All strategies are reported relative to ARRIVE guidelines (https://arriveguidelines.org) for the reporting of pet experiments. Plasmids, antibodies, and cells The C11orf96 gene was cloned in to the pCMV-HA/MYC vector as well as the p3*Flag-10/14 vector (Clontech) utilizing the Clon Express Ultra One Stage Cloning Kit (Vazyme, China) to get the pHA-Felis catus C11orf96 plasmid, pMYC-Felis Rabbit polyclonal to Osteocalcin catus C11orf96 plasmid, pHA-mouse C11orf96 plasmid, pMYC-mouse C11orf96 plasmid, p3*Flag-10-Homo sapiens C11orf96 plasmid, and p3*Flag-14-Homo sapiens C11orf96 plasmid. and it is conserved in various mammals relatively. From bioinformatics evaluation, we discovered that C11orf96 is normally abundant with Ser and provides multiple forecasted phosphorylation sites. Furthermore, proteins connections prediction evaluation revealed which the proteins is connected with many transmembrane family members zinc and protein finger protein. Subsequently, we discovered that C11orf96 is distributed in the cytoplasm strictly. Based on the tissues distribution characteristics, C11orf96 is normally distributed in every organs and tissue, with the best expression amounts in the kidney. These total results indicate that C11orf96 may play a particular natural role in the kidney. Conclusions Summarizing, these data place the building blocks for learning the biological features of C11orf96 as well as for discovering its function in viral replication. Supplementary Gefitinib hydrochloride Details The online version contains supplementary material available at 10.1186/s12917-022-03224-5. gene in the bait vector pEASY?-Blunt Zero Cloning Kit (abbreviated as pEBCK) was obtained by RT-PCR, and 1% agarose gel electrophoresis showed that the size of the cDNA fragment amplified by PCR was in the expected range. The target fragment size of 372?bp (Fig.?1A) was confirmed by sequencing analysis. Sequencing analysis also confirmed that this place was the C11orf96 CDS, indicating Gefitinib hydrochloride successful construction of the bait vector pEBZCKCFelis catus C11orf96, pEBZCK-mouse C11orf96, and pEBZCK-homo sapiens C11orf96. Blast search and comparison with the NCBI nucleotide sequence database revealed that this CDS was completely consistent with the C11orf96 CDS region sequence (“type”:”entrez-nucleotide”,”attrs”:”text”:”XM_006937308.4″,”term_id”:”1304933100″,”term_text”:”XM_006937308.4″XM_006937308.4), mouse C11orf96 (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_001145034.1″,”term_id”:”222537762″,”term_text”:”NM_001145034.1″NM_001145034.1), and C11orf96 (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_001145033.2″,”term_id”:”1547055269″,”term_text”:”NM_001145033.2″NM_001145033.2) used in the design of the cloning primer. Moreover, we successfully constructed eukaryotic expression plasmids of C11orf96 (pHA-fC11orf96, pMYC-fC11orf96, pHA-mC11orf96, pMYC-mC11orf96, pFlag-hC11orf96, and phC11orf96-Flag). After these eukaryotic plasmids were transfected into 293?T cells, the cell lysate was collected for WB assay. The results showed that C11orf96 eukaryotic plasmids were effectively expressed (Fig.?1B). Open in a separate window Fig. 1 Gene cloning and protein expression of the gene from different sources. A?PCR amplification derived from different cDNAs of C11orf96 is 1201?bp, with 3 introns and 3 exons. The untranslated regions (UTRs) are located at 118C131, 615C760, and 780C1201?bp; the CDS region is located at 243C614?bp and is 372?bp long (Fig.?2A). The cloned sequence has 8 ORFs, among which the full-length open reading frame ORF1 (the complete CDS region) is usually 372?bp, which encodes 124 amino acids, including predicted phosphorylation sites (Tyr: 3, Ser: 15). The protein sequence does not contain a transmission Gefitinib hydrochloride peptide and does not have a transmembrane region (Fig.?2B). The top five amino acids are Ser (13.82%)? ?Leu (10.57%)? ?Glu Gefitinib hydrochloride (9.76%)? ?Arg (8.13%)? ?Lys (7.32%). The detailed amino acid composition ratio is usually shown in Figs. ?Figs.2C2C and D. The molecular excess weight is usually 13.80?kDa, the isoelectric point (pI) is 8.4, and the molecular formula is C592H970N174O189S8. The detailed physical and chemical properties are shown in Table S1. The protein secondary structure prediction revealed that this C11orf96 protein consists of four structures: -helix, -change, random coil, and extended chain, which account for 61%, 4%, 33%, and 2% of the protein structure, respectively (Fig.?2E-F). Protein conversation prediction analysis showed that this C11orf96 protein may interact with multiple proteins in the host, including the TMEM117 transmembrane protein that regulates endoplasmic reticulum (ER) stress, several other transmembrane proteins, E3 ubiquitin ligase, and zinc finger proteins (Fig.?2G). These results indicate that this C11orf96 protein may play a role in cellular processes such as ER stress, protein ubiquitination modification, and gene transcription. Open in a separate windows Fig. 2 Analyses of biological characteristics of C11orf96. A?Structural diagram of the gene (sequence data are available from GenBank: accession No. “type”:”entrez-nucleotide”,”attrs”:”text”:”XM_006937308.4″,”term_id”:”1304933100″,”term_text”:”XM_006937308.4″XM_006937308.4). B?The nucleotide and deduced.