Journal: Histochemistry and Cell Biology

Article Title: Spatio-temporal distribution of tubulin-binding cofactors and posttranslational modifications of tubulin in the cochlea of mice

doi: 10.1007/s00418-020-01905-6

Figure Lengend Snippet: Distribution of TBC proteins in the organ of Corti at P1 and P7. a – e age P1; a TBCA expression is found in the inner area of Kölliker’s organ, the inner hair cell and a tectal cell. b TBCB is expressed in the cells of the Kölliker’s organ. c TBCC labelling is detected in the basal half of the two pillar cells. d Similar TBCD is expressed in the pillar cells. e TBCE staining is found in the basal cells of Kölliker’s organ and the basal half of the inner phalangeal cell. Diffuse staining is visible around the nucleus and below the apical surface of the inner hair cells. The three outer hair cells are also marked. f – j age P7: f TBCA is detected in the inner and outer pillar cells, phalangeal extensions of the three Deiters’ cells, tectal cells and Hensen’s cells. g Similar to TBCA, TBCB is detected in the inner and outer pillar cells, the three Deiters’ cells, the tectal cells and Hensen’s cells. h The expression of TBCC is found in the supporting cells (inner and outer pillar cells, the phalangeal extensions of the three Deiters’ cells, the tectal cells and Hensen’s cells). i TBCD staining is found in the basal parts of the inner pillar cell. j In addition to the inner pillar cells, TBCE is also detected in the and the basal half of the inner phalangeal cell phalangeal extensions of Deiters’ cells. ( TM tectorial membrane with unspecific staining) (Scale bar = 25 µm)

Article Snippet: For immunofluorescence analysis, sections were post-fixed in 4% PFA for 10 min, rinsed in 0.05 M phosphate-buffered saline three times and blocked in 10% bovine serum albumin (BSA) in 0.1% Triton X-100 for 30 min. Primary antibodies were incubated in 1% BSA in 0.1% Triton X-100 overnight at 4 °C at the following concentrations: rabbit polyclonal against TBCA (1:50), TBCB (1:100), TBCC (1:200), TBCD (1:100) and TBCE (1:100, all Proteintech), mouse monoclonal against tyrosinated tubulin (1:500, Millipore Merck), rabbit polyclonal against detyrosinated tubulin (1:500, Millipore Merck), mouse monoclonal against acetylated tubulin (1:500, Sigma-Aldrich) or mouse monoclonal against polyglutamylated tubulin (1:500, AdipoGen).

Techniques: Expressing, Staining, Membrane

Journal: Histochemistry and Cell Biology

Article Title: Spatio-temporal distribution of tubulin-binding cofactors and posttranslational modifications of tubulin in the cochlea of mice

doi: 10.1007/s00418-020-01905-6

Figure Lengend Snippet: Distribution of TBC proteins in the organ of Corti at P14. a TBCA labelling is found in both the tectal and Hensen’s cells. β-Tubulin stains the phalangeal extension of the Deiters’ cells. b TBCB expression is detected in basal cell half of the Deiters’ cells, the phalangeal processes are stained by β-tubulin. c The expression of TBCC is found in the apical part of the inner pillar cell, β-tubulin in the Deiters’ cells. d Staining of TBCD is visible in the cell body of outer hair cells. Phalloidin stains the cuticular plate and stereocilia of the outer hair cells and the inner pillar cell. e TBCE is detected in the outer hair cells, whereas Phalloidion marks the cuticular plate and the stereocilia of the outer hair cells and a bundle-like structure extending from the apex to the base of inner pillar cell. (Scale bar = 25 µm)

Article Snippet: For immunofluorescence analysis, sections were post-fixed in 4% PFA for 10 min, rinsed in 0.05 M phosphate-buffered saline three times and blocked in 10% bovine serum albumin (BSA) in 0.1% Triton X-100 for 30 min. Primary antibodies were incubated in 1% BSA in 0.1% Triton X-100 overnight at 4 °C at the following concentrations: rabbit polyclonal against TBCA (1:50), TBCB (1:100), TBCC (1:200), TBCD (1:100) and TBCE (1:100, all Proteintech), mouse monoclonal against tyrosinated tubulin (1:500, Millipore Merck), rabbit polyclonal against detyrosinated tubulin (1:500, Millipore Merck), mouse monoclonal against acetylated tubulin (1:500, Sigma-Aldrich) or mouse monoclonal against polyglutamylated tubulin (1:500, AdipoGen).

Techniques: Expressing, Staining

Journal: PLOS ONE

Article Title: A homogeneous time-resolved fluorescence screen to identify SIRT2 deacetylase and defatty-acylase inhibitors

doi: 10.1371/journal.pone.0305000

Figure Lengend Snippet: (A) Immunofluorescence images of acetylated alpha-tubulin in A549 cells showing enhanced fluorescence in cells treated with 25 μM of SirReal2 (positive control) or 100 μM of 1 . The media for the control contained 2% DMSO, and the scale bar is 10 μm. (B) Comparison of GFP fluorescence intensities from acetylated alpha-tubulin staining from the images in panel A. GFP fluorescence intensities from individual cells were measured and normalized to the DAPI intensities of the same cells, and the intensities were compared with a one-way ANOVA followed by Dunnett’s multiple comparison test (***p < 0.001). (C) Western blot showing knockout of SIRT2 in A549 cells using CRISPR/Cas9. The alpha-tubulin blot, which was performed on a separate membrane, was used as a loading control. (D) Immunofluorescence images of A549 SIRT2-KO cells showing increased acetylated alpha-tubulin levels in cells without the protein, and the ligands SirReal2 and 1 had no effect on the levels of acetylated alpha-tubulin in SIRT2-KO cells. Experiments were performed identical to panel A. (E) Comparison of GFP fluorescence intensities from acetylated alpha-tubulin staining from the images in panel D. These were quantified and compared as described above for panel B. (F) SirReal2 treatment for 72 hours did not cause toxicity in A549 cells, as measured with an MTT assay. The control (Ctrl) cells were treated with 0.7% DMSO to be consistent with the drug treated cells. (G) Treatment of A549 cells with 1 for 72 hours did not cause toxicity, which was determined with an MTT assay as in panel F.

Article Snippet: Antibodies used were a rabbit SIRT2 primary antibody (Cell Signaling Technology catalog #12650), an HRP-conjugated goat anti-rabbit secondary antibody (Invitrogen catalog #65–6120), a mouse alpha-tubulin primary antibody (Novus Biologicals catalog #NB120-11304), and an HRP-conjugated goat anti-mouse secondary antibody (Invitrogen catalog #62–6520).

Techniques: Immunofluorescence, Fluorescence, Positive Control, Control, Comparison, Staining, Western Blot, Knock-Out, CRISPR, Membrane, MTT Assay