mcherry polyclonal antibody  (Proteintech)

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

Journal: The EMBO Journal

Article Title: Hybrid endosomal coats contain different classes of sorting nexins

doi: 10.1038/s44318-026-00716-0

Figure Lengend Snippet: ( A ) Colocalization of Snx3 and SNX-BARs. Wild-type or vps35Δ cells co-expressing Vps5 yomCherry and Vps17 mNG , or Vps5 yomCherry and mNG Snx3 were analysed by a z-series of confocal images taken at a z-distance of 0.3 µm. Maximum projections were generated in ImageJ. Scale bar: 5 μm. ( B ) Pearson correlation coefficient (PCC) values were calculated using the JACoP plugin in the ImageJ software for a total of >80 cells from three independent experiments. See Fig. for biological replicate variability. Bars represent the mean values and standard deviation. A two-tailed unpaired t test was used to evaluate significance. **** P < 0.0001; *** P < 0.001; ** P < 0.01 and * P < 0.1. ( C ) Immunoprecipitation of mCherry-tagged Vps35 wt or Vps35 QGRE . Logarithmically growing vps35Δ cells expressing Vps35 mCherry (WT) or Vps35 QGRE-mCherry (Mut) from an integrative plasmid, and genomically tagged Snx3 V5 and Vps5 HA , were lysed in detergent. Proteins were pulled down with an affinity matrix to mCherry and analysed by SDS-PAGE and Western blotting against the indicated tags. ( D ) Quantification of the amount of Vps5 and Snx3 pulled down by mCherry-labelled Vps35 wt or Vps35 QGRE . The ratios between the signals for Vps35 and Snx3 or Vps5 were calculated. For Vps35 WT the ratio was set to 1 as a reference. Mean and standard deviation from five independent experiments are shown. A two-tailed unpaired t test was used to evaluate significance. **** P < 0.0001; *** P < 0.001; ** P < 0.01 and * P < 0.1. ( E ) Snx3 V5 was pulled down in an experiment as in ( C ), using vps35Δ cells expressing Vps35 mCherry (WT) or Vps35 QGRE-mCherry (Mut) from a plasmid and genomically tagged Snx3 V5 and Vps5 HA in the indicated combinations. ( F ) Quantification of Vps35 mCherry and Vps5 HA pulled down by Snx3 V5 . The ratios between the signals for Snx3 V5 and Vps35 mCherry or Vps5 HA were calculated. For Vps35 wt , the ratio was set to 1 as a reference. Mean and standard deviation from three to five independent experiments are shown. A two-tailed unpaired t test was used to evaluate significance. **** P < 0.0001; *** P < 0.001; ** P < 0.01 and * P < 0.1. .

Article Snippet: mCherry Polyclonal antibody , Chromotek , 26765-1-AP.

Techniques: Expressing, Generated, Software, Standard Deviation, Two Tailed Test, Immunoprecipitation, Plasmid Preparation, SDS Page, Western Blot

Journal: The EMBO Journal

Article Title: Hybrid endosomal coats contain different classes of sorting nexins

doi: 10.1038/s44318-026-00716-0

Figure Lengend Snippet: Logarithmically growing vps35Δ cells expressing genomically tagged Vps10 mNG were transformed with integrative plasmids expressing Vps35 mCherry (WT), Vps35 QGRE-mCherry , or nothing. The cells were logarithmically grown overnight and analysed by spinning disc microscopy. Scale bar: 5 µm.

Article Snippet: mCherry Polyclonal antibody , Chromotek , 26765-1-AP.

Techniques: Expressing, Transformation Assay, Microscopy