma a1-antitrypsin (AAT) levels. (A) Liver lysates were prepared from WT and Lman1 mice and analyzed by immunoblotting with the indicated antibodies. The relative levels were quantified and Normalized to -actin. No significant differences were observed for CatC and CatZ levels in WT and Lman1 mouse livers. (B) Equal amounts of mouse and human plasma proteins were separated by SDS-PAGE and immunoblotted with anti-aat antibodies. The relative levels of ATT in the blots were quantified and plotted. Error bars show standard deviations. Each lane contains a sample from an individual mouse or human. AAT levels are not altered in Lman1 mice and in F5F8D patients with an LMAN1 mutation.

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