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a Representative immunofluorescence images of RPE1 cells in different cell cycle stages stained against γ-tubulin (yellow) and HAUS1-SNAP-S with anti-Strep antibodies (HAUS1-Strep, magenta). DNA was visualized with DAPI (blue). Green arrows indicate the centrioles shown in the close-up view. b , c Quantification of fluorescence intensity at centrosomes for the indicated proteins shown in a) and Supplementary Fig. . n = 125 (interphase), 132 (prophase), 121 (prometaphase), 125 (metaphase), 162 (anaphase), 128 (telophase), 126 (+ STLC), and 117 (+ <t>STLC&BI2536)</t> centrioles were analyzed for HAUS4 intensity. n = 108 (interphase), 127 (prophase), 121 (prometaphase), 126 (metaphase), 121 (anaphase), 122 (telophase), 120 (+ STLC), and 120 (+ STLC&BI2536) centrioles were analyzed for γ-tubulin intensity. n = 127 (interphase), 118 (prophase), 130 (prometaphase), 123 (metaphase), 121 (anaphase), 127 (telophase), 131 (+ STLC), and 119 (+ STLC&BI2536) centrioles were analyzed for POC5 intensity. d Immunoblot showing protein levels of HAUS4 and γ-tubulin in wild-type control, POC5 -/- , POC5 and POC5 mut cells. e Quantification of relative HAUS4 and γ-tubulin levels in ( d ) (normalized to actin). f Representative immunoblots of POC5 -/- cells expressing POC5 or POC5 mut , treated with 50 µg/ml CHX for the indicated durations, using anti-HAUS4, anti-γ-tubulin and anti-GAPDH (loading control) antibodies. g ) Quantification of panel f ). Relative protein levels were normalized to protein levels at time 0. Linear regression was performed; R 2 values are provided in the Source Data. h Representative images of wild-type control, POC5 -/- , POC5, and POC5 mut cells in prophase and metaphase stained against the centrosomal marker γ-tubulin (green). DNA was visualized with DAPI. Green arrows indicate misaligned chromosomes. i Quantification of cells with amplified γ-tubulin foci and misaligned chromosomes for h). N = 139 (control), 136 ( POC5 -/- ), 129 ( POC5 ), and 137 ( POC5 mut ) cells were analyzed for amplified γ-tubulin foci. n = 135 (control), 133 ( POC5 -/- ), 125 ( POC5 ) and 131 ( POC5 mut ) cells were analyzed for misaligned chromosomes. ( a ), ( h ), scale bars: 10 µm, magnification scale bars: 1 µm; in ( b , c , e , i ), data are presented as mean ± SD, and all statistics were derived from ordinary one-way ANOVA analysis of N = 3 biologically independent experiments. Source data are provided as a Source Data file.
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a Representative immunofluorescence images of RPE1 cells in different cell cycle stages stained against γ-tubulin (yellow) and HAUS1-SNAP-S with anti-Strep antibodies (HAUS1-Strep, magenta). DNA was visualized with DAPI (blue). Green arrows indicate the centrioles shown in the close-up view. b , c Quantification of fluorescence intensity at centrosomes for the indicated proteins shown in a) and Supplementary Fig. . n = 125 (interphase), 132 (prophase), 121 (prometaphase), 125 (metaphase), 162 (anaphase), 128 (telophase), 126 (+ STLC), and 117 (+ STLC&BI2536) centrioles were analyzed for HAUS4 intensity. n = 108 (interphase), 127 (prophase), 121 (prometaphase), 126 (metaphase), 121 (anaphase), 122 (telophase), 120 (+ STLC), and 120 (+ STLC&BI2536) centrioles were analyzed for γ-tubulin intensity. n = 127 (interphase), 118 (prophase), 130 (prometaphase), 123 (metaphase), 121 (anaphase), 127 (telophase), 131 (+ STLC), and 119 (+ STLC&BI2536) centrioles were analyzed for POC5 intensity. d Immunoblot showing protein levels of HAUS4 and γ-tubulin in wild-type control, POC5 -/- , POC5 and POC5 mut cells. e Quantification of relative HAUS4 and γ-tubulin levels in ( d ) (normalized to actin). f Representative immunoblots of POC5 -/- cells expressing POC5 or POC5 mut , treated with 50 µg/ml CHX for the indicated durations, using anti-HAUS4, anti-γ-tubulin and anti-GAPDH (loading control) antibodies. g ) Quantification of panel f ). Relative protein levels were normalized to protein levels at time 0. Linear regression was performed; R 2 values are provided in the Source Data. h Representative images of wild-type control, POC5 -/- , POC5, and POC5 mut cells in prophase and metaphase stained against the centrosomal marker γ-tubulin (green). DNA was visualized with DAPI. Green arrows indicate misaligned chromosomes. i Quantification of cells with amplified γ-tubulin foci and misaligned chromosomes for h). N = 139 (control), 136 ( POC5 -/- ), 129 ( POC5 ), and 137 ( POC5 mut ) cells were analyzed for amplified γ-tubulin foci. n = 135 (control), 133 ( POC5 -/- ), 125 ( POC5 ) and 131 ( POC5 mut ) cells were analyzed for misaligned chromosomes. ( a ), ( h ), scale bars: 10 µm, magnification scale bars: 1 µm; in ( b , c , e , i ), data are presented as mean ± SD, and all statistics were derived from ordinary one-way ANOVA analysis of N = 3 biologically independent experiments. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: Structural mechanisms for centrosomal recruitment and organization of the microtubule nucleator γ-TuRC

doi: 10.1038/s41467-025-57729-2

Figure Lengend Snippet: a Representative immunofluorescence images of RPE1 cells in different cell cycle stages stained against γ-tubulin (yellow) and HAUS1-SNAP-S with anti-Strep antibodies (HAUS1-Strep, magenta). DNA was visualized with DAPI (blue). Green arrows indicate the centrioles shown in the close-up view. b , c Quantification of fluorescence intensity at centrosomes for the indicated proteins shown in a) and Supplementary Fig. . n = 125 (interphase), 132 (prophase), 121 (prometaphase), 125 (metaphase), 162 (anaphase), 128 (telophase), 126 (+ STLC), and 117 (+ STLC&BI2536) centrioles were analyzed for HAUS4 intensity. n = 108 (interphase), 127 (prophase), 121 (prometaphase), 126 (metaphase), 121 (anaphase), 122 (telophase), 120 (+ STLC), and 120 (+ STLC&BI2536) centrioles were analyzed for γ-tubulin intensity. n = 127 (interphase), 118 (prophase), 130 (prometaphase), 123 (metaphase), 121 (anaphase), 127 (telophase), 131 (+ STLC), and 119 (+ STLC&BI2536) centrioles were analyzed for POC5 intensity. d Immunoblot showing protein levels of HAUS4 and γ-tubulin in wild-type control, POC5 -/- , POC5 and POC5 mut cells. e Quantification of relative HAUS4 and γ-tubulin levels in ( d ) (normalized to actin). f Representative immunoblots of POC5 -/- cells expressing POC5 or POC5 mut , treated with 50 µg/ml CHX for the indicated durations, using anti-HAUS4, anti-γ-tubulin and anti-GAPDH (loading control) antibodies. g ) Quantification of panel f ). Relative protein levels were normalized to protein levels at time 0. Linear regression was performed; R 2 values are provided in the Source Data. h Representative images of wild-type control, POC5 -/- , POC5, and POC5 mut cells in prophase and metaphase stained against the centrosomal marker γ-tubulin (green). DNA was visualized with DAPI. Green arrows indicate misaligned chromosomes. i Quantification of cells with amplified γ-tubulin foci and misaligned chromosomes for h). N = 139 (control), 136 ( POC5 -/- ), 129 ( POC5 ), and 137 ( POC5 mut ) cells were analyzed for amplified γ-tubulin foci. n = 135 (control), 133 ( POC5 -/- ), 125 ( POC5 ) and 131 ( POC5 mut ) cells were analyzed for misaligned chromosomes. ( a ), ( h ), scale bars: 10 µm, magnification scale bars: 1 µm; in ( b , c , e , i ), data are presented as mean ± SD, and all statistics were derived from ordinary one-way ANOVA analysis of N = 3 biologically independent experiments. Source data are provided as a Source Data file.

Article Snippet: To test the function of PLK1 in mitosis, cells were treated with 5 μM of STLC 4 hr, followed by 2 h of 50 nM of BI2536 (Cell signaling) treatment.

Techniques: Immunofluorescence, Staining, Fluorescence, Western Blot, Control, Expressing, Marker, Amplification, Derivative Assay