rabbit anti rab11  (Cell Signaling Technology Inc)

Journal: Journal of Cell Science

Article Title: Syntaxin-2 balances phagocytic uptake and phagolysosomal clearance in macrophages

doi: 10.1242/jcs.263855

Figure Lengend Snippet: Stx2 exhibits multiple subcellular distributions in macrophages, and inhibits engagement and uptake of IgG-opsonized particles. (A–F) DIC and confocal images of endogenous Stx2 with endogenous (A) β-actin (ACTB), (B) RAB5A, (C) Rab11, (D) VAMP4, (E) VAMP7 and (F) LAMP1 at steady-state. (G) DIC and confocal images of RAW 264.7 macrophages fed with OPZ (white asterisks) for 1h and probed for endogenous Stx2 and LAMP1. Boxed areas enlarged in the insets. ‘ r ’ values show degree of colocalization. Scale bars: 10 μm (main images) and 5 μm (insets). DAPI denotes the nucleus. All images representative of three independent repeats. (H) Western blot of Stx2 and β-actin (ACTB, loading control) from RAW 264.7 macrophages (WT) and macrophages transduced with scrambled (sc) shRNA (control) or shRNAs targeted to Stx2 (Stx2-KD). (I) Quantified Stx2 band density normalized to ACTB (loading control). N =3 independent experiments. Results are mean±s.e.m. ns, not significant; * P <0.05. (J) DIC and stacked (three consecutive optical sections) confocal images from 15, 30 and 60 min OPZR-incubated macrophages also probed for Stx2. DAPI marks the nucleus. Scale bars: 10 μm. (K) Quantification of engaged OPZR per macrophage (15 min: control, n =61; Stx2-KD, n =82; 30 min: control, n =56; Stx2-KD, n =109; 60 min: control, n =111; Stx2-KD, n =86). N =3 independent experiments. Results are mean±s.e.m. **** P <0.0001. (L) Maximum intensity projection confocal images of inside-outside stained macrophages incubated with OPZR for 15, 30 and 60 min. Scale bars: 10 µm. (M,N) Quantification of (M) phagocytic index and (N) phagocytic rate for OPZR analyzed at 15 min (control, n =75; Stx2-KD, n =68), 30 min (control, n =273; Stx2-KD, n =344) and 60 min (control, n =165; Stx2-KD, n =190). N =3 independent experiments. M shown as violin plots with median and quartiles marked. For N, results are mean±s.e.m. ** P <0.01, **** P <0.0001. All statistical tests were two-tailed unpaired Student's t -tests. See also Fig. S1 .

Article Snippet: Cells intended for surface staining were skipped for permeabilization and directly used for blocking after fixation and PBS wash. Primary antibodies diluted in blocking buffer were used for 1 h to immunostain cells for Stx2 (Synaptic Systems, catalog #110123, 1:200 dilution; R&D Systems, catalog #AF2568, 1:200 dilution), Rab5A (Cell Signaling Technology, catalog #46449, 1:100 dilution), Rab11 (Cell Signaling Technology, catalog #46449, 1:200 dilution), VAMP4 (Thermo Fisher Scientific, catalog #PA1-768, 1:100 dilution), VAMP7 (Thermo Fisher Scientific, catalog #PA5-116892, 1:100 dilution), LAMP1 (Thermo Fisher Scientific, catalog #14-1071-81, 1:500 dilution), Fcγ (Thermo Fisher Scientific, catalog #14-0161-82, 1: 100 dilution), TfR (Abcam, catalog #ab214039, 1:200 dilution), Cat L (R&D Systems, catalog #AF1515, 1:100 dilution), Cat B (Cell Signaling Technology, catalog #31718, 1:100 dilution), Cat D (Cloud-Clone Corporation, catalog #MAB280Hu22, 1:100 dilution) and Phalloidin647 (Thermo Fisher Scientific, catalog # A22287, 1:500 dilution).

Techniques: Western Blot, Control, Transduction, shRNA, Incubation, Staining, Two Tailed Test

Journal: mBio

Article Title: RAB5 is a host dependency factor for the generation of SARS-CoV-2 replication organelles

doi: 10.1128/mbio.03314-24

Figure Lengend Snippet: Early endosomal membranes highly co-localize with SARS-CoV-2 dsRNA. VeroE6 cells were infected with SARS-CoV-2 HK (MOI = 1) in a synchronized manner. Six hours later, cells were fixed, blocked, and stained for the virus dsRNA and the intracellular markers RAB5, CD63, TGN46, GOSR1, CD81, RAB11, ADRP, MitoTracker, and calnexin. Images: representative pictures of three independent experiments. White scale bar: 10 µm. Graph: the Pearson’s correlation coefficient ( R ) value for the co-localization of SARS-CoV-2 dsRNA and the intracellular markers listed above was calculated from eight randomly selected fields. Raw values with their mean and SEM are represented. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: RAB11 , Mouse monoclonal (1A12B9D8) to RAB11 , 1:100 , ThermoFisher Scientific, MA5-31876.

Techniques: Infection, Staining, Virus