dync1i1 polyclonal antibody  (Proteintech)

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 ) Venn diagram showing four shared interactors between TIA1 and DIC1B interactomes (DYNC1i1 in BioGRID, see Methods and Dataset ). ( B ) Mass spectrometry analysis of proteins interacting with GST-TIA1 in rat brain lysates, using GST tag as a control. Red dots indicate significantly enhanced interactors ( p < 0.05 and log 2 fold change >1.2). Data from three replicates; statistical significance assessed by paired t -test. ( C ) Immunoblots of ANXA7 in proteins pulled down by GST-TIA1 from rat brain. ( D ) Mass spectrometry analysis of proteins interacting with GST-ANXA7 in rat brain lysates, using GST tag as a control. Red dots indicate significantly enhanced interactors ( p < 0.05 and log 2 fold change >1.2). Data from three replicates; statistical significance assessed by paired t -test. ( E ) Immunoblots of TIA1 and DIC1B in proteins pulled down by GST-ANXA7 from rat brain. ( F ) Key frames from time-lapse images showing retrograde co-trafficking of light-induced Opto-TIA1 (red) and ANXA7-EGFP (green) granules in DIV9 rat hippocampal neurons. Scale bar = 2 µm. Arrowheads indicate co-trafficking. ( G ) Purified recombinant Myc-ANXA7 (rMyc-ANXA7) protein enhances rTIA1 and rFlag-DIC1B interaction, shown by increased rTIA1 pulled down by rFlag-DIC1B. The arrow indicates the weak interaction between TIA1 and DIC1B observed in the absence of rANXA7. ( G’ ) Quantification of ( G ), data from three biological replicates (Myc-ANXA7 vs. TIA1: P = 0.0088; TIA1 vs. TIA1 + Myc-ANXA7: P = 0.0463). ( H ) Co-IP assay examining the interaction between endogenous DIC1B and HA-tagged TIA1 using anti-HA magnetic beads in DIV11 rat cortical neurons. The interaction is studied under endogenous ANXA7 knockdown (shANXA7) or Myc-ANXA7 overexpression conditions. ( H ’) Quantifying TIA1-DIC1B interaction from ( H ) shows the effects of different ANXA7 levels ( n = 5 technical replicates from four biological replicates. Control vs. Myc-ANXA7: P = 0.0267; Control vs. shANXA7: P = 0.0474). ( I ) Schematic diagram of FLIM-FRET to examine the affinity between EGFP-TIA1 (donor) and DIC1B-mRFP (acceptor) under varying levels of ANXA7 (A7). ( J ) Represented images showing color-coded EGFP-TIA1 lifetime in axon shafts of transfected neurons, with lifetime ( J’ ) and FRET efficiency ( J” ) quantified and compared across indicated groups. Scale bar = 2 μm ( n = 29, 37, 35, and 49 axons from four biological replicates. (1) vs. (2): P = 0.0266; (1) vs. (4): P < 0.0001). ( K ) Left: schematic illustrating PLA detection of endogenous TIA1 and DIC1B interaction. Right: representative confocal images showing TIA1/DIC1B PLA signals in neurons with varying ANXA7 (A7) levels; bracketed axons are enlarged below. Scale bars = 50 µm (top), 10 µm (bottom). ( K’ ) Quantification of axonal PLA density from ( K ) ( n = 172, 90, 172, and 90 ROIs from six biological replicates. All P < 0.0001). Data represent mean ± SEM; one-sample t -test in ( G’ , H’ ); one-way ANOVA in ( J’ , J” , K’ ). .

Article Snippet: DYNC1I1 Polyclonal antibody , Proteintech , Cat#13808-1-AP; RRID: AB_2093492.

Techniques: Mass Spectrometry, Control, Western Blot, Purification, Recombinant, Co-Immunoprecipitation Assay, Magnetic Beads, Knockdown, Over Expression, Transfection