rabbit anti apmap  (Proteintech)

Journal: Frontiers in Immunology

Article Title: Endoplasmic reticulum stress-induced CRELD2 promotes APMAP-mediated activation of TGF-β/SMAD and NF-κB pathways in esophageal squamous cell carcinoma

doi: 10.3389/fimmu.2025.1616201

Figure Lengend Snippet: CRELD2 increases the membrane localization of APMAP. (A) The interaction between CRELD2 and APMAP was detected in the indicated cells by co-immunoprecipitation assay using anti-CRELD2 followed by immunoblot with anti-APMAP or anti-CRELD2. (B) The expression profile of APMAP was systematically analyzed in tumor tissues and paired normal counterparts using the UALCAN database. (C, D) qRT-PCR (C) and Western blot (D) analysis of APMAP expression in HEEC and ESCC cell lines (TE1, KYSE150, and KYSE170). (E) qRT-PCR and Western blot analysis of CRELD2 and APMAP expression in TE1 and KYSE150 cells with CRELD2 overexpression or knockdown. (F) qRT-PCR assay of KYSE150 and TE1 cells treated with Tg (+) or DMSO (–) for 12 h. (G) The subcellular localization of APMAP in KYSE150 cells treated as indicated was measured by immunofluorescence staining. Nuclei are stained with DAPI. Arrows indicate plasma membrane localization of APMAP. Scale bar, 25 μm. (H) Western blot showing APMAP expression in the membrane fraction from each treatment condition. The protein levels were quantified by band densitometry. Data represent the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, n.s., not significant.

Article Snippet: The cells were fixed in 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 for 15 min. After washing with PBS, the cells were blocked with 2% bovine serum albumin, and then incubated with the rabbit anti-human polyclonal antibody APMAP (Cusabio, Wuhan, China, dilution at 1:100) at 4°C for 24 h. Finally, the cells were incubated with a fluorescent secondary antibody for 1 h at room temperature.

Techniques: Membrane, Co-Immunoprecipitation Assay, Western Blot, Expressing, Quantitative RT-PCR, Over Expression, Knockdown, Immunofluorescence, Staining, Clinical Proteomics

Journal: Frontiers in Immunology

Article Title: Endoplasmic reticulum stress-induced CRELD2 promotes APMAP-mediated activation of TGF-β/SMAD and NF-κB pathways in esophageal squamous cell carcinoma

doi: 10.3389/fimmu.2025.1616201

Figure Lengend Snippet: Silencing of APMAP alleviates the malignant phenotype of ESCC cells. (A) The transfection efficiency of si-APMAP in KYSE150 and TE1 cells was detected by qRT-PCR method. (B, C) The proliferation ability of the indicated cells was assessed by MTS (B) and colony formation (C) assays. (D, E) Cell migration and invasion of the indicated cells were verified through wound healing (D) and transwell invasion (E) assays. Scale bar, 100 μm. Data represent the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01.

Article Snippet: The cells were fixed in 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 for 15 min. After washing with PBS, the cells were blocked with 2% bovine serum albumin, and then incubated with the rabbit anti-human polyclonal antibody APMAP (Cusabio, Wuhan, China, dilution at 1:100) at 4°C for 24 h. Finally, the cells were incubated with a fluorescent secondary antibody for 1 h at room temperature.

Techniques: Transfection, Quantitative RT-PCR, Migration

Journal: Frontiers in Immunology

Article Title: Endoplasmic reticulum stress-induced CRELD2 promotes APMAP-mediated activation of TGF-β/SMAD and NF-κB pathways in esophageal squamous cell carcinoma

doi: 10.3389/fimmu.2025.1616201

Figure Lengend Snippet: APMAP activates TGF-β/SMAD and NF-κB signaling during ER stress-induced EMT and proliferation. (A) qRT-PCR analysis of EMT and proliferation marker expression in KYSE150 and TE1 cells transfected with control siRNA (si-NC) or si-APMAP. (B) Western blot analysis of the indicated proteins expression in KYSE150 and TE1 cells transfected with si-NC or si-APMAP. (C) The protein expression of key genes of the TGF-β/SMAD, NF-κB, IL6/STAT3, Wnt/β-catenin, and PI3K/AKT signaling pathways in APMAP-knockdown KYSE150 and TE1 cells was examined by Western blot assay. (D) The effect of APMAP knockdown on the protein expression of SMAD2/3 and p-SMAD2/3 in Tg (100 nM, 12 h) treated KYSE150 and TE1 cells. (E, F) Co-immunoprecipitation assay was conducted with anti-TGFBR1 or anti-APMAP antibodies followed by immunoblot with anti-TAK1, anti-TGFBR1 or anti-APMAP in the indicated cells. (G) Western blot analysis of p-TAK1 and TAK1 expression in APMAP-manipulated KYSE150 cells and TE1 cells (overexpression or knockdown). (H) Co-immunoprecipitation assay was conducted with anti-APMAP antibody followed by immunoblot with anti-TAB1, anti-TAB2, or anti-APMAP in the indicated cells. (I) The proposed model for ER stress-induced CRELD2 promotes membrane the localization of APMAP, which increases its interaction with TAK1, TAB1, and TAB2, thereby promoting the activation of the TGF-β/SMAD and NF-κB pathways and inducing the EMT and proliferation of ESCC cells, is shown. The protein levels were quantified by band densitometry. Data represent the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01.

Article Snippet: The cells were fixed in 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 for 15 min. After washing with PBS, the cells were blocked with 2% bovine serum albumin, and then incubated with the rabbit anti-human polyclonal antibody APMAP (Cusabio, Wuhan, China, dilution at 1:100) at 4°C for 24 h. Finally, the cells were incubated with a fluorescent secondary antibody for 1 h at room temperature.

Techniques: Quantitative RT-PCR, Marker, Expressing, Transfection, Control, Western Blot, Protein-Protein interactions, Knockdown, Co-Immunoprecipitation Assay, Over Expression, Membrane, Activation Assay

Journal: bioRxiv

Article Title: Paraoxonase-like APMAP maintains endoplasmic reticulum-associated lipid and lipoprotein homeostasis

doi: 10.1101/2024.01.26.577049

Figure Lengend Snippet: A. Schematic diagram of APMAP domains. APMAP FL depicts the full-length human APMAP consisting of N-terminal transmembrane domain (TM) and C-terminal 6-bladed β-propeller region. PON1 and PON2 are full-length Paraoxonase enzymes with 6-bladed β-propeller domain B. Tertiary structural prediction of human APMAP using AlphaFold (Q9HDC9), highlighting the TM and 6-bladed β-propeller region. The prediction is colored-coded based on model confidence score and assigned color is labelled on the right C. Superposition of the 6-bladed β-propeller regions of AlphaFold predicted APMAP (Q9HDC9) and experimental crystal structure of PON1 (PDB code:1v04), generated using PyMOL (The PyMOL Molecular Graphics System, Version 2.5.5 Schrodinger, LLC) D. Live cell confocal micrographs of C-terminally mNeonGreen (mNG) tagged endogenous APMAP in U2OS cell line showing ER-localization. Cells were transfected with DsRed-KDEL to mark ER network. Scale bar = 10 μm E. Airyscan confocal micrographs of Huh7 cells overexpressed with APMAP-EGFP (Top) and untagged APMAP (Bottom), IF stained using α-HSP90B1 antibody (ER marker, red). Untagged APMAP was co-IF stained using α-APMAP antibody (green) and α-HSP90B1 antibody (ER marker, red). Scale bar = 10 μm F. Airyscan confocal micrographs of Huh7 cells transfected with APMAP TM -EGFP and APMAP AD -EGFP IF stained with α-HSP90B1 (ER marker, red). Scale bar = 10 μm G. Confocal micrographs of EGFP-tagged Drosophila APMAP (dAPMAP) expressed specifically in fat body (FB) using Dcg-Gal4 > UAS- dAPMAP-EGFP. Tissue was co-transfected with ER marker TdTomato-Sec61 (Red) and LDs were stained with MDH (blue). Scale Bar = 10 μm Line scans represent spatial distribution of APMAP (green) with respect to ER maker (red) and LDs (blue). Straight line of 5 pixel width were drawn at ER tubular network and quantified using ‘plot profile’ function in ImageJ.

Article Snippet: The primary antibodies used are mouse anti-HSP90B1 (1:100; Sigma-Aldrich; AMAb91019), rabbit anti-HSP90B1 (1:100; Sigma-Aldrich; HPA008424), rabbit anti-APMAP (1:100; Sigma-Aldrich; HPA012863), mouse anti-APMAP (1:150; Novus biologicals; NBP2-01716), rabbit anti-GFP (1:200; Abcam; ab290), rabbit anti-mCherry (1:200; Invitrogen; PA534974), rabbit anti-calnexin (1:500; Abcam; ab22595), mouse anti-CLIMP63 (1:100, Enzo, G1-296), Rabbit anti-calnexin (1:200; Abcam; ab22595), goat anti-Apolipoprotein B (1:200, Rockland immunochemicals, 600-101-111) and mouse anti-ceramide (1:50; Enzo; ALX-804-196).

Techniques: Generated, Transfection, Staining, Marker

Journal: bioRxiv

Article Title: Paraoxonase-like APMAP maintains endoplasmic reticulum-associated lipid and lipoprotein homeostasis

doi: 10.1101/2024.01.26.577049

Figure Lengend Snippet: A. Cartoon demonstration of semi-permeabilization and immunolabeling experiment for validation of APMAP topology. Cells overexpressed with either APMAP FL -EGFP or mCherry-KDEL is treated with either digitonin (permeabilizes only PM) or Triton X-100 (permeabilizes both PM and ER) and are IF stained using α-EGFP and α-mCherry respectively B. Confocal micrographs of U2OS cells expressing APMAP-EGFP or mCherry-KDEL. Cells were treated with either 0.05% digitonin or 0.1% Triton X-100 and IF stained with either α-EGFP (red) and α-mCherry (green) to access exposed C-terminal tag. Scale bar = 10 μm C. Cartoon representation of fluorescence protease protection assay. U2OS cells overexpressed with C-terminally GFP tagged APMAP or Reticulon4a are treated with digitonin to permit selective permeabilization of PM, which is followed by treatment with Proteinase K to allow proteolytic digestion of GFP tag exposed to cytoplasm D. Live cell confocal micrographs of U2OS cells expressing APMAP-EGFP or Reticulon4a-EGFP subjected to subsequent treatments with 0.01% digitonin and 50 μg/ml proteinase K. Images shown are taken 30 sec after treatments up to 6 mins. Scale bar = 10 μm E. Cartoon depiction of the type II membrane topology of human-APMAP, showing N-terminal region facing the cytoplasm and the C-terminal region facing the ER lumen F. Volcano plot of TurboID-based proteomics showing enriched proteins in the APMAP overexpressed Huh7 cells compared to control (untransfected) cells, with Log2 of fold enrichment on X-axis relative to -Log10 of p-values on the Y-axis G. List of selected top protein hits from APMAP Turbo-ID based proteomics, detailing the proposed functions and topology of each gene.

Article Snippet: The primary antibodies used are mouse anti-HSP90B1 (1:100; Sigma-Aldrich; AMAb91019), rabbit anti-HSP90B1 (1:100; Sigma-Aldrich; HPA008424), rabbit anti-APMAP (1:100; Sigma-Aldrich; HPA012863), mouse anti-APMAP (1:150; Novus biologicals; NBP2-01716), rabbit anti-GFP (1:200; Abcam; ab290), rabbit anti-mCherry (1:200; Invitrogen; PA534974), rabbit anti-calnexin (1:500; Abcam; ab22595), mouse anti-CLIMP63 (1:100, Enzo, G1-296), Rabbit anti-calnexin (1:200; Abcam; ab22595), goat anti-Apolipoprotein B (1:200, Rockland immunochemicals, 600-101-111) and mouse anti-ceramide (1:50; Enzo; ALX-804-196).

Techniques: Immunolabeling, Staining, Expressing, Fluorescence, Membrane

Journal: bioRxiv

Article Title: Paraoxonase-like APMAP maintains endoplasmic reticulum-associated lipid and lipoprotein homeostasis

doi: 10.1101/2024.01.26.577049

Figure Lengend Snippet: A. Confocal micrographs of larval fat-body (FB) tissue sections from control ( Dcg-Gal4 line) and dAPMAP-RNAi line with FB-specific RNAi depletion. LDs are stained with MDH (Magenta). Scale bar = 10 μm B. TLC quantification of relative whole-larval triglyceride (TG) levels from control ( Dcg-Gal4 ) and dAPMAP RNAi line with FB-specific RNAi depletion. N=3; **P<0.0021; Two tailed unpaired t-test C. TLC quantification of relative whole adult female and male fly triglyceride (TG) levels from control ( Dcg-Gal4 ) and dAPMAP RNAi line with FB-specific RNAi depletion. N=3; ***P<0.0002; Two tailed unpaired t-test D. TLC quantification of relative whole-larval TG levels control ( Da-Gal4 ) and dAPMAP RNAi line with global depletion. N=3; *P<0.0322; Two tailed unpaired t-test E. TLC quantification of whole adult fly TG levels from control ( Da-Gal4 ) and dAPMAP RNAi line with global depletion. N=3; *P<0.0322; Two tailed unpaired t-test F. Confocal micrographs of larval gut tissue sections from control ( Da-Gal4 line) and dAPMAP-RNAi line with global depletion. LDs are stained with MDH (Magenta). Scale bar = 10 μm G. TLC quantification of relative whole-larval gut triglyceride (TG) levels from control ( Da-Gal4 ) and dAPMAP RNAi line with global depletion. N=3; *P<0.0322; Two tailed unpaired t-test H. Airyscan confocal micrographs of siCtrl and siAPMAP treated Huh7 cells, followed by 16 h of OA treatment. Cells were co-IF stained with anti-HSP90B1 (ER marker, Red) and α-ApoB (Green). LDs were stained with MDH (Blue). Red arrows highlight the ApoB crescents situated around LDs. Scale Bar = 10 μm I. ApoB rescue experiments in APMAP depleted cells by re-addition of APMAP FL , APMAP AD (cytoplasmic) and APMAP AD -KDEL (ER-lumen targeted), all tagged with EGFP. Violin scatter dot plot represents average area covered by ApoB per cell (n >40 cells; N=3; ****P<0.0001, ***P<0.0002, **P<0.0021, *P<0.0332; One-way ANOVA with Sidak’s multiple comparisons; α = 0.05) J. Fold change of ApoB secreted in the culture medium from siCtrl and siAPMAP treated Huh7 cells incubated with OA, determined by ELISA assay. K. LipoGlo luminescence micrographs of 5 days post fertilization (5dpf) showing distribution of ApoB-containing lipoproteins (LipoGlo) in larval zebrafish vasculature. Animals were either treated with non-targeting CRISPR-based guides (scrambled) or gRNAs directed to delete zebrafish APMAP or MTP. Luminescence quantifications for head, trunk, and yolk displayed from N=4 independent experiments. L. Total LipoGlo signal from 5dpf whole-animal lysates. N=3, ****P<0.0001.

Article Snippet: The primary antibodies used are mouse anti-HSP90B1 (1:100; Sigma-Aldrich; AMAb91019), rabbit anti-HSP90B1 (1:100; Sigma-Aldrich; HPA008424), rabbit anti-APMAP (1:100; Sigma-Aldrich; HPA012863), mouse anti-APMAP (1:150; Novus biologicals; NBP2-01716), rabbit anti-GFP (1:200; Abcam; ab290), rabbit anti-mCherry (1:200; Invitrogen; PA534974), rabbit anti-calnexin (1:500; Abcam; ab22595), mouse anti-CLIMP63 (1:100, Enzo, G1-296), Rabbit anti-calnexin (1:200; Abcam; ab22595), goat anti-Apolipoprotein B (1:200, Rockland immunochemicals, 600-101-111) and mouse anti-ceramide (1:50; Enzo; ALX-804-196).

Techniques: Staining, Two Tailed Test, Marker, Incubation, Enzyme-linked Immunosorbent Assay, CRISPR