mouse anti rab4  (Santa Cruz Biotechnology)

Journal: European journal of cell biology

Article Title: Receptor-mediated internalization promotes increased endosome size and number in a RAB4- and RAB5-dependent manner

doi: 10.1016/j.ejcb.2023.151339

Figure Lengend Snippet: The number of EE/SE per cell is regulated by select endosomal RAB proteins. (A) HeLa cells were grown on cover-slips and 6-well plates and either mocktreated (transfection reagent only) or incubated with the following amounts of siRNA oligonucleotides specific for RAB4, RAB5, RAB8A, RAB10 or RAB11A per 2 ml well for 48 h: RAB4, 280 nmol; RAB5, 400 nmol; RAB8A, 600 nmol, RAB10, 400 nmol; RAB11A, 600 nmol. Cells from the 6-well plates were collected, lysed and subjected to immunoblot analysis with antibodies to RAB4, RAB5, RAB8A, RAB10 and RAB11 (to validate knock-down efficacy), and to vinculin (loading control). The solid line separating mock and RAB11A from the other RAB proteins indicates that this pair of lanes was run on a separate gel due to space constraints. Gels and images are representative of 3 independent experiments. (B-G) Representative images of cells immunostained for EEA1 upon mock-treatment (B), RAB4 siRNA-treatment (C), RAB5 siRNA-treatment (D), RAB8A siRNA-treatment (E), RAB10 siRNA-treatment (F), RAB11A siRNA-treatment (G). (H) Graph illustrating the number of EE/SE observed per field of mock-treated and siRNA knock-down cells, with or without 15 min transferrin uptake. Data provided is a mean and standard deviation from 3 independent experiments. 3–5 images were obtained from each treatment, and Imaris software was used to measure EE/SE numbers. The p-values for EE/SE numbers were calculated with a Mann-Whitney test, because the data did not meet the assumption of normality by the D’Agostino Pearson or Kolmogorov-Smirnov tests. (I) Graph depicting the area of EEA1 endosomes observed per field of mock-treated and siRNA knock-down cells, with or without 15 min transferrin uptake. Data provided are mean and standard deviations from 3 independent experiments. 3–5 images were obtained from each treatment, and Imaris software was used to measure endosome area. The p-values for EEA1 endosome areas were calculated by t-test, as the data met the assumption of normality by the Kolmogorov-Smirnov test.

Article Snippet: Primary and secondary antibodies used in this study were rabbit anti-EEA1 (Cell Signaling cat. no. C45810), mouse anti-RAB5 (BD Transduction Labs cat. no. 610724), mouse anti-RAB4 (BD Transduction Labs cat. no. 610889), rabbit anti-RAB8 (Abcam cat no. 237702), rabbit anti-RAB10 (Abcam cat. no. 237703), mouse anti-RAB11 (BD Transduction Labs cat. no. 610657), HRP-conjugated anti-vinculin (Cell Signaling cat no. E1E9V XP), goat anti-mouse horseradish peroxidase (HRP), (cat. no. 115–035–003; Jackson ImmunoResearch Laboratories, West Grove, PA), donkey anti-rabbit HRP (cat. no. NA934V; GE Healthcare, Pittsburgh, PA), Alexa 568–conjugated goat anti-mouse (cat. no. A11031), and Alexa 488–conjugated goat anti-rabbit (cat. no. A11034; Life Technologies, Carlsbad, CA).

Techniques: Transfection, Incubation, Western Blot, Standard Deviation, Software, MANN-WHITNEY

Journal: European journal of cell biology

Article Title: Receptor-mediated internalization promotes increased endosome size and number in a RAB4- and RAB5-dependent manner

doi: 10.1016/j.ejcb.2023.151339

Figure Lengend Snippet:

Article Snippet: Primary and secondary antibodies used in this study were rabbit anti-EEA1 (Cell Signaling cat. no. C45810), mouse anti-RAB5 (BD Transduction Labs cat. no. 610724), mouse anti-RAB4 (BD Transduction Labs cat. no. 610889), rabbit anti-RAB8 (Abcam cat no. 237702), rabbit anti-RAB10 (Abcam cat. no. 237703), mouse anti-RAB11 (BD Transduction Labs cat. no. 610657), HRP-conjugated anti-vinculin (Cell Signaling cat no. E1E9V XP), goat anti-mouse horseradish peroxidase (HRP), (cat. no. 115–035–003; Jackson ImmunoResearch Laboratories, West Grove, PA), donkey anti-rabbit HRP (cat. no. NA934V; GE Healthcare, Pittsburgh, PA), Alexa 568–conjugated goat anti-mouse (cat. no. A11031), and Alexa 488–conjugated goat anti-rabbit (cat. no. A11034; Life Technologies, Carlsbad, CA).

Techniques: Sequencing, Concentration Assay

Journal: BMC Cell Biology

Article Title: R-Ras regulates β 1 -integrin trafficking via effects on membrane ruffling and endocytosis

doi: 10.1186/1471-2121-11-14

Figure Lengend Snippet: GFP-R-Ras colocalizes with Rab11, but not Rab5- or Rab4-positive endosomal compartments . Cos7 cells were transfected with GFP-R-Ras-(wt), -(38V), or -(41A) and then fixed and stained with antibodies against: (A-F) Rab11; (G-I) Rab5; or (J-L) Rab4. The largest vesicle compartments in 85% of -(wt) and 92% of -(38V) transfected cells were positive for Rab11 (arrows, for -(wt) n = 13 cells and -(38V), n = 12), but not Rab5 or Rab4 (<5%, n = 15 each). Intracellular GFP-R-Ras localized to smaller compartments and PM fluorescence lacked correlation with Rab fluorescence. Scale bar is 20 μm. (D-F) Insets of cells shown in A-C are shown at 3× zoom to highlight features.

Article Snippet: The following primary antibodies were used: mouse anti-Rab11 at 1:100; mouse anti-Rab5 at 1:100; mouse anti-Rab4 at 1:100 (all from BD Biosciences Pharmingen); rabbit anti-caveolin-1 [Tyr-14 phospho-specific] (Cell Signaling Technologies, 1:100); human anti-β 1 -integrin (clone N29, Chemicon, 1:100; which recognizes an activation-dependent epitope and is described in Wilkins et al. [ ]); human anti-R-Ras, custom made by Antibodies by Design (see below), 30 μg/mL.

Techniques: Transfection, Staining, Fluorescence