rabbit anti rab5 c8b1  (Cell Signaling Technology Inc)

Journal: The EMBO Journal

Article Title: Annexin A7 enhances TIA1 axonal trafficking to counteract pathological aggregation in neurons

doi: 10.1038/s44318-025-00609-8

Figure Lengend Snippet: ( A ) Schematic diagram illustrating RNP labeling in neurons co-transfected with EGFP-TIA1 and Cy5-UTP. ( B ) Live-cell imaging of DIV8 rat hippocampal neurons reveals directed axonal trafficking of RNPs. Kymographs of bracketed axons show retrograde (triangles) and stationary (asterisks) RNPs. Scale bars = 50 µm (left), 20 µm (right); y-axis = 180 s. ( B’ ) Percentage of TIA1-positive RNPs and all RNPs undergoing retrograde trafficking ( n = 22, 42 neurons derived from four biological replicates, P < 0.0001). ( C ) Schematic of the microfluidic device. Scale bars = 1 cm (left), 200 µm (right). ( D ) Dynamics of TIA1-mCherry granules in the axons of DIV8 neurons. Magnified boxed regions on the right show distinct TIA1 granules, marked by colored arrowheads. Scale bars = 50 μm (left top) and 10 μm (left bottom, right). ( D’ ) Distribution profile of average trafficking speeds for TIA1 granules: retrograde (≤ −0.05 μm/s), anterograde (≥ 0.05 μm/s), stationary (−0.05 to 0.05 μm/s). ( D” ) Percentage of retrograde and anterograde TIA1 granules ( n = 53 axons from four biological replicates, P < 0.0001). ( E – F’ ) Key frames from time-lapse images showing co-trafficking of TIA1 granules with LysoTracker ( E ) and DIC1B-mRFP ( F ) in axons. Arrowheads in different colors mark distinct mobile particles. Scale bar = 5 µm. ( E’ ) Quantification of TIA1 granules co-trafficking with axon-derived CTB, BoNT/A-Hc, or LysoTracker ( n = 14, 12, 11 axons from three biological replicates). ( F’ ) Quantification of TIA1 granules co-trafficking with whole cell–applied MitoTracker, or with co-expressed EGFP-Rab5 and DIC1B-mRFP ( n = 25, 25 and 18 axons from at least 3 biological replicates). ( G ) Key frames from time-lapse images showing TIA1-mCherry axon trafficking in DIC1B knockdown neurons. Scale bars = 10 μm. ( G’ ) Speed quantification of ( G’ ) ( n = 216, 51 and 54 granules from four biological replicates. Control vs. shDIC1B-1#: P = 0.0044; Control vs. shDIC1B-2#: P = 0.0011). ( H ) Key frames from time-lapse images showing axonal TIA1 granules (indicated by arrowheads). Disassembly of these granules is marked by red arrows. Right: Kymograph of the indicated axonal segment. Scale bar = 5 µm; y-axis = 10 s. ( H’-H” ) Quantification shows TIA1 intensity heterogeneity along axons ( H’ ) before and 180 s post-1,6-Hex ( H” ) ( n = 40 axons from three biological replicates, P < 0.0001). ( I ) Design of Opto-Control and Opto-TIA1 constructs (top). Schematics illustrating light-induced formation of Opto-TIA1 granules in neurons (bottom). ( I’ ) Key frames from time-lapse images showing movement of Opto-TIA1 granules in axons. The timeline on the left indicates the onset of light activation. Intensity profiles of the bracketed area shown on the right. Moving and fusing granules are indicated by arrowheads. Scale bars = 10 µm (left), 5 µm (right); y-axis = 25% (Normalized to ( F Max -F 0 )). Data represent mean ± SEM; two-tailed unpaired t -test in ( B’ , D” , H” ); one-way ANOVA in ( G’ ). .

Article Snippet: Rab5 (C8B1) Rabbit mAb , Cell Signaling Technology , Cat#3547; RRID: AB_2300649.

Techniques: Labeling, Transfection, Live Cell Imaging, Derivative Assay, Knockdown, Control, Construct, Activation Assay, Two Tailed Test

Journal: The EMBO Journal

Article Title: Annexin A7 enhances TIA1 axonal trafficking to counteract pathological aggregation in neurons

doi: 10.1038/s44318-025-00609-8

Figure Lengend Snippet: ( A ) Cy5-UTP co-localization with indicated RNP markers. Arrowheads indicate RNPs with these markers. Scale ba = 5 µm. ( B ) Schematic diagram of the pulse-chase labeling assay to specifically label retrograde membranous axonal organelles in neurons cultured in a microfluidic device. See also the Methods for details. Scale bar = 50 µm. ( C , D ) Key frames from time-lapse images showing TIA1 granules trafficking with axon-derived CTB ( C ) or BoNT/A-Hc ( D ) in a microfluidic device. Arrowheads indicate moving TIA1 granules. Scale bar = 10 µm. ( E , F ) Key frames from time-lapse images showing TIA1 granules trafficking with whole cell stained MitoTracker ( E ) or co-expressed EGFP-Rab5 ( F ). Arrowheads indicate moving TIA1 granules and organelles. Scale bar = 5 µm. ( G ) Representative confocal images of endogenous TIA1 with organelle markers (Rab5 for endosomes, LC3 for autophagosomes, LAMP1 for lysosomes, DIC1B for dynein) in axons, with intensity profiles shown below. Scale bars = 10 µm (top), 5 µm (bottom). ( G’-G”’ ) Quantification of ( G ), with ( G’ ) showing the Pearson’s coefficient of endogenous TIA1 granules with indicated markers, and ( G” ) showing the ratio of TIA1 co-localized with the indicated markers, and ( G”’ ) showing the ratio of the markers co-localized with TIA1 ( n = 56, 56, 53, 56 axons from three biological replicates). ( H ) Screening of shDIC1B constructs for knockdown efficiency in cultured rat cortical neurons. shRNA sequences of 1# and 2# are available in Table . ( I–I’ ) Key frames from time-lapse images showing axon trafficking of TIA1-mCherry granules with or without 10 μM, 15 min nocodazole treatment, with arrowheads in different colors marking distinct granules ( I ), and corresponding quantification ( I’ ) ( n = 334 and 497 granules from three biological replicates). Scale bar = 10 µm. Data represent mean ± SEM; two-tailed unpaired t -test in ( I’ ); one-way ANOVA in ( G’ , G” , G”’ ); ** P < 0.01, *** P < 0.001. See appendix for exact P values. .

Article Snippet: Rab5 (C8B1) Rabbit mAb , Cell Signaling Technology , Cat#3547; RRID: AB_2300649.

Techniques: Pulse Chase, Labeling, Cell Culture, Derivative Assay, Staining, Construct, Knockdown, shRNA, Two Tailed Test

Journal: Current Biology

Article Title: Systems mapping of bidirectional endosomal transport through the crowded cell

doi: 10.1016/j.cub.2024.08.026

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Article Snippet: Rabbit monoclonal anti-Rab5 (C8B1) , Cell signaling Technology , Cat#3547; RRID: AB_2300649.

Techniques: Recombinant, CRISPR, Sequencing, Introduce, Cloning, Software