ap2m1  (MedChemExpress)

Journal: mBio

Article Title: Candida albicans -induced ubiquitination of EGFR reveals novel host–fungal interaction pathways

doi: 10.1128/mbio.03448-25

Figure Lengend Snippet: C. albicans induces EGFR phosphorylation at Y1045 and EGFR ubiquitination in TR146 oral epithelial cells in an ECE1 -driven manner. ( A ) Schematic of EGFR phosphorylation and ubiquitination. Created with BioRender.com. ( B ) TR146 cells were infected with wild-type (WT) C. albicans (BWP17), an ECE1 null strain ( ece1 Δ/Δ), an ECE1 re-integrant strain ( ece1 Δ/Δ + ECE1 ), or a candidalysin null strain ( ece1 Δ/Δ + ECE1 Δ184–279 ) for 4 h at an MOI of 10 or stimulated with PBS as a vehicle control. Lysates (10 µg) were electrophoresed on gradient gels to detect pEGFR Y1045 and α-actin via Western blot. Data are representative of three independent experiments. ( C ) TR146 cells were infected with WT C. albicans (SC5314), ece1 Δ/Δ, ece1 Δ/Δ + ECE1 , ece1 Δ/Δ+ ECE1 Δ184–279 , and als3 Δ/Δ for 6 h at an MOI of 5 or stimulated with PBS as a vehicle control. EGF 100 ng/mL was used as a positive control. Lysates were collected and EGFR was immunoprecipitated. Immunoprecipitation (IP) samples were electrophoresed on gradient gels to detect total EGFR and ubiquitin via Western blot. Data are representative of three independent experiments. Dashed vertical lines indicate omitted, extraneous portions of blot images. ( D ) Relative protein levels in panel C were quantified using densitometry. Ubiquitin was normalized to total EGFR and expressed relative to the control (PBS), which was set to 1. Data represent the average of three to seven independent experiments and are presented as mean values with standard error of the mean (SEM) error bars. Statistical significance was determined using the Kruskal–Wallis test with Dunn’s multiple comparison test. P < 0.05, *** P < 0.001. ( E ) TR146 cells were infected with WT C. albicans (SC5314), ece1 Δ/Δ, and als3 Δ/Δ for 6 h at an MOI of 5 or stimulated with PBS as a vehicle control. Lysates were collected and EGFR was immunoprecipitated. IP samples were electrophoresed on gradient gels to detect total EGFR, Grb2, AP2M1, and HRS via Western blot. Dashed vertical lines indicate omitted, extraneous portions of blot images. Data are representative of three independent experiments. ( F–H ) Relative protein levels in panel E were quantified using densitometry. Grb2, AP2M1, and HRS were normalized to total EGFR and expressed relative to the control (PBS), which was set to 1. Data represent the average of three to four independent experiments and are presented as mean values with SEM error bars. Statistical significance was determined using a parametric test (ordinary one-way ANOVA with Dunnett’s multiple comparison test). * P < 0.05. ns, not significant.

Article Snippet: AP2M1 , Rabbit , 1:1,000 , Cell Signaling Technology , 68196.

Techniques: Phospho-proteomics, Ubiquitin Proteomics, Infection, Control, Western Blot, Positive Control, Immunoprecipitation, Comparison

Journal: mBio

Article Title: Candida albicans -induced ubiquitination of EGFR reveals novel host–fungal interaction pathways

doi: 10.1128/mbio.03448-25

Figure Lengend Snippet: C. albicans promotes EGFR degradation through Ece1p and Als3p, predominantly mediated by the lysosomal pathway, and induces the recruitment of adaptor proteins Grb2, AP2M1, and HRS. ( A–C ) TR146 cells were infected with different C. albicans strains (wild-type [WT] SC5314, ece1 Δ/Δ and als3 Δ/Δ) or stimulated with PBS as a vehicle control for 2 h (MOI 10), 8 h (MOI 1), and 12 h (MOI 1). Cell lysates were electrophoresed on gradient gels to detect total EGFR and α-actin via Western blot. Data are representative of three independent experiments. ( D and E ) TR146 cells were pre-treated for 1 h with 0.25 µM of lysosomal inhibitor Bafilomycin A1 (BafA1) and 10 µM of proteasomal inhibitor MG-132 or DMSO as a vehicle control, and then treated with 100 ng/mL EGF as a positive control for 1 h or infected with SC5314 (MOI 5) for 10 h. Cell lysates were electrophoresed on gradient gels to detect total EGFR and α-actin via Western blot. Data are representative of three independent experiments. Dashed vertical lines indicate omitted, extraneous portions of blot images. ( F and G ) Relative protein levels in panels D and E were quantified using densitometry. Total EGFR was normalized to α-actin and expressed relative to the control (DMSO + water or DMSO + PBS), which was set to 100. Data are presented as mean values with standard error of the mean error bars. Statistical significance was determined using a parametric test (ordinary one-way ANOVA with Dunnett’s multiple comparison test). * P < 0.05. ns, not significant.

Article Snippet: AP2M1 , Rabbit , 1:1,000 , Cell Signaling Technology , 68196.

Techniques: Infection, Control, Western Blot, Positive Control, Comparison

Journal: European Journal of Medical Research

Article Title: The role of AP2M1 in oncofetal characteristics: integrative in silico, in vitro, and in vivo analyses using zebrafish models

doi: 10.1186/s40001-025-03583-3

Figure Lengend Snippet: AP2M1 and oncofetal genes are increased in hepatocellular carcinoma patients. A Expression levels of AP2M1 , SOX2 , and POU5F1 across different stages of HCC in TCGA datasets. The box plots represent the expression distribution of AP2M1 (left), SOX2 (middle), and POU5F1 (right) across Stage I (green), Stage II (yellow), and Stage III (purple) of HCC. B AP2M1 (green) expression levels were confirmed by immunohistochemistry analysis in normal and tumor cells. Images were obtained by confocal microscopy. Photographs are representative of 15 independent experiments. C The scatter plots showed the relationship between AP2M1 expression levels and FOXM1 and IGF2BP1 expression levels. The blue line represents the linear regression fit with the shaded area indicating the 95% confidence interval (CI)

Article Snippet: AP2M1 siRNA oligonucleotides were synthesized by Bioneer (Daejeon, Korea).

Techniques: Expressing, Immunohistochemistry, Confocal Microscopy

Journal: European Journal of Medical Research

Article Title: The role of AP2M1 in oncofetal characteristics: integrative in silico, in vitro, and in vivo analyses using zebrafish models

doi: 10.1186/s40001-025-03583-3

Figure Lengend Snippet: AP2M1 is associated with the cell proliferation of the HCC cell line and embryonic hepatocytes. A HepG2 cells were transfected with AP2M1 siRNA for 72 h. The expression levels of AP2M1 were determined by western blotting using β-actin as an internal control. Data were quantified and expressed as the mean ± SEM of 5 independent experiments. ***p < 0.001 vs. value in the negative control. B HepG2 cell proliferation determined by an MTT assay was quantified and expressed as the mean ± SEM of 36 independent experiments. ***p < 0.001 vs. value in the negative control. C Hep3B cells were transfected with AP2M1 siRNA for 72 h. The expression levels of AP2M1 were determined by western blotting using β-actin as an internal control. Data were quantified and expressed as the mean ± SEM of 5 independent experiments. ***p < 0.001 vs. value in the negative control. D Hep3B cell proliferation determined by an MTT assay was quantified and expressed as the mean ± SEM of 36 independent experiments. ***p < 0.001 vs. value in the negative control. E HepG2 cell migration was determined by a wound-healing assay. Representative photomicrographs of cell monolayers at 48 h post-wounding. Cell migration area (%) was calculated from the ratio of changes in the wound area. Data were quantified and expressed as the mean ± SEM of 10 independent experiments. ***p < 0.001 vs. value in the negative control

Article Snippet: AP2M1 siRNA oligonucleotides were synthesized by Bioneer (Daejeon, Korea).

Techniques: Transfection, Expressing, Western Blot, Control, Negative Control, MTT Assay, Migration, Wound Healing Assay

Journal: European Journal of Medical Research

Article Title: The role of AP2M1 in oncofetal characteristics: integrative in silico, in vitro, and in vivo analyses using zebrafish models

doi: 10.1186/s40001-025-03583-3

Figure Lengend Snippet: ap2m1a is crucial for hepatocyte proliferation in zebrafish embryo model. A The structure of human AP2M1 (top) and zebrafish ap2m1a (bottom). The black arrow indicates structural differences between the two. B The amino acid sequence of human AP2M1 and zebrafish ap2m1a. The MHD (Mu homology domain) was represented by red box. C The expression of ap2m1a mRNA was detected by RT-qPCR in various concentration of ap2m1a -MO. Data were quantified and expressed as the mean ± SEM of 3 independent experiments. *** p < 0.001 vs. value in the negative control. D The expression of fabp10a mRNA was detected by RT-PCR in uninjected embryos and ap2m1a -MO 2.5 ng/nl injected embryos. Data were quantified and expressed as the mean ± SEM of 3 independent experiments. *** p < 0.001 vs. value in the negative control. E Quantitative analysis of the sizes of 27 uninjected embryos and 26 ap2m1a -MO 2.5 ng/nl injected embryos. fabp10a expression in those embryos was detected by WISH. The black arrow demonstrates zebrafish hepatocytes. Data were quantified and expressed as the mean ± SEM of each embryo. **** p < 0.0001 vs. value in the negative control

Article Snippet: AP2M1 siRNA oligonucleotides were synthesized by Bioneer (Daejeon, Korea).

Techniques: Sequencing, Expressing, Quantitative RT-PCR, Concentration Assay, Negative Control, Reverse Transcription Polymerase Chain Reaction, Injection

Journal: European Journal of Medical Research

Article Title: The role of AP2M1 in oncofetal characteristics: integrative in silico, in vitro, and in vivo analyses using zebrafish models

doi: 10.1186/s40001-025-03583-3

Figure Lengend Snippet: AP2M1 confers stemness to hepatocytes. A The scatter plots depict the relationship between AP2M1 expression levels and SOX2 and POU5F1 expression levels. The blue line represents the linear regression fit with the shaded area indicating the 95% CI. B AP2M1 siRNA decreased the expression levels of DLK-1, Nanog, IGF-2, CK-19, SOX-2, SOX-9, SALL-4, EPCAM, AFP and PROM-1 in HepG2 cells as analyzed by qPCR. Data were quantified and expressed as the mean ± SEM of 5–6 independent experiments. *p < 0.05 and ***p < 0.001 vs. value in the negative control. C HepG2 cells were stained with crystal violet staining using the Matrigel invasion assay as described. Data were quantified and expressed as the mean ± SEM of 10 independent experiments. ***p < 0.001 vs. value in the negative control. D Expression of dlk1, nanog, igf2a, keratin93, sox2, sox9, sall4, and prom1a in 48hpf zebrafish embryos was detected by RT-PCR and samples were prepared in triplicates. Particularly, dlk1, nanog, and prom1a were decreased by **p < 0.01, ***p < 0.001, *p < 0.05, respectively

Article Snippet: AP2M1 siRNA oligonucleotides were synthesized by Bioneer (Daejeon, Korea).

Techniques: Expressing, Negative Control, Staining, Invasion Assay, Reverse Transcription Polymerase Chain Reaction

Journal: European Journal of Medical Research

Article Title: The role of AP2M1 in oncofetal characteristics: integrative in silico, in vitro, and in vivo analyses using zebrafish models

doi: 10.1186/s40001-025-03583-3

Figure Lengend Snippet: AP2M1 is associated with Wnt signaling pathway. A Relative mRNA expression of AXIN2 (Wnt), HES1 (Notch), and GLI1 (Hedgehog) following AP2M1 knockdown in HepG2 cells. Data were quantified and expressed as the mean ± SEM of 3 independent experiments. **p < 0.01, ***p < 0.001 vs. value in the negative control. B Relative mRNA expression of AXIN2 (Wnt), HES1 (Notch), and GLI1 (Hedgehog) following AP2M1 knockdown in Hep3B cells. Data were quantified and expressed as the mean ± SEM of 3 independent experiments. **p < 0.01, ***p < 0.001 vs. value in the negative control. C Relative mRNA expression of axin2 (Wnt), her6 (Notch), and gli1 (Hedgehog) in 48hpf zebrafish embryos was detected by RT-PCR. Data were quantified and expressed as the mean ± SEM of 3 independent experiments. **p < 0.01 and ***p < 0.001 vs. value in the negative control

Article Snippet: AP2M1 siRNA oligonucleotides were synthesized by Bioneer (Daejeon, Korea).

Techniques: Expressing, Knockdown, Negative Control, Reverse Transcription Polymerase Chain Reaction

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A cytoplasmic motif in HLA-E that drives clathrin-mediated endocytosis and VCP-associated postendocytic trafficking

doi: 10.1073/pnas.2514956122

Figure Lengend Snippet: The unconventional endosomal transport of HLA-E depends on CME and VCP-dependent surface return. ( A ) Immunoblot for AP2M1 in HEK293T cells with AP2M1 knocked out. ( B ) HLA internalization rates after 1 h in WT HEK293T cells or HEK293T cells with AP2M1 knockout ( AP2 -KO), which were transiently transfected with the indicated HLA constructs, or cotransfected with HLA constructs and WT AP2M1 (AP2 WT) or its dominant-negative mutant (AP2 D176A). Internalization assays were carried out as described in Fig. 1 D . ( C ) Immunoblot for CLTC in HEK293T cells with CLTC knocked out with two different gRNAs (KO.1, KO.2). ( D ) HLA internalization rates after 1 h in WT HEK293T cells or HEK293T cells with CLTC knocked out were transiently transfected with the indicated HLA constructs. Internalization assays were carried out as described in Fig. 1 D . ( E ) Diagram illustrating the ELISA-based peptide binding assay (created with BioRender.com). ( F ) The binding of the cytoplasmic tails of CD1, HLA-E, and their respective mutated versions lacking the binding motif to the AP-2 complex was assessed by ELISA, as illustrated in E . Data were collected for five replicates (three technical repeats/run) and are shown as mean ± SEM. ( G ) Sequences of the cytoplasmic tail of different constructs. Sequence identity is indicated by dots and mutated positions are highlighted in red. ( H ) Immunoblot for VCP in HEK293T cells with siRNA-mediated VCP knockdown or after overnight incubation with NMS-873 (5 nM) and CB-5083 (2 nM). ( I ) Recycling of HLA-E after 1 h in HEK293T cells with siRNA-mediated VCP depletion or following overnight incubation with NMS-873 (5 nM) or CB-5083 (2 nM) with or without VL9 peptide (50 µM) during internalization. Recycling assays were carried out as in Fig. 3 A . ( J ) Recycling of HLA-E after 1 h in HEK293T cells incubated with chloroquine (CQ, 20 µM) overnight with or without VL9 peptide (50 µM) during internalization. Recycling assays were carried out as Fig. 3 A . Data were collected from six ( B and D ) or five ( H and I ) replicates and are presented as mean ± SD (error bars). Statistical analyses were performed using one-way ANOVA with Tukey’s post hoc test ( B , D , and H ) or paired two-tailed t test with Welch’s correction (F, I). Asterisks denote statistical significance: ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Plasmids expressing the WT AP2M1 (μ2-HA-WT,Addgene plasmid #32752; http://n2t.net/addgene:32752 ; RRID: Addgene_32752) or the mutated AP2M1 (μ2-HA-D176A, Addgene plasmid #32754; http://n2t.net/addgene:32754 ; RRID: Addgene_32754) were gifts from Alexander Sorkin.

Techniques: Western Blot, Knock-Out, Transfection, Construct, Dominant Negative Mutation, Enzyme-linked Immunosorbent Assay, Binding Assay, Sequencing, Knockdown, Incubation, Two Tailed Test

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A cytoplasmic motif in HLA-E that drives clathrin-mediated endocytosis and VCP-associated postendocytic trafficking

doi: 10.1073/pnas.2514956122

Figure Lengend Snippet: The unconventional endosomal transport of HLA-E depends on CME and VCP-dependent surface return. ( A ) Immunoblot for AP2M1 in HEK293T cells with AP2M1 knocked out. ( B ) HLA internalization rates after 1 h in WT HEK293T cells or HEK293T cells with AP2M1 knockout ( AP2 -KO), which were transiently transfected with the indicated HLA constructs, or cotransfected with HLA constructs and WT AP2M1 (AP2 WT) or its dominant-negative mutant (AP2 D176A). Internalization assays were carried out as described in Fig. 1 D . ( C ) Immunoblot for CLTC in HEK293T cells with CLTC knocked out with two different gRNAs (KO.1, KO.2). ( D ) HLA internalization rates after 1 h in WT HEK293T cells or HEK293T cells with CLTC knocked out were transiently transfected with the indicated HLA constructs. Internalization assays were carried out as described in Fig. 1 D . ( E ) Diagram illustrating the ELISA-based peptide binding assay (created with BioRender.com). ( F ) The binding of the cytoplasmic tails of CD1, HLA-E, and their respective mutated versions lacking the binding motif to the AP-2 complex was assessed by ELISA, as illustrated in E . Data were collected for five replicates (three technical repeats/run) and are shown as mean ± SEM. ( G ) Sequences of the cytoplasmic tail of different constructs. Sequence identity is indicated by dots and mutated positions are highlighted in red. ( H ) Immunoblot for VCP in HEK293T cells with siRNA-mediated VCP knockdown or after overnight incubation with NMS-873 (5 nM) and CB-5083 (2 nM). ( I ) Recycling of HLA-E after 1 h in HEK293T cells with siRNA-mediated VCP depletion or following overnight incubation with NMS-873 (5 nM) or CB-5083 (2 nM) with or without VL9 peptide (50 µM) during internalization. Recycling assays were carried out as in Fig. 3 A . ( J ) Recycling of HLA-E after 1 h in HEK293T cells incubated with chloroquine (CQ, 20 µM) overnight with or without VL9 peptide (50 µM) during internalization. Recycling assays were carried out as Fig. 3 A . Data were collected from six ( B and D ) or five ( H and I ) replicates and are presented as mean ± SD (error bars). Statistical analyses were performed using one-way ANOVA with Tukey’s post hoc test ( B , D , and H ) or paired two-tailed t test with Welch’s correction (F, I). Asterisks denote statistical significance: ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Plasmids expressing the WT AP2M1 (μ2-HA-WT,Addgene plasmid #32752; http://n2t.net/addgene:32752 ; RRID: Addgene_32752) or the mutated AP2M1 (μ2-HA-D176A, Addgene plasmid #32754; http://n2t.net/addgene:32754 ; RRID: Addgene_32754) were gifts from Alexander Sorkin.

Techniques: Western Blot, Knock-Out, Transfection, Construct, Dominant Negative Mutation, Enzyme-linked Immunosorbent Assay, Binding Assay, Sequencing, Knockdown, Incubation, Two Tailed Test