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. 2009 Aug 4;18(19):3553–3566. doi: 10.1093/hmg/ddp304

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© 2009 The Author(s)

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 4. — Analysis of PKD1L2 protein in skeletal muscle. (A) Domain composition of PKD1L2 with the annotated position of peptides used to raise antibodies APKD1L2_1 and APKD1L2_2. Functional domains as predicted by SMART (http://smart.embl-heidelberg.de/). C-LECT, C-type lectin (CTL) or carbohydrate-recognition domain (CRD); Pfam Gal_Lectin, galactose-binding lectin domain; PKD, PKD domain; GPS, G-protein-coupled receptor proteolytic site domain; LH2, lipoxygenase homology 2 (beta barrel) domain; blue rectangles, transmembrane domains; pink dots, low-complexity region. (B) Western blot analysis using PKD1L2 antibodies. Both antibodies APKD1L2_1 and APKD1L2_2 recognize a ∼268 kDa protein in wild-type soluble skeletal muscle extracts. APKD1L2_2 recognizes other bands of smaller size. WB, antibody used for western blot. (C) Immunoprecipitation and peptide analysis using APKD1L2_1 antibody on wild-type skeletal muscle extract. APKD1L2_1 specifically immunoprecipitates a ∼268 kDa band (left panel) that is also recognized by APKD1L2_2 (middle panel). The right panel shows a Coomassie-stained gel in which the immunoprecipitated protein complex has been resolved. Proteins identified by mass spectrometry are indicated. IP, immunoprecipitation; WB, western blot; Lys, lysate; IP+, immunoprecipitated sample; IP−, negative control. (D) Reciprocal co-immunoprecipitation of PKD1L2 and FASN in skeletal muscle. Western blot analysis using AFASN antibody specifically recognizes a ∼268 kDa band from protein complexes immunoprecipitated using APKD1L2_1 antibody in skeletal muscle soluble fraction, from both wild-type and ostes/ostes mice. Western blot analysis using APKD1L2 antibody specifically recognizes a ∼268 kDa band from protein complexes immunoprecipitated using AFASN antibody in skeletal muscle soluble fraction, from both wild-type and ostes/ostes mice. PKD1L2 and FASN co-immunoprecipitation was also observed in membrane fractions (bottom panels). IP, immunoprecipitation; WB, western blot; Lys, lysate; IP+, immunoprecipitated sample; IP−, negative control.