npc1 antibody  (Sanying Ltd)

Journal: The Journal of Biological Chemistry

Article Title: Real-time monitoring of secretory protein traffic for investigating trafficking regulators of ACE2 in living cells

doi: 10.1016/j.jbc.2025.110593

Figure Lengend Snippet: Identification of NPC1 as the lysosomal adaptor for ACE2 through HiBiT-integrated RUSH. A , schematics of the screening strategy for ACE2 trafficking cofactors using Str–Ii_HiBiT-ACE2-SBP-FLAG-transfected 293T cells by coimmunoprecipitation/mass spectrometry (co-IP/MS). B , Gene Ontology (GO) enrichment analysis of secretion-related cellular compartment using the Enrichr platform . C , immunoblotting analysis of ACE2 levels in ACE2-GFP-transfected 293T treated with 200 nM bafilomycin A1 (Baf A1) for 6 h. Anti-GFP antibody was used to detect ACE2-GFP. D , heatmap illustrating the ACE2 trafficking cofactors at the lysosomal membrane (GO: 0005765). E , ACE2–NPC1 interaction network based on a previous study . F , immunoblotting analysis of ACE2 and NPC1 using co-IP in Huh7 cells. The indicated plasmids were transfected into Huh7 cells for 24 h, and NPC1 was pulled down by GFP-Vector or ACE2-GFP with GFP antibody. G , immunofluorescence analysis of ACE2 and NPC1 colocalization in Huh7 cells. ACE2-GFP and NPC1-mScarlet were cotransfected into Huh7 cells for 24 h and imaged using confocal microscopy. Colocalization plot profile was generated with ImageJ software. H , immunoblotting analysis of 293T cells transfected with scrambled siRNA (siNC) or NPC1 siRNA (siNPC1) for 48 h. I , Huh7 cells expressing ACE2-GFP and Lamp1-RFP were transfected with siNC or siNPC1 for 48 h, and the colocalization of ACE2 and Lamp1 was analyzed by confocal microscopy. Colocalization plot profiles were generated with ImageJ software. J , secretion dynamics assay to assess the function of NPC1 in ACE2 trafficking in Str–Ii_HiBiT-ACE2-SBP-FLAG-transfected 293T cells cotransfected with siNC or siNPC1. ACE2, angiotensin-converting enzyme 2; NPC1, Niemann–Pick complex 1; RUSH, Retention Using Selective Hooks; SBP, streptavidin-binding peptide; Str, streptavidin.

Article Snippet: The primary antibodies used in this study were as follows: HA tag antibody (51064-2-AP, Lot: 00101521; Sanying), NPC1 antibody (13926-1-AP, Lot: 00100574; Sanying), β-Tubulin (AC030, Lot: 9100030001; ABclonal).

Techniques: Transfection, Mass Spectrometry, Co-Immunoprecipitation Assay, Western Blot, Membrane, Plasmid Preparation, Immunofluorescence, Confocal Microscopy, Generated, Software, Expressing, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Real-time monitoring of secretory protein traffic for investigating trafficking regulators of ACE2 in living cells

doi: 10.1016/j.jbc.2025.110593

Figure Lengend Snippet: Cholesterol (CHO) induces ACE2 lysosomal escape by downregulating NPC1. A , representative of three quantitative RT–PCR analysis of SREBP2 and NPC1 levels in Huh7 cells treated with 50 μM CHO for 12 h. Data are mean ± SD (n = 3 technical replicates). B , immunoblotting analysis of NPC1 levels in Huh7 cells treated with 50 μM CHO for 12 h. C , Huh7 cells expressing ACE2-GFP and Lamp1-RFP were treated with 50 μM CHO for 0 or 12 h, and the colocalization of ACE2 and Lamp1 was analyzed by confocal microscopy. Colocalization plot profiles were generated with ImageJ software. D , secretion dynamics assay to assess the ACE2 trafficking in Str–Ii_HiBiT-ACE2-SBP-FLAG-transfected 293T cells treated with 50 μM CHO for 12 h. E , diagram showing mechanisms of CHO-regulated ACE2 secretory pathway. F , representative of three flow cytometry analysis of cell surface ACE2 expression in Huh7 cells treated with 50 μM CHO for 12 h. G , mean fluorescence intensity (MFI) analysis of cell surface ACE2 expression. Data are mean ± SD (n = 3 independent biological replicates). Statistical significance was determined using an unpaired two-tailed Student’s t test. (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, and “ns” means not significant). ACE2, angiotensin-converting enzyme 2; NPC1, Niemann–Pick complex 1; SREBP2, sterol regulatory element–binding protein 2.

Article Snippet: The primary antibodies used in this study were as follows: HA tag antibody (51064-2-AP, Lot: 00101521; Sanying), NPC1 antibody (13926-1-AP, Lot: 00100574; Sanying), β-Tubulin (AC030, Lot: 9100030001; ABclonal).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Confocal Microscopy, Generated, Software, Transfection, Flow Cytometry, Fluorescence, Two Tailed Test, Binding Assay