Journal: Aging Cell
Article Title: Late‐onset retinal degeneration pathology due to mutations in CTRP5 is mediated through HTRA1
doi: 10.1111/acel.13011
Figure Lengend Snippet: CTRP5 interacts with HTRA1. (a) Clone 53 (CL53) containing a partial sequence of HTRA1 was identified as a CTRP5 interactor during Y2H screening of a normalized human cDNA library cloned into a prey vector, pGADT7‐RecAB, containing the activation domain (AD) of GAL4. Full‐length WT‐CTRP5 fused to the binding domain (BD) of GAL4 (BD‐WT‐CTRP5) was used as bait. An empty bait plasmid was used as a control to exclude false‐positive interactions. Yeast cells carrying the proper combinations of bait and prey plasmids were plated on semi‐selective (SD‐Leu‐Trp, i.e., selective only for the bait and prey plasmids) and selective (SD‐Leu‐Trp‐His, i.e., selective for the bait and prey plasmids and the protein–protein interaction) media supplemented with 3‐AT (1, 2, and 5 mM). (b) Schematic of full‐length HTRA1 with domain organization in comparison with the corresponding sequence (aa 306–480) of clone 53 obtained from Y2H library screening for CTRP5 interactors. The follistatin (FS) domain is a combination of the IGF‐binding protein (IGFBP) and the Kazal‐type serine protease inhibitor (KI) domains.The position of HTRA1 catalytic triad (Ser328, His220, and Asp250) is indicated. (c) HTRA1 interacts with both WT‐CTRP5 and S163R‐CTRP5 with similar binding affinities in the Y2H assay. Yeast cells containing AD‐CL53 HTRA1 (306–480aa) and BD‐WT‐CTRP5 or BD‐S163R‐CTRP5 constructs were plated at serial 10‐fold dilutions with a starting concentration of OD 600 of 0.8 on semi‐selective (SD‐Leu‐Trp) and on selective (SD‐Leu‐Trp‐His) media supplemented with 10 mM 3‐AT. (d) Co‐Immunoprecipitation assay for confirmation of CTRP5 and HTRA1 interaction. Lysates from ARPE‐19 cells overexpressing WT‐CTRP5‐V5 or S163R‐CTRP5‐V5 constructs were used for immunoprecipitation (IP) using HTRA1 antibody. Immunoprecipitates were resolved on 10% SDS‐PAGE followed by immunoblotting (IB). Blots were probed with anti‐V5 and HTRA1 antibodies. Normal rabbit IgG (IgG control) was used as a negative control to determine the antibody specificity. Lysates of untransfected cells were used as control for overexpression. Input has 10% of total lysates. Cells transfected with the empty vector were used as a transfection control in the experiment. (e) Reverse co‐immunoprecipitation was performed using lysates from cells overexpressing WT‐CTRP5‐V5 or S163R‐CTRP5‐V5 constructs. Immunoprecipitation was done using anti‐V5 antibody, and proteins were detected in immunoblots using anti‐V5 and HTRA1 antibodies
Article Snippet: Thus, the BD‐CTRP5 construct was used in Y2H screening against a universal, normalized human cDNA library (TaKaRa).
Techniques: Sequencing, cDNA Library Assay, Clone Assay, Plasmid Preparation, Activation Assay, Binding Assay, Library Screening, Protease Inhibitor, Y2H Assay, Construct, Concentration Assay, Co-Immunoprecipitation Assay, Immunoprecipitation, SDS Page, Western Blot, Negative Control, Over Expression, Transfection
