Journal: mBio

Article Title: Quantitative proteomic analysis identifies the unfolded protein response as a host pathway co-opted by ASFV to promote replication

doi: 10.1128/mbio.03242-25

Figure Lengend Snippet: D117L activates three UPR pathways through targeting key UPR components. ( A ) Heatmap of ASFV proteins identified by proteomics analysis across infection. ( B ) PPI network analysis of the virus-host interaction. ( C ) HEK293T cells were transfected with vector or Flag-D117L for 24 h, and mRNA expression of Ddit3, Dnajc3, and Dnajb9 was detected by RT-qPCR. ( D ) HEK293T cells were transfected with vector, Flag-D117L, Flag-D117LΔ38-60, Flag-D117LΔ67-76, or Flag-D117LΔ96-117; each group was also co-transfected with ATF6-LUC and Renilla-TK. After 24 h post-transfection, luciferase activities were examined. ( E ) Mass spectrometry was performed to identify the interacting proteins of D117L and D117LΔ38-60, and the top 10 proteins were listed. ( F ) Kyoto Encyclopedia of Gene and Genomes pathway enrichment analysis of D117L interacting proteins. ( G and H ) Immunofluorescence analysis of the colocalization of Flag-tagged D117L (green) and Myc-tagged or endogenous HSPA5 (red) in iPAM cells. ( I ) Immunoprecipitation (IP) assay examining the interaction between HSPA5 and D117L. ( J and K ) IP assays assessing the interaction of D117L (WT) or D117LΔ38-60, D117LΔ67-76, and D117LΔ96-117 mutants with CANX or HSPA5 in HEK293T cells.Data are presented as mean + SD. PP values were calculated by a two-tailed unpaired t-test. ** P < 0.01, *** P < 0.001.

Article Snippet: For immunoprecipitation analysis, the HEK293T cells were homogenized with IP lysis buffer (containing 50 mM Tris-Cl [pH = 7.4], 150 mM NaCl, 1%Triton-100, 0.1% SDS, 1% sodium deoxycholate, and protease inhibitor cocktail), centrifuged to remove cell debris, and then the supernatant was collected for immunoprecipitation with Myc (HY-K0206, MCE) and DYKDDDDK magnetic beads (b26103; Selleck Chemicals, USA) at 4°C for 2 h. After washing five times with IP lysis buffer, the protein-bound beads were finally resuspended in SDS-PAGE loading buffer.

Techniques: Infection, Virus, Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Luciferase, Mass Spectrometry, Immunofluorescence, Immunoprecipitation, Two Tailed Test