Some chickens have silky-feather because of a loss of hooklets in pennaceous feathers, while most chickens have the wild-type normal feather. The feather is an excellent model for evolution and development due to its complex structure and vast diversity. Red staining indicates that PDSS2 protein is localized in the cytoplasm. The cPDSS2 antibody also recognizes the endogenous cPDSS2 protein with weak signals (red), as indicated by arrowheads in GFP-negative DF1 (D, F) cells. The eGFP-cPDSS2 fusion protein (green) is fully consistent with cPDSS2 antibody pattern (red), as indicated by arrows in transfected GFP-positive 293T (C) and DF1 (D) cells. (C–F) Immunofluorescence experiments in 293T and DF1 cells. 293T-WT, wild-type 293T cells DF1-WT, wild-type DF1 cells 293T-Trans, 293T cells transfected with pcDNA-eGFP-cPDSS2. The cPDSS2 antibody also binds to the endogenous PDSS2 protein from the DF1 (predicted molecular weight of cPDSS2: 41.2 kDa) and 293T cells (predicted molecular weight of human PDSS2: 44.1 kDa). (B) Western blotting analysis demonstrates that both cPDSS2 and GFP antibodies bind to the eGFP-cPDSS2 fusion protein (predicted molecular weight: 68.1 kDa) from the transfected 293T cells. (A) The chicken amino acid residues used for generating the anti-chicken PDSS2 antibody and its homologous sequence in human. Determining the exact transcription start site of PDSS2 with 5′ RACE is technically challenging due to the high GC content (75.8%) in 339-bp sequence of the presumed 5′ UTR and exon 1 at 70,486,623–70,486,961 bp.įigure S8: The chicken PDSS2 antibody is specific to the PDSS2 protein. Furthermore, PDSS2(-103C-G) is contained in a few clones. Most of the 5′ ends are located between position −81 and −96 around the most common position −90. The most common start site (70,486,636 bp) is highlighted in bold and indicated by the black arrow. The causative mutation PDSS2(-103C-G) (70,486,623 bp) is highlighted in uppercase and indicated by the red arrow. The nucleotide positions relative to the ATG are marked with solid circle under each site. The A of the translation start site (ATG) is defined as position +1. The 5′ ends of clones are indicated by the vertical lines with Arabic numerals to indicate the number of clones isolated for each site. The total number of random RACE clones for each sample is included in the bracket. Three wild-type ( H/H) and three silky-feather ( h/h) dorsal skin tissues are used for 5′ RACE analysis as described in Materials and Methods. Figure shows the DNA, mRNA and protein information of part of PDSS2 in skin. Figure S3: The 5′ RACE analysis of PDSS2 in skin tissue.
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