ls columns  (Miltenyi Biotec)

Journal: The Journal of Biological Chemistry

Article Title: Epitope mirroring between the malaria surface proteins PfGARP and PIESP2 identifies a knob-associated complex in infected erythrocytes

doi: 10.1016/j.jbc.2026.113291

Figure Lengend Snippet: Identification and validation of PIESP2 by GM7mAb. A , the codon-optimized PIESP2 construct was synthesized lacking two predicted transmembrane domains and a signal peptide. Nomenclature of defined domains, expression construct, and putative topology are shown. B TRX-PIESP2 protein containing a His-tag, located after TRX protein, was used for affinity purification and fusion protein was purified by Mono Q FPLC. C , Ponceau S-stained image of dose-dependent TRX and TRX-PIESP2, followed by immunoblotting with GM7mAb are shown. D , ELISA-based detection of TRX and TRX-PIESP2 by serial dilution of GM7mAb. E , immunoblotting with GM7mAb using lysates from wild-type 3D7 parasites and PfGARP knockout lines generated at NIH and HMS (only the NIH knockout lane is shown in the left panel ). An anti-Pfaldolase antibody was used as a loading control ( right panel ). F , immunoblot analysis of PfGARP knockout and PIESP2 knockout parasite lysates probed with GM7mAb. The left panel shows the corresponding Ponceau S-stained blot for the immunoblots presented in the right panels . The 48 kDa band was detected in the PfGARP knockout lysate but was absent in the PIESP2 knockout lysate.

Article Snippet: High parasitemia cultures of mature-stage wild-type 3D7, two independent PfGARP knockouts (NIH and HMS), and PIESP2 knockout parasites were purified on Miltenyi Biotech LS columns (MACS) and resuspended in distilled water (1:25 v/v) to lyse the cells.

Techniques: Biomarker Discovery, Construct, Synthesized, Expressing, Affinity Purification, Purification, Staining, Western Blot, Enzyme-linked Immunosorbent Assay, Serial Dilution, Knock-Out, Generated, Control

Journal: The Journal of Biological Chemistry

Article Title: Epitope mirroring between the malaria surface proteins PfGARP and PIESP2 identifies a knob-associated complex in infected erythrocytes

doi: 10.1016/j.jbc.2026.113291

Figure Lengend Snippet: Localization of PIESP2 in P . falciparum infected erythrocytes. A , using GM7mAb, ring stage and mixed trophozoite/schizont stage infected RBCs were tested by immunoblotting for temporal expression of PIESP2. B , multiplex immunoblotting of RIPA-lysed wild-type 3D7 and PfGARP KO (NIH and HMS) parasite lysates. Blots were probed with GM7mAb and rabbit anti-Pfaldolase. Signals were detected by Odyssey CLx using conjugated anti-mouse and anti-rabbit fluorescent secondary antibodies. C , IFA of GM7mAb reactivity was performed in wild-type 3D7, PfGARP knockout (HMS), and PIESP2 knockout erythrocytes. Both iRBCs and uninfected RBC s (data not shown) were fixed and permeabilized with cold methanol (−20 o C for 45 min), labeled with GM7mAb and visualized with Alexa Fluor 488-conjugated anti-mouse IgG. Parasite nuclei were labeled with DAPI. D , five reverse motifs found in PfGARP-M1 and PIESP2 are shown.

Article Snippet: High parasitemia cultures of mature-stage wild-type 3D7, two independent PfGARP knockouts (NIH and HMS), and PIESP2 knockout parasites were purified on Miltenyi Biotech LS columns (MACS) and resuspended in distilled water (1:25 v/v) to lyse the cells.

Techniques: Infection, Western Blot, Expressing, Multiplex Assay, Knock-Out, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Epitope mirroring between the malaria surface proteins PfGARP and PIESP2 identifies a knob-associated complex in infected erythrocytes

doi: 10.1016/j.jbc.2026.113291

Figure Lengend Snippet: PIESP2 is a component of knobs. A–C , immunofluorescence (IFA) colocalization of PIESP2 using GM7mAb with two known knob markers (PfEMP1 and KAHRP) in wild-type 3D7 iRBCs. MSP1 was used as a negative control. Parasite nuclei were stained with DAPI. iRBCs were fixed and permeabilized with cold methanol (−20 o C for 45 min). D , immunoblotting with anti-KAHRP rabbit pAb and anti-PIESP2 (GM7mAb) using uRBC ghosts, iRBC ghosts, and biochemically enriched knobs. PIESP2 is detected in iRBCs and detergent-resistant knobs. E , immunogold transmission electron microscopy of wild-type 3D7 parasite infected erythrocytes with GM7mAb. Signal was detected using colloidal gold-labeled secondary antibody. Green arrowheads denote PIESP2 signal at the tip of knobs. Letter P refers to intracellular parasite and E shows erythrocyte cytoplasm. Scale bars 100 nm.

Article Snippet: High parasitemia cultures of mature-stage wild-type 3D7, two independent PfGARP knockouts (NIH and HMS), and PIESP2 knockout parasites were purified on Miltenyi Biotech LS columns (MACS) and resuspended in distilled water (1:25 v/v) to lyse the cells.

Techniques: Immunofluorescence, Negative Control, Staining, Western Blot, Transmission Assay, Electron Microscopy, Infection, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Epitope mirroring between the malaria surface proteins PfGARP and PIESP2 identifies a knob-associated complex in infected erythrocytes

doi: 10.1016/j.jbc.2026.113291

Figure Lengend Snippet: Localization of PfGARP and PIESP2 on the surface of iRBCs. A , differential reactivity of GM7mAb was measured in non-permeabilized wild-type 3D7, PfGARP knockout, and PIESP2 knockout erythrocytes by immunofluorescence assays (IFA). B , mAb7899 served as a negative control. C and D , a montage of representative IFA images for each genotype is shown in panel C . The image displayed in the second WT 3D7/GM7 mAb panel of A is identical to the image shown in the first WT 3D7 panel of C . To quantify the GM7mAb signal observed in panel A, a larger set of images was analyzed. For quantification, individual infected erythrocytes were manually outlined in ImageJ based on visible fluorescence signal. For each cell, mean fluorescence intensity (MFI) was calculated by subtracting the corresponding background signal. Five representative cells per group were analyzed. Data are presented as mean ± SD. Additional details of IFA quantification are described in the Methods section.

Article Snippet: High parasitemia cultures of mature-stage wild-type 3D7, two independent PfGARP knockouts (NIH and HMS), and PIESP2 knockout parasites were purified on Miltenyi Biotech LS columns (MACS) and resuspended in distilled water (1:25 v/v) to lyse the cells.

Techniques: Knock-Out, Immunofluorescence, Negative Control, Infection, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Epitope mirroring between the malaria surface proteins PfGARP and PIESP2 identifies a knob-associated complex in infected erythrocytes

doi: 10.1016/j.jbc.2026.113291

Figure Lengend Snippet: Quantification of GM7mAb reactivity by flow cytometry. Distinct localization of PIESP2 and skeleton binding protein 1. A , flow cytometry was performed on live and unfixed iRBCs from wild-type 3D7, PfGARP knockout, and PIESP2 knockout parasites by GM7mAb to measure surface reactivity. GM7mAb detected robust signal in wild-type 3D7 and PfGARP knockout iRBCs and relatively reduced signal in PIESP2 knockout iRBCs. The numerical values at the right of each panel represent mature parasites. Details of data analysis by FlowJo Software and gating strategies are described in the Methods section. B and C , mAb7899 and mouse IgG3 isotype were used as negative controls. D , immunofluorescence assays (IFA) were used to compare the localization of GM7mAb reactivity (PIESP2) with rabbit anti-SBP1 pAb antibody in wild-type 3D7 and PfGARP knockout iRBCs by confocal microscopy. For confocal microscopy using anti-SBP1 pAb, iRBCs were air-dried and fixed at room temperature with 100% acetone for two minutes (details in ). Representative images of wild-type 3D7 and PfGARP KO parasites subject to co-staining are shown in . The IFA signals were detected by Alexa Fluor 488 anti-mouse secondary antibody and Alexa Fluor 568 anti-rabbit secondary antibody, respectively. Images were acquired on a Zeiss LSM 880 confocal microscope equipped with a 63 × /1.4 NA Plan-Apochromat oil-immersion objective. Scale bar: 2 μm.

Article Snippet: High parasitemia cultures of mature-stage wild-type 3D7, two independent PfGARP knockouts (NIH and HMS), and PIESP2 knockout parasites were purified on Miltenyi Biotech LS columns (MACS) and resuspended in distilled water (1:25 v/v) to lyse the cells.

Techniques: Flow Cytometry, Binding Assay, Knock-Out, Software, Immunofluorescence, Confocal Microscopy, Staining, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Epitope mirroring between the malaria surface proteins PfGARP and PIESP2 identifies a knob-associated complex in infected erythrocytes

doi: 10.1016/j.jbc.2026.113291

Figure Lengend Snippet: Detection of antibodies in human malaria plasma samples by PIESP2 immunoassay. A , blue color-coded three amino acids denote the boundaries of TRX-PIESP2 construct in the full-length PIESP2 (PF3D7_0501200) sequence. Red color-coded amino acids denote the PIESP2 segment recognized by GM7mAb. Bold RTLAD sequence denotes the location of PEXEL motif. B , mapping of GM7mAb recognition segment within PIESP2-2-2 construct (amin acids 155–228). C , Ponceau-S staining of PIESP2 constructs shown in panel B . D , immunoblotting of PIESP2 constructs (panel C) by GM7mAb. E , alphaFold predicted location of PIESP2-2-2 segment recognized by GM7mAb. F , development of an ELISA for the detection of antibodies against PIESP2. Guided by the presence of reverse motifs in PIESP2, a 25-amino acid peptide (PSP-25) contained within PIESP2-2-2 was synthesized and conjugated to biotin via a flexible linker. The specificity of the peptide was validated by ELISA using GM7mAb and TRX-PIESP2 fusion protein . G , ELISA screens of human plasma samples from a malaria-endemic region in Mali, Africa. Negative controls of PBS and uninfected human serum (H1) were included. The reactivity cutoff value is shown in each ELISA panel. Several plasma samples including 169, 179, 190, 199, 223, 228 were reconfirmed by multiple ELISA screens. Additional details are described in the section.

Article Snippet: High parasitemia cultures of mature-stage wild-type 3D7, two independent PfGARP knockouts (NIH and HMS), and PIESP2 knockout parasites were purified on Miltenyi Biotech LS columns (MACS) and resuspended in distilled water (1:25 v/v) to lyse the cells.

Techniques: Clinical Proteomics, Construct, Sequencing, Staining, Western Blot, Enzyme-linked Immunosorbent Assay, Synthesized

Journal: The Journal of Biological Chemistry

Article Title: Epitope mirroring between the malaria surface proteins PfGARP and PIESP2 identifies a knob-associated complex in infected erythrocytes

doi: 10.1016/j.jbc.2026.113291

Figure Lengend Snippet: Cytoadhesion and schematic representation of knob topology. A , wild-type 3D7, PfGARP knockout, and PIESP2 knockout parasites were evaluated for static binding to human cerebral microvascular endothelial cells (HBEC-5i-CRL-3245 line. ATCC). Bound iRBCs were fixed, stained with Wright-Giemsa, and quantified by microscopy. Effect of GM7mAb, mAb7899, and mouse IgG3 isotype was measured as outlined in the section. Each parasite line was evaluated in a minimum of three independent experiments. B , schematic representation of PIESP2 trafficking to knobs via Maurer’s clefts in infected erythrocytes. C and D , schematic representation of parasite-derived adhesion proteins including PfEMP1, PIESP2, and PfGARP anchored on knobs as putative ligands for host endothelial receptors.

Article Snippet: High parasitemia cultures of mature-stage wild-type 3D7, two independent PfGARP knockouts (NIH and HMS), and PIESP2 knockout parasites were purified on Miltenyi Biotech LS columns (MACS) and resuspended in distilled water (1:25 v/v) to lyse the cells.

Techniques: Knock-Out, Binding Assay, Staining, Microscopy, Infection, Derivative Assay