File S1 - Insight into Buffalo (Bubalus bubalis) RIG1 and MDA5 Receptors: A Comparative Study on dsRNA Recognition and In-Vitro Antiviral Response

Supporting Text, Tables, and Figures. Text S1: Methodology for isolation and culture of buffalo foetal fibroblast cells. Text S2: Procedures for model construction and simulation analysis. Text S3: Deduced amino acid sequence of buffalo RIG1 and MDA5. Table S1: PCR cycling parameters used for the amplification of RLR genes. Table S2: Sequences (NCBI Accession numbers) used for phylogenetic analysis. Table S3: Comparative analysis of the stereo-chemical parameters of RIG1, MDA5 and their corresponding templates. Figure S1: Agarose gel electrophoresis of amplified products of different fragments of buffalo RIG1 and MDA5 genes. Figure S2: Overall tertiary structure of helicase and C-terminal domains of (A) RIG1 and (B) MDA5 proteins of buffalo. The helicases and C terminal domains of buffalo RIG1 and MDA5 were modeled based on the X-Ray crystallographic structures of human RIG1 and MDA5 (termed as templates). The pair-wise alignment of buffalo and human sequences showed 82 and 84 percent sequence identities for RIG1 and MDA5, respectively. Due to this striking homology between target and template proteins, the built models retained all the key structural features of the templates that included HEL-1, HEL-2, HEL-2i, and the C-terminal domains. Figure S3: Ramachandran plot of buffalo (A) RIG1 and (B) MDA5 receptors. Stereochemical qualities of modeled proteins were found to be highly comparable to those of templates, indicating quality of the models were reasonably good to carry out further studies. Figure S4: Stability parameters of RIG1 and MDA5 receptors as a function of simulation time. (A) RMSD of RIG1. (B) RMSD of MDA5. (C) RMSF of RIG1. (D) RMSF of MDA5. In each panel, red color indicates stability parameters of dsRNA bound receptors and black color denotes receptors without dsRNA. Figure S5: Western analysis of intestinal tissue to determine specificity and cross reactivity of primary antibodies to RIG1 and MDA5. Dilution of primary antibodies 1∶500; Dilution of secondary anti goat IgG 1∶50000. The chemiluminescence was detected on x-ray films using Immobilon Western Chemiluminescent HRP (Millipore Corporation, MA, USA) as substrate. (PDF)