mouse anti rab7  (Developmental Studies Hybridoma Bank)

Journal: Cell Communication and Signaling : CCS

Article Title: The intracellular domains of the DSL ligands Serrate and Delta provide different activities

doi: 10.1186/s12964-025-02472-w

Figure Lengend Snippet: The intracellular Ks are essential for the Mib1-mediated endocytosis of Ser. A - F’ Subcellular localisation of the Ser-variants revealed by anti-HA antibody staining. a: apical; b: basal. Rab7 staining was used to mark endosomes. Nrx staining in ( E - F’ ) labels the apical side of the epithelium. A - A’’ , E , E’ Subcellular localisation of Ser in wildtype wing imaginal discs. Ser localises at the apical membrane and Rab7-positive endosomes (red arrow and arrowheads, respectively). The frame in A shows an enlarged view of the area marked with the rectangle. The arrowheads point to some of the Ser-HA- and Rab7-positive vesicles. B - B'' In mib1 mutant cells, endocytosis of Ser is strongly reduced (see also Fig. H - H’’ ). Most of the HA signal can be observed at the apical and the basal membrane (red and yellow arrow in B' , respectively). Moreover, Ser is virtually absent from endosomes. Note the accumulation of Ser also in the basal membrane (yellow arrow). C - C'' , F , F’ SerK2R localisation in wild-type cells. Ser localises to the apical and to the basal membrane, but is hardly seen in endosomes (red and yellow arrow, respectively). D Expression of SerRQRL in wildtype cells results in its accumulation in the apical membrane (red arrow). This indicates that the ICD of Ser is not required for the transport of Ser to the apical membrane. G - N Antibody uptake assay to analyse the endocytosis of Ser, SerK2R and Ser K1362R in S2R+ cells. G Design of the assay. The antibody is raised against the ECD of Ser. H , I , J , K , L At time point 0, all antibody labelled Ser-variants were located in the plasma membrane. (H1-H2, J1-J2, L1-L2). In absence of Mib1, Ser, SerK2R, Ser K1362R were present in plasma membrane after 30 and even 60 min., indicating that they are not efficiently endocytosed in the absence of Mib1. ( I -I2’) Co-expression of Ser with Mib1. The presence of Mib1 results in the efficient internalisation of Ser, which localises to Rab7-positive endosomes after 30 and 60 min. No Ser was observed in the plasma membrane already after 30 min. ( K -K2’) In contrast to Ser, the presence of Mib1 does not induce the internalisation of SerK2R, indicated by the presence of SerK2R in the plasma membrane, even after a chase of 60 min. ( L -M2’) Ser K1362R can be endocytosed in the presence of Mib1. However, in contrast to Ser, a fraction is still present at the plasma after a chase of 30’, suggesting that it is less efficiently endocytosed than Ser. N Quantification of the endocytosis of the Ser-variants. For the quantification, the cells were counted in which anti Ser-ECD was detected either at the plasma membrane (PM), at the plasma membrane and in vesicles (PM/V) or in vesicles only (V). The corresponding cell number was calculated in relation to the total cell number. The analysis reveals that Ser K1362R is less efficiently endocytosed than Ser, but more efficiently than SerK2R

Article Snippet: Anti Rab7 (mouse) , DSHB CG5915 , 1:100.

Techniques: Staining, Membrane, Mutagenesis, Expressing, Clinical Proteomics