cell culture hek 293t  (ATCC)

Journal: Journal of Virology

Article Title: ALDH1L1 suppresses the replication of porcine epidemic diarrhea virus by degrading viral nucleocapsid and envelope proteins

doi: 10.1128/jvi.01933-25

Figure Lengend Snippet: ALDH1L1 interacts with and degrades PEDV N and E proteins via the selective autophagy pathway. ( A and B ) HEK 293T cells were transfected with plasmids coding HA-N or HA-E and Flag-ALDH1L1. Co-IP assay was performed using anti-Flag-bound beads. ( C and D ) After inducing Flag-ALDH1L1 and GST-N or GST-E expression in the BL21(DE3) bacterial strain, the associations of ALDH1L1 with N or E proteins were analyzed via the GST pull-down assay. ( E and F ) After transfection with HA-N or HA-E and Flag-ALDH1L1, HeLa cells were labeled with antibodies and nuclear DAPI for confocal immunofluorescence microscopy. Scale bars = 100 µm. ( G and H ) After transfecting HEK 293T cells with varying concentrations of Flag-ALDH1L1, HA-N, or HA-E, western blotting was performed to assess N and E protein expression. ( I and J ) HEK 293T cells were transfected with plasmids coding the Flag-ALDH1L1 plasmid and HA-N or HA-E, followed by treatment with BafA1 (50 µM), CQ (50 µM), MG132 (5 µM), or 3-MA (1 mM) for 10 h. The resulting cellular lysates were subjected to western blotting.

Article Snippet: Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, D6429) supplemented with 10% fetal bovine serum (Gibco, 10099141) was used to culture human embryonic kidney HEK 293T cells (ATCC, CRL-11268) and African green monkey kidney Vero cells (ATCC, CCL-81).

Techniques: Transfection, Co-Immunoprecipitation Assay, Expressing, Pull Down Assay, Labeling, Immunofluorescence, Microscopy, Western Blot, Plasmid Preparation

Journal: Journal of Virology

Article Title: ALDH1L1 suppresses the replication of porcine epidemic diarrhea virus by degrading viral nucleocapsid and envelope proteins

doi: 10.1128/jvi.01933-25

Figure Lengend Snippet: ALDH1L1 inhibits PEDV replication via the E3 ubiquitin ligase STUB1. ( A ) After HEK 293T cells were cotransfected with plasmids encoding HA-STUB1 and Flag-ALDH1L1, the Co-IP assay was performed using anti-Flag-bound beads. ( B ) After HEK 293T cells were transfected with plasmids coding Flag-ALDH1L1, Co-IP assays were performed using an anti-Flag antibody. ( C ) After inducing GST-STUB1 and ALDH1L1 expression in the BL21(DE3) strain, the interaction between ALDH1L1 and STUB1 was examined via the GST pull-down assay. ( D ) After cotransfecting HeLa cells with Flag-ALDH1L1 and STUB1-HA for 24 h via incubation with an anti-Flag Mab, ALDH1L1 and STUB1 colocalization was confirmed via confocal immunofluorescence microscopy. Scale bars = 100 µm. ( E and F ) After cotransfection with Myc-ALDH1L1, HA-Ub, HA-STUB1, and Flag-N, or Flag-E, HEK 293T cells were treated with CQ and collected. The ubiquitinated N and E proteins were subjected to western blotting after immunoprecipitation with an anti-Flag antibody. ( G and H ) After cotransfecting HEK 293T cells with siRNAs (negative control or STUBA siRNA) and HA-N or HA-E and Flag-ALDH1L1-coding plasmids, western blotting was performed with an anti-HA antibody.

Article Snippet: Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, D6429) supplemented with 10% fetal bovine serum (Gibco, 10099141) was used to culture human embryonic kidney HEK 293T cells (ATCC, CRL-11268) and African green monkey kidney Vero cells (ATCC, CCL-81).

Techniques: Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Transfection, Expressing, Pull Down Assay, Incubation, Immunofluorescence, Microscopy, Cotransfection, Western Blot, Immunoprecipitation, Negative Control

Journal: Journal of Virology

Article Title: ALDH1L1 suppresses the replication of porcine epidemic diarrhea virus by degrading viral nucleocapsid and envelope proteins

doi: 10.1128/jvi.01933-25

Figure Lengend Snippet: ALDH1L1 suppresses PEDV replication via the cargo receptor TOLLIP. ( A ) After cotransfecting HEK 293T cells with plasmids coding HA-TOLLIP and Flag-ALDH1L1, a Co-IP assay was performed with anti-Flag-bound beads. ( B ) After transfecting HEK 293T cells with plasmids encoding Flag-ALDH1L1, Co-IP assays were performed using an anti-Flag antibody. ( C ) After inducing GST-TOLLIP and ALDH1L1 expression in the BL21(DE3) strain, the interaction between ALDH1L1 and TOLLIP was examined via the GST pull-down assay. ( D ) HeLa cells were transfected with plasmids coding Flag-ALDH1L1 and HA-TOLLIP. Then, the cells were incubated with specific antibodies and visualized under a confocal immunofluorescence microscope. Scale: 100 µm. ( E and F ) After cotransfecting HEK 293T cells with siRNAs (negative control or TOLLIP siRNA) and HA-N or HA-E and Flag-ALDH1L1-coding plasmids, western blotting was performed using an anti-HA antibody.

Article Snippet: Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, D6429) supplemented with 10% fetal bovine serum (Gibco, 10099141) was used to culture human embryonic kidney HEK 293T cells (ATCC, CRL-11268) and African green monkey kidney Vero cells (ATCC, CCL-81).

Techniques: Co-Immunoprecipitation Assay, Expressing, Pull Down Assay, Transfection, Incubation, Immunofluorescence, Microscopy, Negative Control, Western Blot