rabbit anti rab14 antibody  (Proteintech)

Journal: bioRxiv

Article Title: Mast cell-mediated inflammation relies on insulin-regulated aminopeptidase controlling cytokine export from the Golgi

doi: 10.1101/2022.01.21.477149

Figure Lengend Snippet: (A) Representative immunofluorescent images of resting BMMCs stained for IRAP and the endosomal markers Rab14, Stx6, EEA1, or 15min endocytized transferrin-AF647. (B) BMMC were stimulated for 30min with ionomycin/PMA prior to intracellular staining for serotonin and IRAP. (C) Beta-hexosaminidase secretion of peritoneal mast cells after 30min stimulation with ionomycin/PMA or 48/80 or without stimulation. Shown are mean values ± SEM of three independent experiments. (D) Degranulation analysis by flow cytometry via LAMP-1 surface staining of live peritoneal mast cells stimulated for 30min with ionomycin/PMA or 48/80 or left unstimulated. Shown are mean values ± SEM of three independent experiments. (E) Histamine secretion by peritoneal mast cells stimulated for 30min with ionomycin/PMA or 48/80. Shown are mean values ± SEM of the secretion of IRAPko cells normalized to wt from three independent experiments. (F) Resting BMMCs were fixed and immunostained for IRAP and VAMP8 or TNF-α. Images are representative of four independent experiments. (G) BMMCs were stimulated with ionomycin/PMA for 15min prior to fixation and immunostaining for VAMP8 and IRAP. Images are representative of two independent experiments. (H) Imaging flow cytometry quantification of VAMP8 mean pixel intensity (MPI) in the Stx4 mask of ionomycin/PMA-stimulated BMMC. Analysis was performed on >300 cells. (I) IRAPwt and ko mice or (J) mast-cell deficient Wsh mice reconstituted with IRAPwt and ko BMMC were challenged on one ear with 30mg/ml arachidonic acid while the control ear was left untreated. Histamine concentration was quantified in ear tissue homogenates and normalized to total protein concentration. Data are shown from one out of two experiments. *p<0.05,** p<0.01, ***p<0.001 for all figures.

Article Snippet: Mouse monoclonal IRAP antibody clone 3E, rabbit monoclonal anti-IRAP XP clone D7C5, rabbit anti-EEA1 (all Cell Signaling); rat antimouse lysosome-associated membrane protein (LAMP)1 clone 1D4B, mouse monoclonal anti-STX6, mouse monoclonal anti-GM130 (BD Pharmingen); rabbit polyclonal anti-STX6 (ProteinTech Group, Chicago, IL, United States); mouse monoclonal anti-Stx4 clone QQ-17 (Santa Cruz); rabbit polyclonal anti-TNF (abcam 34674 for confocal imaging and imaging flow cytometry), PE-PerCP5.5 anti-mouse TNF clone MP6-XT22 (eBiosciences for FACS); rat anti-IL-6 and rat anti-IL-10 (eBiosciences); rabbit polyclonal anti-VAMP3 (abcam 2102); rabbit polyclonal anti-VAMP8 (novus); goat anti-serotonin (abcam 66047), rabbit polyclonal anti-Rab14 (Sigma Aldrich).

Techniques: Staining, Flow Cytometry, Immunostaining, Imaging, Concentration Assay, Protein Concentration

Journal: Small GTPases

Article Title: Rab22a regulates the establishment of epithelial polarity

doi: 10.1080/21541248.2020.1754104

Figure Lengend Snippet: Rab22a-GFP and Rab14-RFP localization in non-polarized MDCK cells. MDCK cells were plated on glass coverslips and transfected with Rab22a-GFP (green) and Rab14-RFP (red). DAPI labels the nucleus (blue). Enlarged vesicles in the perinuclear region are both Rab22a and Rab14 positive (arrows). Scale bar, 20 μm. Lower panel is an enlargement of the inset outlined in the upper panel. Rab22a-GFP positive puncta in the cytoplasm do not contain Rab14 (arrowheads). Scale bar, 5 μm

Article Snippet: The following primary antibodies were used: mouse anti-podocalyxin was a gift from Dr. Karl Matlin (University of Chicago), rabbit anti-Rab22a (Abcam, EPR9486), rabbit anti-Rab14 (Sigma, R0656-200UL), mouse anti-Rab5 (BD Biosciences, 610,725), rabbit anti-Rab6 (Cell Signalling Technology, 9625), rabbit anti-Rab7 (Cell Signalling Technologies, 9367), rabbit anti-Rab11 (Cell Signalling Technology, 5589).

Techniques: Transfection

Journal: Small GTPases

Article Title: Rab22a regulates the establishment of epithelial polarity

doi: 10.1080/21541248.2020.1754104

Figure Lengend Snippet: Rab22 localizes to the cell:cell interface in MDCK cell pairs. Cell pairs in three-dimensional culture expressing Rab22a-WT-GFP or constitutively active (Rab22a-Q64 L-GFP) together with Rab14-WT-RFP. Transfected cells were embedded in Matrigel into a chamber slide and fixed after 16 hours. Rab22a-GFP localizes to the cell:cell interface (arrows) and to puncta in the cytoplasm (arrowheads). Rab14-RFP and Rab22a-WT-GFP and Rab22a-Q64 L-GFP colocalize in the cytoplasmic puncta (arrowheads). Scale bar, 10 μm

Article Snippet: The following primary antibodies were used: mouse anti-podocalyxin was a gift from Dr. Karl Matlin (University of Chicago), rabbit anti-Rab22a (Abcam, EPR9486), rabbit anti-Rab14 (Sigma, R0656-200UL), mouse anti-Rab5 (BD Biosciences, 610,725), rabbit anti-Rab6 (Cell Signalling Technology, 9625), rabbit anti-Rab7 (Cell Signalling Technologies, 9367), rabbit anti-Rab11 (Cell Signalling Technology, 5589).

Techniques: Expressing, Transfection

Journal: Small GTPases

Article Title: Rab22a regulates the establishment of epithelial polarity

doi: 10.1080/21541248.2020.1754104

Figure Lengend Snippet: Knockdown of Rab22a results in multi-lumen formation and overexpression of Rab22a rescues Rab14 knockout. (a) MDCK Rab22a shRNA cells were embedded in Matrigel on a chamber slide and fixed after 48 hours. Podocalyxin (PDCX, red) was labelled to identify the forming apical domains. DAPI labels the nucleus (blue). Knockdown of Rab22a results in the formation of multiple lumens. Scale bar, 10 μm (b) Quantification of single lumen formation. The average percent of cysts with single lumen was recorded for shRNA control (n = 1050) and Rab22 knockdown (n = 817) cysts, across three independent repetitions with four replicates per plating. Rescue of single lumen with Rab22a shRNA resistant plasmid in cysts. MDCK cells that were transduced with Rab22a shRNA were transfected with Rab22a plasmid that was engineered to be resistant to the shRNA. Single cells were embedded in Matrigel and grown for 24 hours and the formation of single lumen cysts was quantified. The average percent of cysts with single lumen was recorded for shRNA control (n = 513), Rab22a knockdown (n = 606) and Rab22a shRNA with resistant plasmid (n = 190) cysts, across two independent repetitions with four replicates per plating. Expression of the Rab22a shRNA resistant plasmid rescues single lumen formation. (c) Rescue of single lumen formation in Rab14 KD cells. Rab14 KD cells were transfected with Rab22a-WT-GFP and Rab22a-Q67 L-GRP and single lumen formation was quantified. The average percent of cysts with single lumen was recorded for shRNA control (n = 304), Rab14 shRNA (n = 483), Rab14 shRNA with Rab22a-WT-GFP (n = 125), and Rab14 shRNA with Rab22a-Q64 L-GFP (n = 149) cysts across two independent repetitions with four replicates per plating

Article Snippet: The following primary antibodies were used: mouse anti-podocalyxin was a gift from Dr. Karl Matlin (University of Chicago), rabbit anti-Rab22a (Abcam, EPR9486), rabbit anti-Rab14 (Sigma, R0656-200UL), mouse anti-Rab5 (BD Biosciences, 610,725), rabbit anti-Rab6 (Cell Signalling Technology, 9625), rabbit anti-Rab7 (Cell Signalling Technologies, 9367), rabbit anti-Rab11 (Cell Signalling Technology, 5589).

Techniques: Over Expression, Knock-Out, shRNA, Plasmid Preparation, Transduction, Transfection, Expressing