α rab7  (Developmental Studies Hybridoma Bank)

Journal: bioRxiv

Article Title: Smoothened turnover regulated by Hedgehog signaling in Drosophila

doi: 10.64898/2026.01.08.698469

Figure Lengend Snippet: (A) Schematic of frontal depiction of the wing pouch of the wing disc with the A (gray) and P (green) compartments indicated by gray and green, respectively; boxes indicate positions of (F-K) images. (B) White dotted lines delineate wing pouch and compartment border of disc expressing BAC encoded Hh:GFP. (C) Sagittal section of the wing disc expressing BAC encoded Hh:GFP (α-GFP antibody staining). Septate junction stained with α-DLG (red) marks septate junction; phalloidin marks adherens junction (blue). (D) Graph of Hh:GFP fluorescence in A and P compartments. (E) Schematic of sagittal wing pouch section with apical and basolateral compartments, Hh:GFP (green) and Rab7 (red) indicated. (F-G”) Frontal views of region of the A compartment of wing discs with BAC-encoded Hh:GFP detected with α-GFP antibody (green) and stained with α-Rab7 antibody (red) and Phalloidin (blue). (H,H’) Sagittal section showing total Hh signal (H) and Hh not colocalized with Rab7. White and yellow arrows indicate peripodial membrane and apical compartment, respectively. (I-K’) Same as (F-H’) for P compartment. (K) Graph showing the proportion of BAC encoded Hh:GFP signal that overlaps with Rab7 in apical/basolateral optical sections.

Article Snippet: The following antibodies were used: mouse α-GFP (Roche), rabbit α-RFP (Rockland), mouse α-Ptc (DSHB, Apa1), mouse α-En (DSHB, 4D9), DAPI, Phalloidin, mouse α-Dlg1, (DSHB, 4F3), α-Rab7 , α-Smo (DSHB, 20C6), Alexa633 conjugated-Phalloidin (Invitrogen), DAPI (Invitrogen), AbN ( ).

Techniques: Expressing, Staining, Fluorescence, Membrane

Journal: bioRxiv

Article Title: Smoothened turnover regulated by Hedgehog signaling in Drosophila

doi: 10.64898/2026.01.08.698469

Figure Lengend Snippet: (A-E) Frontal views of unfixed wing discs, optical section close to the apical surface; left, anterior; right, posterior. (A,B) Wing discs expressing BAC-encoded GFP:Cherry:Smo without (A) and with ammonium chloride (B); GFP channel (green), RFP channel (red). (C) Wing disc expressing BAC-encoded GFP:Smo (green) and Rbcn3A RNAi (expressed during L3 at 29°C) in the dorsal region (below white dashed line) using ( ap-Gal4/tub-Gal80 ts ; GFP:Smo/Rbcn3A-RNAi ). (D,E) Unfixed wing discs expressing Cherry:Smo (red) and Rab5:YFP (green) (D) or Cherry:Smo (red) and Rab7:YFP (green). (F) Amount of Cherry:Smo/Rab7 double positive and Cherry:Smo /Rab5 double positive puncta.

Article Snippet: The following antibodies were used: mouse α-GFP (Roche), rabbit α-RFP (Rockland), mouse α-Ptc (DSHB, Apa1), mouse α-En (DSHB, 4D9), DAPI, Phalloidin, mouse α-Dlg1, (DSHB, 4F3), α-Rab7 , α-Smo (DSHB, 20C6), Alexa633 conjugated-Phalloidin (Invitrogen), DAPI (Invitrogen), AbN ( ).

Techniques: Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: tsCRISPR based identification of Rab proteins required for the recycling of Drosophila TRPL ion channel

doi: 10.3389/fcell.2024.1444953

Figure Lengend Snippet: tsCRISPR mediated knock-out of specific Rab proteins results in a TRPL recycling defect. Of the 22 Rab proteins screened, 6 showed strong defects in TRPL recycling. (A) Water immersion micrographs showing TRPL::eGFP localization in Drosophila eyes of Ey-uS-Cas9-TRPL::eGFP/+ and of Ey-uS-Cas9-TRPL::eGFP/sgRNA-norpA control flies, as well as the indicated Rab CRISPR mutants. Flies were kept in the dark for 72 h after eclosure, were exposed to orange light for 16 h and were subsequently returned to darkness for another 24 h. Scale bar: 20 µm. (B) Localization of TRPL in Ey - uS-Cas9/+ control flies and Rab4, Rab7, Rab32 and RabX5 CRISPR mutants in d, d-l, and d-l-d conditions. Flies were dark-adapted for 72 h (d), subsequently exposed to orange light for 16 h (d-l), and were subsequently returned to darkness for another 2 h (d-l-d). Cross sections through ommatidia were probed with α-TRPL antibodies and α-Cnx99A antibodies, as indicated. Rhabdomeres were visualized using phalloidin and nuclei were stained with DAPI. Scale bar: 10 μm.

Article Snippet: The concentrations of the primary antibodies used were as follows: rabbit α-TRPL , 1:20; guinea pig α-TRPL , 1:10; mouse α-Calnexin99A (DSHB Cat# Cnx99A 6-2-1, RRID: AB2722011), 1:10; goat α-Golgin245 (DSHB Cat# Golgin245, RRID: AB_2569587), 1:2000; rabbit α-Myc (invitrogen, Cat# PA1-981), 1:500; mouse α-Myc (Cell Signaling Technology, Cat# 2276), 1:500 and mouse α-Rab7 (DSHB, Cat# Rab7, RRID: AB_2722471), 1:20.

Techniques: Knock-Out, Control, CRISPR, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: tsCRISPR based identification of Rab proteins required for the recycling of Drosophila TRPL ion channel

doi: 10.3389/fcell.2024.1444953

Figure Lengend Snippet: Colocalization analysis of TRPL, Rab3 and Rab7. (A) Colocalization of TRPL and Rab7-Myc positive vesicles in longitudinal sections of Rab7-Myc ommatidia. Flies were initially kept in the dark for 1 day and were then exposed to orange light for 4 h. TRPL was labeled with an α-TRPL antibody, Rab7-Myc was labeled with an α-Myc antibody. Rhabdomeres were visualized with phalloidin. Arrowheads indicate colocalization of TRPL with Rab7-Myc at time point 4 h of orange light illumination. Scale bar: 10 μm. (B) Colocalization of Rab7 and Rab3-Myc in longitudinal cryosections of Rab3-Myc ommatidia. Rab7 was labeled with an α-Rab7 antibody, Rab3-Myc was labeled with an α-Myc antibody. Rhabdomeres were visualized using phalloidin. Arrowheads indicate colocalization of Rab7 with Rab3-Myc. (C) Colocalization of Rab3-Myc and Calnexin in longitudinal cryosections of Rab3-Myc ommatidia. Calnexin was labeled with an α-Cnx99A antibody, Rab3-Myc was labeled with an α-Myc antibody. Rhabdomeres were visualized using phalloidin and nuclei using DAPI. Scale bar: 10 μm (D) Quantification of colocalization of TRPL with Rab7-Myc after 4 h of orange light illumination. The TRPL channel was chosen for the selection of the areas to be quantified. Quantification of colocalization of Rab7 with Rab3-Myc, and Cnx99A with Rab3-Myc. The Rab3-Myc channel was chosen for the selection of the areas to be quantified. Colocalization was assessed using Pearson correlation. Error bars: SEM (n = 3–5).

Article Snippet: The concentrations of the primary antibodies used were as follows: rabbit α-TRPL , 1:20; guinea pig α-TRPL , 1:10; mouse α-Calnexin99A (DSHB Cat# Cnx99A 6-2-1, RRID: AB2722011), 1:10; goat α-Golgin245 (DSHB Cat# Golgin245, RRID: AB_2569587), 1:2000; rabbit α-Myc (invitrogen, Cat# PA1-981), 1:500; mouse α-Myc (Cell Signaling Technology, Cat# 2276), 1:500 and mouse α-Rab7 (DSHB, Cat# Rab7, RRID: AB_2722471), 1:20.

Techniques: Labeling, Selection

Journal: Journal of Extracellular Vesicles

Article Title: Extracellular vesicle isolation methods identify distinct HIV‐1 particles released from chronically infected T‐cells

doi: 10.1002/jev2.12476

Figure Lengend Snippet: J1.1 LAV large extracellular vesicles (Frac‐A) isolation and characterization. (a) Total RNA from Frac‐A pellets collected at 6 and 24 h was extracted and analyzed by RT‐qPCR for HIV‐1 transcripts (TAR, TAR‐ gag and env ); (b) Frac‐A samples collected post‐release from J1.1 cells at 6 and 24 h were used to treat naïve CEM and U937 cells. A total of 10 6 naïve cells were resuspended in 100 µL supernatant and 200 µL of media and incubated for 8 h, then the supernatants were removed, and cells were placed in 1 mL fresh media and incubated for 48 h The cells were then collected and processed for Western blot and probed for HIV‐1 proteins; (c) Large J1.1 EPs (Frac‐A) were loaded onto Izon qEV 70 nm single columns. Forty fractions were collected and pooled in sets of 5. Pooled fractions were then nanotrapped and Western‐blotted for Lamp1, Rab5, Rab7, LC3, gp120, Nef, p24, GAPDH, and actin using Western blot; (d) HIV‐1 RNA content of pooled SEC fractions derived from large EVs population produced by J1.1 cells. Next, the fractions 1‐5 were used to treat naïve Jurkat (e), and U937 cells (f). A total of 10 6 naïve cells were resuspended in 100 µL supernatant, and 200 µL of media and 50 µL of Infectin™ and incubated for 8 h, then the supernatants were removed, and cells were placed in 1 mL fresh media and incubated for 2 days. The cells were then harvested and pelleted for Western blot analysis. Separately the pooled fractions 1‐5 were tested on infectivity with the use of Infectin™ as a control (g). Densitometry percent count of HIV‐1 p24 expression was determined relative to actin. (h) Next, infectivity of large EPs from HIV‐1‐infected primary cells was assessed. PBMCs from three donors were treated with PHA and IL‐2 and allowed to grow for 5 days in culture. The PBMCs were then infected with HIV‐1 89.6 (MOI: 10) with Infectin™ and cultured for another 4 days. Cells were then removed, and supernatants were centrifuged at 2,000 × g for 45 min to collect large EPs, that were fractionated by sizing columns and used to infect recipient cells. The recipient cells were separated in half and collected in 3‐ and 10 days post‐infection, respectively, and F#2 represents the starting material from fraction #2 used for infection of Jurkat cells; total RNA from fractionated EPs and recipient cells was isolated. Using 3′‐end primers specific to TAR, TAR‐ gag and env regions, cDNA was produced. RNA levels were assessed by RT‐qPCR with TAR‐specific primers. Student's t‐tests compared HIV‐1 RNA copy numbers from the fractions used for infection and from the recipient cells collected at 3‐ and 10‐days post‐infection. * P < 0.05; ** P < 0.01, *** P < 0.001. Error bars, SD.

Article Snippet: Next, Frac‐A material was equally separated into five parts and treated with antibodies against amphisome markers—α‐Lamp1, α‐Rab5, α‐Rab7, α‐ MAP LC3 α/β and α‐IgG (Santa Cruz Biotechnology, cat#sc‐5275) as isotype control.

Techniques: Isolation, Quantitative RT-PCR, Incubation, Western Blot, Derivative Assay, Produced, Infection, Control, Expressing, Cell Culture

Journal: Journal of Extracellular Vesicles

Article Title: Extracellular vesicle isolation methods identify distinct HIV‐1 particles released from chronically infected T‐cells

doi: 10.1002/jev2.12476

Figure Lengend Snippet: Immunoprecipitation from J1.1 LAV Frac‐A. (a) Diagram of the immunoprecipitation performed with a‐Rab7, a‐Rab5, a‐Lamp1, and a‐LC3 antibodies using fractions 1‐5 from qEV 70 size exclusion chromatography; (b) IP‐ed samples were pulled‐down washed and probed for the presence of viral proteins gp120/160, p24, Nef, and GAPDH by WB and (c) viral RNAs such as TAR, TAR‐ gag and env by qRT‐PCR where IgG served as control. Data represents mean ± standard deviation (SD) of three technical replicate measurements. Statistical significance was calculated with a two‐tailed unpaired Student's t‐test * P < 0.05, ** P < 0.01 significance level.

Article Snippet: Next, Frac‐A material was equally separated into five parts and treated with antibodies against amphisome markers—α‐Lamp1, α‐Rab5, α‐Rab7, α‐ MAP LC3 α/β and α‐IgG (Santa Cruz Biotechnology, cat#sc‐5275) as isotype control.

Techniques: Immunoprecipitation, Size-exclusion Chromatography, Quantitative RT-PCR, Control, Standard Deviation, Two Tailed Test