Journal: The EMBO Journal

Article Title: Microcephaly-associated protein WDR62 supports purine metabolism by interacting with co-chaperone BAG2

doi: 10.1038/s44318-026-00724-0

Figure Lengend Snippet: Representative confocal micrographs of AD293 cells treated with 0.5 M sorbitol for 1 h. Cells transiently transfected with WDR62-mCherry and either the HSP co-chaperones ( A ) BAG2-EGFP, ( B ) EGFP-DNAJC7, ( C ) EGFP-STIP1, or de novo purine biosynthesis (DNPB) enzymes ( D ) PFAS-EGFP, ( E ) PPAT-EGFP, ( F ) GART-EGFP or ( G ) PAICS-EGFP. Co-localisation of each signal is indicated by fluorescence intensity plots to the right of each set of images ( y -axis represents fluorescence intensity (a.u.), x -axis (µm) represents the length of the white line drawn on the ROI). ( H ) Bar graph representing the co-localisation between WDR62-mCherry and EGFP signal for each respective protein (mean ± SD). Each dot represents the Person’s correlation coefficient for a single ROI. ( I ) Confocal micrographs of AD293 cells co-transfected with WDR62-mCherry and PFAS-EGFP, in either control (top) or purine-depleted (bottom) conditions. Bar graph on the right represents co-localisation between WDR62-mCherry and PFAS-EGFP (mean ± SD) ( ***P = 0.0004). ( J ) Co-immunoprecipitation and immunoblot of myc-PFAS and HA-WDR62. ( K ) The association of endogenous WDR62 with BAG2 and PFAS as measured by PLA (grey spots) with DAPI-stained nuclei in blue. PLA signal is observed under basal (control) and sorbitol-treated (0.5 M, 1 h) conditions. ( L ) Quantification of the number of PLA puncta per cell for WDR62/BAG2 and WDR62/PFAS. Data represent n = 3 biological replicates. P values calculated based on mean values using a one-way ANOVA for ( H ) and a two-tailed unpaired T test for ( I , L ) (* P < 0.05, ** P < 0.005, *** P < 0.001, **** P < 0.0001, n.s. is P > 0.05). All scale bars represent 20 µm. .

Article Snippet: DNAJC7 (rabbit polyclonal) , Proteintech , 11090-1-AP.

Techniques: Transfection, Fluorescence, Control, Immunoprecipitation, Western Blot, Staining, Two Tailed Test

Journal: Molecular and Cellular Biology

Article Title: Role of the nucleophosmin (NPM) portion of the non-Hodgkin's lymphoma-associated NPM-anaplastic lymphoma kinase fusion protein in oncogenesis

doi: 10.1128/mcb.17.4.2312

Figure Lengend Snippet: FIG. 4. Focus formation assays of NPM-ALK, TPR-MET, and engineered mutant proteins. Fr3T3 fibroblasts were infected with retroviral stock prepared with the construct indicated below, seeded into 100-mm-diameter tissue culture dishes, and then stained with Giemsa stain after 2 weeks in culture. Shown are uninfected parental Fr3T3 cells (A), the empty pSRaMSVtkneo retroviral vector (B), NPM-ALK (C), TPR-MET (D), TPR-ALK (E), NPM-MET (F), M5/7/9LNPM-ALK (G), K210RNPM-ALK (H), DMBNPM-ALK (I), D64NPM-ALK (J), D65–103NPM-ALK (K), and DC154NPM-ALK (L).

Article Snippet: Anti-MET rabbit polyclonal antibody Ab143 recognizes the carboxy-terminal portion of TPR-MET (residues 514 to 523) (54), whereas the h-MET (C28) antibody is a purified rabbit polyclonal immunoglobulin G (IgG) that recognizes the 28 carboxy-terminal amino acids of the human c-MET p140 b chain (Santa Cruz Biotechnology, Santa Cruz, Calif.).

Techniques: Mutagenesis, Infection, Retroviral, Construct, Staining, Giemsa Stain, Plasmid Preparation

Journal: Molecular and Cellular Biology

Article Title: Role of the nucleophosmin (NPM) portion of the non-Hodgkin's lymphoma-associated NPM-anaplastic lymphoma kinase fusion protein in oncogenesis

doi: 10.1128/mcb.17.4.2312

Figure Lengend Snippet: FIG. 5. Schematic of NPM-ALK mutants. Diagrammatic representations of wild-type NPM-ALK, the NPM-ALK mutant proteins analyzed in this study, and the wild-type TPR-MET protein (54) are illustrated. Indicated motifs include the putative metal binding (MB) domain of NPM-ALK and the leucine zipper (LZ) regions of TPR-MET. The transforming capability (as indicated by average number of foci per 100-mm-diameter dish), ability to oligomerize, and subcellular location of each protein are indicated to the right. aa, amino acid; Y, yes; N, no; ND, not determined.

Article Snippet: Anti-MET rabbit polyclonal antibody Ab143 recognizes the carboxy-terminal portion of TPR-MET (residues 514 to 523) (54), whereas the h-MET (C28) antibody is a purified rabbit polyclonal immunoglobulin G (IgG) that recognizes the 28 carboxy-terminal amino acids of the human c-MET p140 b chain (Santa Cruz Biotechnology, Santa Cruz, Calif.).

Techniques: Mutagenesis, Binding Assay

Journal: Molecular and Cellular Biology

Article Title: Role of the nucleophosmin (NPM) portion of the non-Hodgkin's lymphoma-associated NPM-anaplastic lymphoma kinase fusion protein in oncogenesis

doi: 10.1128/mcb.17.4.2312

Figure Lengend Snippet: FIG. 6. In vitro catalytic activities of NPM-ALK, TPR-MET, and engineered mutants. (A) Cos7 cells were transiently transfected with the indicated cDNA constructs subcloned into the pSRaMSVtkneo retroviral vector, and the ex- pressed proteins were tested for catalytic activities by in vitro kinase assays. Lysates from Cos7 cells transfected with the empty vector (lane 1), from the retro- viral vector expressing NPM-ALK, TPR-ALK, M5/7/9LNPM-ALK, D65–103NPM-ALK, or DC154NPM-ALK, and from the SUP-M2 cell line were immunoprecipitated with the ALK1 monoclonal antibody (1:10). Lysates from Cos7 cells transfected with the empty vector (lane 2) or the retroviral vector expressing TPR-MET or NPM-MET were immunoprecipitated with the anti-MET antibody Ab143 (1:1,000). (B) Because the in vitro autokinase activities of the DMBNPM-ALK and D64NPM-ALK proteins expressed in Cos7 cells were difficult to demonstrate for unknown reasons, in vitro- transcribed and -translated proteins were immunoprecipitated with anti-ALK 11 and tested for in vitro kinase activities (lanes 2 and 3), together with in vitro-transcribed and -translated NPM-ALK as a positive control (lane 1). All proteins were resolved by SDS–7.5% PAGE. Each protein is indicated by an arrowhead. Molecular mass markers are indicated between the gels. Molecular masses of the precipitated pro- teins are as follows: NPM-ALK, 75 kDa; TPR-MET, 65 kDa; TPR-ALK, 78 kDa; NPM-MET, 55 kDa; M5/7/9LNPM-ALK, 75 kDa; DMBNPM-ALK, 74 kDa; D64NPM- ALK, 68 kDa; D65–103NPM-ALK, 71 kDa; and DC154NPM-ALK, 58 kDa.

Article Snippet: Anti-MET rabbit polyclonal antibody Ab143 recognizes the carboxy-terminal portion of TPR-MET (residues 514 to 523) (54), whereas the h-MET (C28) antibody is a purified rabbit polyclonal immunoglobulin G (IgG) that recognizes the 28 carboxy-terminal amino acids of the human c-MET p140 b chain (Santa Cruz Biotechnology, Santa Cruz, Calif.).

Techniques: In Vitro, Transfection, Construct, Retroviral, Plasmid Preparation, Expressing, Immunoprecipitation, Positive Control

Journal: Molecular and Cellular Biology

Article Title: Role of the nucleophosmin (NPM) portion of the non-Hodgkin's lymphoma-associated NPM-anaplastic lymphoma kinase fusion protein in oncogenesis

doi: 10.1128/mcb.17.4.2312

Figure Lengend Snippet: FIG. 10. Subcellular localization of NPM-ALK, TPR-MET, and engineered mutant proteins. Cells were immunostained with either the ALK-specific monoclonal antibody ALK1 (51) (all panels except D, D9, F, and F9) or the h-MET (C28) antibody (Santa Cruz Biotechnology). Simultaneous staining with DAPI was performed to visualize the nuclei of the cells (panels A9 to J9). Shown are the t(2;5)-positive lymphoma cell line SUP-M2 (A and A9), the t(2;5)-negative chronic myelogenous leukemia cell line K562 (B and B9), NPM-ALK (C and C9), TPR-MET (D and D9), TPR-ALK (E and E9), NPM-MET (F and F9), DMBNPM-ALK (G and G9), D64NPM-ALK (H and H9), D65–103NPM-ALK (I and I9), and M5/7/9LNPM-ALK (J and J9). All cDNA constructs were expressed in Cos7 cells (panels C and C9 through J and J9, inclusive).

Article Snippet: Anti-MET rabbit polyclonal antibody Ab143 recognizes the carboxy-terminal portion of TPR-MET (residues 514 to 523) (54), whereas the h-MET (C28) antibody is a purified rabbit polyclonal immunoglobulin G (IgG) that recognizes the 28 carboxy-terminal amino acids of the human c-MET p140 b chain (Santa Cruz Biotechnology, Santa Cruz, Calif.).

Techniques: Mutagenesis, Staining, Construct