rabbit anti cep152  (Bethyl)

Journal: bioRxiv

Article Title: Binding of CEP152 to PLK4 stimulates kinase activity to promote centriole assembly

doi: 10.1101/2024.11.12.623205

Figure Lengend Snippet: (A) Scheme of transfections and selection strategy to generate HeLa and U2OS cell lines expressing CEP152 fused to an AID. Sequencing result confirming the inactivation of CEP152 alleles in HeLa CEP152-AID cells by a deletion mutation. * point mutation. (C) HeLa CEP152-AID cells were treated with or without IAA for 2 h. Cells were lysed and analyzed by Western blotting. (D) HeLa CEP152-AID cells treated with or without IAA for indicated time points. Cells were lysed and analyzed by Western blotting. (E) HeLa CEP152-AID cells were treated with or without IAA for 2 h. IAA was washed-out for indicated time points and cells were lysed for Western blot analysis. The ratio of CEP152 / a-tubulin is indicated below the blot (F) Immunofluorescence staining of HeLa CEP152-AID cells after fixation depicts fast degradation of CEP152 upon 2 h IAA treatment. Scale bar: 1 μm. Values were normalized to the non-treated control and individual values were presented in the graph with ± SD. n = 2, ** < 0.01. (G) HeLa CEP152-AID cells were treated with IAA for 2 h and processed for U-ExM. Gels were stained with acetylated tubulin and the length and the diameter of centrioles were measured. The measured numbers were divided by expansion factor of the gels to obtain biological values. Scale bar: 1 μm, biological scale: 0.6 μm. Graphs represent the quantification of centriole length and diameter, n = 2, ns > 0.05. (H) Representative U-ExM images of HeLa CEP152-AID cells treated with IAA for the indicated times. Centriole schemes depicting approximate centriole orientation are included (left). Quantification graph shows the accumulation of defects on centriole structure in HeLa CEP152-AID cells treated with IAA for indicated time points with +SD. n = 2. * = 0.05, *** < 0.001, **** < 0.0001, scale bar: 1 μm, biological scale: 0.25 μm. (right). (I) HeLa CEP152-AID cells treated with IAA for 2 h and 72 h, fixed and stained with the indicated antibodies. Centriole number is counted by using centrin signal and results are shown in the graph with ± SD. ns > 0.05, ** < 0.01, **** < 0.0001. Scale bar: 1 μm.

Article Snippet: Mouse anti-centrin-2 (1:500 IF; 20H5) from Merck, mouse anti-CEP152 (1:1000 WB and 1:2000 IF; P1285) from Invitrogen, mouse anti-CEP192 (1:500 WB; A302-324A), rabbit anti-STIL (1:1000 IF; A302-442A) from Bethyl, mouse anti-MYC (1:1000 WB; 9E10), rabbit anti-GST (1:1000 WB; Z-5) mouse anti-ninein (1:1000 IF; F5), mouse anti-SAS-6 (1:500 IF, 1:100 U-ExM) from Santa Cruz Biotechnology, and rabbit anti-pericentrin (1:2000 IF; ab4448) from Abcam were used in different techniques.

Techniques: Transfection, Selection, Expressing, Sequencing, Mutagenesis, Western Blot, Immunofluorescence, Staining, Control

Journal: bioRxiv

Article Title: Binding of CEP152 to PLK4 stimulates kinase activity to promote centriole assembly

doi: 10.1101/2024.11.12.623205

Figure Lengend Snippet: (A) HeLa CEP152-AID cells were treated with or without IAA for 2 h and cells were then fixed for immunofluorescence analysis. Scale bar: 1 μm. PLK4 signal was normalized to the non-treated control and values were shown in the graph with ± SD. ns > 0.05. (B) HeLa CEP152-AID cells were treated with IAA for 0 h or 2 h, fixed and stained with the indicated antibodies for U-ExM analysis. Arrows indicate PLK4 foci. Quantification graph of U-ExM images shows the percentage of the cells with either proximal or scattered PLK4 distribution at the centrosomes after rapid degradation of CEP152. n = 2, Scale bar: 1 μm, biological scale: 0.2 μm. (C) HeLa CEP152-AID cells transfected with control (siGL2) and CEP63 siRNAs for 48 h were treated or not with IAA for 2 h. Samples were stained with indicated antibodies. Images show the change in PLK4 levels at the centrosome. Scale bar: 1 μm. PLK4 signal was normalized to the non-treated control and values were shown in the graph with ± SD n = 3. * = 0.05, ** < 0.01. (D) HEK293T cells were transfected with siRNAs against GL2 (control) and CEP152. Cells were then transfected with empty Flag vector or Flag PLK4 for 48h. Cells were harvested 72 h after the first siRNA transfections for immunoprecipitation and Western Blot analysis using the indicated antibodies. n = 3, * = 0.05.

Article Snippet: Mouse anti-centrin-2 (1:500 IF; 20H5) from Merck, mouse anti-CEP152 (1:1000 WB and 1:2000 IF; P1285) from Invitrogen, mouse anti-CEP192 (1:500 WB; A302-324A), rabbit anti-STIL (1:1000 IF; A302-442A) from Bethyl, mouse anti-MYC (1:1000 WB; 9E10), rabbit anti-GST (1:1000 WB; Z-5) mouse anti-ninein (1:1000 IF; F5), mouse anti-SAS-6 (1:500 IF, 1:100 U-ExM) from Santa Cruz Biotechnology, and rabbit anti-pericentrin (1:2000 IF; ab4448) from Abcam were used in different techniques.

Techniques: Immunofluorescence, Control, Staining, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: bioRxiv

Article Title: Binding of CEP152 to PLK4 stimulates kinase activity to promote centriole assembly

doi: 10.1101/2024.11.12.623205

Figure Lengend Snippet: (A) Experimental work-flow to yield non-detectable CEP152 signals at centrosomes in HeLa CEP152-AID cells. (B) HeLa CEP152-AID cells were transfected with control or CEP152 siRNAs for 48 h and treated with - / + IAA for 2 h. Samples were fixed as in A . and stained. Scale bar: 1 μm. p-S305 PLK4 and PLK4 signals were quantified in cells with no detectable CEP152 and normalized to the control after background signal subtraction. Graphs show individual values n = 2, ns > 0.05, * = 0.05, ** < 0.01. (C) Schematic representation of GFP tagged full length (FL) CEP152, CEP152 1-512aa (N-term), 508-1710aa (C-term) and CEP152 D43/44A constructs and their ability to bind PLK4 and to localize at the centrosome. (D) HeLa WT cells were transfected with pEGFP-CEP152 constructs or pEGFP for 36 h. Cells were fixed after pre-extraction for immunofluorescence analysis. Scale bar: 1 μm. n = 3, ns > 0.05, * = 0.05, *** < 0.001. (E) HEK293T cells were co-transfected with Flag-PLK4 and different pEGFP-CEP152 constructs shown in C for 36 h and then harvested for co-immunoprecipitation and Western Blot analysis. Graph shows the ratio of p-S305 PLK4 / Flag-PLK4 with ± SD. n = 3, * = 0.05, ** < 0.01. (F) GST-CEP152 (1-512aa) or GST alone was incubated with Zz-PLK4-His in the presence and the absence of 0.5 mM ATP for in vitro kinase assay and then analyzed by Western blotting.

Article Snippet: Mouse anti-centrin-2 (1:500 IF; 20H5) from Merck, mouse anti-CEP152 (1:1000 WB and 1:2000 IF; P1285) from Invitrogen, mouse anti-CEP192 (1:500 WB; A302-324A), rabbit anti-STIL (1:1000 IF; A302-442A) from Bethyl, mouse anti-MYC (1:1000 WB; 9E10), rabbit anti-GST (1:1000 WB; Z-5) mouse anti-ninein (1:1000 IF; F5), mouse anti-SAS-6 (1:500 IF, 1:100 U-ExM) from Santa Cruz Biotechnology, and rabbit anti-pericentrin (1:2000 IF; ab4448) from Abcam were used in different techniques.

Techniques: Transfection, Control, Staining, Construct, Extraction, Immunofluorescence, Immunoprecipitation, Western Blot, Incubation, In Vitro, Kinase Assay

Journal: bioRxiv

Article Title: Binding of CEP152 to PLK4 stimulates kinase activity to promote centriole assembly

doi: 10.1101/2024.11.12.623205

Figure Lengend Snippet: (A) HEK293T cells were transfected with siRNAs against GL2 (control) or CEP152. Cells were then co-transfected with empty Flag vector and Myc-PLK4 or Flag-PLK4 and Myc-PLK4 for 48h. Cells were harvested 72 h after the first siRNA transfections for immunoprecipitation (IP) analysis and immunostaining. Graph on the left shows the quantification. n = 3, * = 0.05. (B) HEK293T cells were co-transfected with Flag-PLK4 and pEGFP or increasing amounts of pEGFP-CEP192 for 36 h followed by Western blot detection. n = 3, * = 0.05, ** < 0.01.

Article Snippet: Mouse anti-centrin-2 (1:500 IF; 20H5) from Merck, mouse anti-CEP152 (1:1000 WB and 1:2000 IF; P1285) from Invitrogen, mouse anti-CEP192 (1:500 WB; A302-324A), rabbit anti-STIL (1:1000 IF; A302-442A) from Bethyl, mouse anti-MYC (1:1000 WB; 9E10), rabbit anti-GST (1:1000 WB; Z-5) mouse anti-ninein (1:1000 IF; F5), mouse anti-SAS-6 (1:500 IF, 1:100 U-ExM) from Santa Cruz Biotechnology, and rabbit anti-pericentrin (1:2000 IF; ab4448) from Abcam were used in different techniques.

Techniques: Transfection, Control, Plasmid Preparation, Immunoprecipitation, Immunostaining, Western Blot

Journal: bioRxiv

Article Title: Binding of CEP152 to PLK4 stimulates kinase activity to promote centriole assembly

doi: 10.1101/2024.11.12.623205

Figure Lengend Snippet: (A) U2OS WT cells were synchronized in early S-phase with 5 mg/ml Aphidicolin for 16 h and active PLK4 was enriched by treatment with 0.1 μM centrinone for 16 h and wash-out for 3 h. Fixed samples were used for U-ExM analysis. (B) Experimental workflow of S-phase synchronization, centrinone wash-out and IAA treatments of U2OS CEP152-AID cells. U2OS CEP152-AID cells were treated as indicated in (A) and fixed samples were used for U-ExM analysis. Gels were stained with the indicated antibodies in - / + IAA treated U2OS CEP152-AID cells. The number of p-S305 PLK4 foci was counted and the percentage of centrioles was shown in the graph. * shows p-S305 PLK4. (D) Representative U-ExM images of different p-S305 localizations in U2OS CEP152-AID cells after the protocol shown in ( B) . Arrowheads and arrows mark distal and proximal localization of p-S305 PLK4, respectively. Graph shows the percentage of the centrioles with different p-S305 PLK4 localizations in - / + IAA treated U2OS CEP152-AID cells. (E) Representative U-ExM images of SAS-6 localization in - / + IAA treated U2OS CEP152-AID cells. Graph shows the percentage of centrioles with SAS-6 recruitment in - / + IAA treatments. Scale bars: 1 μm for 4-fold expansion images.

Article Snippet: Mouse anti-centrin-2 (1:500 IF; 20H5) from Merck, mouse anti-CEP152 (1:1000 WB and 1:2000 IF; P1285) from Invitrogen, mouse anti-CEP192 (1:500 WB; A302-324A), rabbit anti-STIL (1:1000 IF; A302-442A) from Bethyl, mouse anti-MYC (1:1000 WB; 9E10), rabbit anti-GST (1:1000 WB; Z-5) mouse anti-ninein (1:1000 IF; F5), mouse anti-SAS-6 (1:500 IF, 1:100 U-ExM) from Santa Cruz Biotechnology, and rabbit anti-pericentrin (1:2000 IF; ab4448) from Abcam were used in different techniques.

Techniques: Staining