beta I Tubulin Antibody (OTI4A3) NBP2-46245 from Novus Biologicals, a Bio-Techne Brand

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Cellular and molecular life sciences : CMLS
October 29 2022

Fig 1: Upregulation of Tubb4 gene expression following Tubb3 knockdown. a Cultured hippocampal neurons (DIV12) transfected with control or Tubb3 knockdown constructs. Upper: Detection with a pan-alpha antibody to probe for total-tubulin. Lower: Detection with a pan-beta tubulin antibody. Scale bar, 50 µm. Middle panels: Enlargements of cell somata boxed regions. Tubulin intensities were measured inside the soma, indicated by the small colored boxes. DAPI staining of the nucleus (not shown) was used to verify that Tubb3 intensity measurements were performed in the cytoplasm. Scale bar, 5 µm. b Quantification of a, depicting median values (N = 3 experiments). The intensity levels of pan-Tuba and pan-Tubb show no significant (n.s.) decrease, following transfection of Tubb3 KD construct, as compared to control, set to 1. Each data point in the graph represents one cell (panTuba: Control (n = 46), Tubb3 KD (n = 47). panTubb: Control (n = 64), Tubb3 KD (n = 60). For graphical representation, data were binned with a bin size of 0.08 a.u. Statistics: Mann–Whitney Rank Sum Test. c Cultured hippocampal neurons (DIV12) transfected with control or Tubb3 knockdown constructs. Left: Detection with a specific Tubb3 antibody. Right: Detection with a specific Tubb4 antibody. Scale bar, 50 µm. Middle panels: Enlargements of cell somata boxed regions. Tubulin intensities were measured inside the soma, indicated by the small colored boxes. DAPI staining of the nucleus (not shown) was used to verify that tubulin intensity measurements were performed in the cytoplasm. Scale bar, 5 µm. d Quantification of c and data shown in Figure S7, depicting median values (N = 3 experiments). **p < 0.01, ***p < 0.001, ns not significant. Each data point in the graph represents one cell. Tubb1: Control (n = 35), Tubb3 KD (n = 36), Tubb2: Control (n = 31), Tubb3 KD (n = 37), Tubb3: Control (n = 8), Tubb3 KD (n = 12), Tubb4: Control (n = 30), Tubb3 KD (n = 39), Tubb5: Control (n = 33), Tubb3 KD (n = 33). For graphical representation, data were binned with a bin size of 0.08 a.u. Statistics: Mann–Whitney Rank Sum Test
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Cellular and molecular life sciences : CMLS
October 29 2022

Fig 2: Microtubule growth and tubulin expression following cLTP induction. a Live cell imaging of DIV 11/12 cultured hippocampal neurons transfected with EB3-Tomato and exposed to chemical induction of LTP. To induce cLTP, neurons transfected at DIV8-9 were subjected to cLTP induction 3 days post transfection. cLTP was induced by treatment with 50 µM Forskolin, 100 nM Rolipram and 100 µM Picrotoxin for 10 min in Ringer solution without Mg2+, following 10 min of recovery in Ringer solution containing Mg2+. Control cells were kept in Ringer solution containing Mg2+. Image acquisition: 1 frame/s over 3 min. Scale bar, 20 µm. EB3 comets in axonal segments in colored boxes of overview images (top panel) are visualized by kymographs (bottom panel). b Quantification of a, depicting median values (N = 3 experiments). ***p < 0.001. Each data point represents one EB3 comet; control (n = 54), cLTP (n = 70). For graphical representation of the distribution, data are binned with a bin size of 0.02 µm/s. Statistics: Mann–Whitney Rank Sum Test. c–j Quantification of tubulin isotype expression in acute hippocampal slices, following chemical induction of LTP. Control slices were kept in ACSF. cLTP slices were incubated in ACSF with TEA for 10 min and further incubated in pure ACSF over 0, 10 or 20 min. (gamma-Adaptin: loading control; N = 3 experiments, each). Representative western blots and the corresponding quantification of (c, d) pan-Tuba, (e, f) pan-Tubb, (g, h) Tubb1 and (i, j) Tubb3. Depicted are median values. Black dots show the mean value. For graphical representation of the distribution, data are binned with a bin size of 0.05 a.u. Statistics: One way ANOVA followed by Student's t-test or Mann–Whitney Rank Sum Test. *p < 0.05, **p < 0.01