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. 2022 Oct 29;79(11):575. doi: 10.1007/s00018-022-04607-5

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© The Author(s) 2022

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — 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