mcherry rabbit polyclonal antibody  (Huabio Inc)

Journal: mBio

Article Title: ATP1A1 enhances porcine reproductive and respiratory syndrome virus type 2 attachment and internalization

doi: 10.1128/mbio.03896-25

Figure Lengend Snippet: PRRSV-2 binds to ATP1A1 via interaction between GP4 C-terminus and ER4. ( A ) The predicted three-dimensional models of the complex of various PRRSV-2 structural proteins and ATP1A1. ( B ) Molecular docking analysis of ATP1A1 (blue) and GP4 (green). The interacting residues on ATP1A1 are highlighted in black dotted boxes and colored in yellow. ( C ) Analysis of the interaction between ATP1A1 and GP4 via Co-IP assay. Marc-145 cells were transfected with plasmids expressing cherry-GP4, and cells transfected with cherry-EV were used as a negative control. Cell lysates were immunoprecipitated with rabbit anti-ATP1A1 pAb, followed by immunoblotting with rabbit anti-mCherry pAb or rabbit anti-ATP1A1 pAb to reveal GP4 and ATP1A1, respectively. ( D ) Domain structure of four ATP1A1 deletion mutants. ( E ) Domain mapping of GP4 interacting with ATP1A1. Marc-145 cells were transfected with plasmids expressing four GP4 mutants. Cell lysates were immunoprecipitated with rabbit anti-ATP1A1 pAb, followed by immunoblotting with rabbit anti-mCherry pAb or rabbit anti-ATP1A1 pAb to reveal GP4 and ATP1A1, respectively. ( F ) The 2D model illustration of green monkey ATP1A1 was determined from the prediction of transmembrane regions ( https://www.novopro.cn/tools/tmhmm.html ). ( G ) Purification of ATP1A1 ER proteins. The above five extracellular regions of ATP1A1 were cloned into expression vector pGEX4T and further transferred into Escherichia coli BL21 (DE3) strain, followed by IPTG induction. After purification, the purified proteins were subjected to SDS-PAGE analysis. ( H ) Identification of the interaction between GP4 and the extracellular regions of ATP1A1 in GST pulldown. The purified recombinant ATP1A1 ER proteins were coupled to GST beads at 4°C for 2 h, where GST served as control. The beads were incubated with soluble cherry-GP4 protein derived from the supernatant of HEK-293T cells lysate (transient transfection in 6-well plates) at 4°C for overnight. The eluted samples were subjected to IB and detected by anti-mCherry pAb and anti-GST mAb. ( I ) The comparison of ATP1A1-ERs effect on PRRSV-2 replication. Indicated peptides (50 μg/mL) were pre-incubated with PRRSV HuN4-F112 (200 TCID 50 ) at 37°C for 2 h. Then, the mixture was added to Marc-145 cells and replaced with DMEM medium containing 2% FBS after 1 h incubation at 37°C. At 24 hpi, virus replication was analyzed by qRT-PCR. ( J and K ) GST-ER4 peptide blocks PRRSV-2 infection in a dose-dependent manner. The GST-ER4 peptide in various concentrations was incubated with PRRSV HuN4-F112 (200 TCID 50 ) at 37°C for 2 h. Then, the mixture was added to Marc-145 cells and replaced with DMEM medium containing 2% FBS after 2 h incubation. At 24 hpi, virus replication was identified in IFA ( J ) and qRT-PCR ( K ). ( L and M ) The pre-entry assay. The GST-ER4 peptide (100 μg/mL) was incubated with PRRSV HuN4-F112 (200 TCID 50 ) at 37°C for 2 h. Then, the mixture was added to Marc-145 cells and replaced with DMEM medium containing 2% FBS after 2 h incubation. At 24 hpi, virus replication was detected by qRT-PCR ( L ), and viral titers were determined at 48 hpi ( M ). ( N and O ) The post-entry assay. Marc-145 cells were infected with PRRSV HuN4-F112 (200 TCID 50 ) at 37°C for 2 h. Then, the cells were inoculated by GST-ER4 peptide (100 μg/mL), followed by washing three times with PBS after 2 h incubation. After this, the peptide-containing medium was replaced with DMEM containing 2% FBS. At 24 hpi, virus replication was detected by qRT-PCR ( N ), and viral titers were determined by detecting TCID 50 at 48 hpi ( O ). Significant differences were indicated as follows: ns ( P > 0.05), * ( P < 0.05), ** ( P < 0.01), *** ( P < 0.001), and **** ( P < 0.0001).

Article Snippet: Phospho-Src-Y419 rabbit mAb (AP1027), EGFR rabbit pAb (A11351), Phospho-EGFR-Y845 rabbit pAb (AP0023), Caveolin-1 rabbit pAb (A1555), Phospho-Caveolin-1-Y14 rabbit pAb (AP0742), rabbit anti-GST-Tag mAb (AE077), β-actin mouse mAb (AC026), and the mCherry rabbit polyclonal antibody ( HA500049 ) was obtained from HUABIO (Hangzhou, China).

Techniques: Co-Immunoprecipitation Assay, Transfection, Expressing, Negative Control, Immunoprecipitation, Western Blot, Purification, Clone Assay, Plasmid Preparation, SDS Page, Recombinant, Control, Incubation, Derivative Assay, Comparison, Virus, Quantitative RT-PCR, Infection