antibody against hs  (AMS Biotechnology)

Journal: Cells

Article Title: Mitotic Maturation Compensates for Premature Centrosome Splitting and PCM Loss in Human cep135 Knockout Cells

doi: 10.3390/cells11071189

Figure Lengend Snippet: Interactome of Cep135, Wdr8, and Ssx2ip in Xenopus egg extracts. ( A ) Schematic drawing illustrating the state of centrosomes in somatic cells, developing oocytes, ovum (egg), and fertilized egg (zygote). ( B , C ) Immunoblotting ( B ) and corresponding quantification ( C ) of Ssx2ip, Cep135, and α-tubulin to evaluate protein expression at different Xenopus oocyte stages. The total amount of protein was controlled by glycerol aldehyde 3 phosphate dehydrogenase (Gapdh). ( D , E ) Identification of proteins specifically precipitated with antibodies against Cep135 ( D ) or Wdr8 ( E ) using tandem mass spectrometry. The list shows only a fraction of proteins identified. ( F ) Immunoprecipitations using antibodies against Wdr8 (elu1 Wdr8, elu1 IgG: control) analyzed by immunoblotting of Cep135 and Ssx2ip. Input (total extract) and supernatants (sup1/sup2) of extracts after IP are shown from two sequential precipitations.

Article Snippet: Antibodies against human (hs) WDR8 were generated in rabbits against the N-terminal (MNFSEVFKLSSLLCK) and C-terminal (ETEAVVGTACRQLGGHT) peptides (Seramun Diagnostica GmbH, Heidesee, Germany) and used for immunofluorescence (1:50).

Techniques: Western Blot, Expressing, Mass Spectrometry, Control

Journal: Cells

Article Title: Mitotic Maturation Compensates for Premature Centrosome Splitting and PCM Loss in Human cep135 Knockout Cells

doi: 10.3390/cells11071189

Figure Lengend Snippet: cep135 , wdr8 , and ssx2ip KOs generated by CRISPR/Cas9 in human RPE-1 cells line are vital and proliferate. ( A ) Genomic organization of human CEP135 , WDR8 , or SSX2IP loci with indicated targeting sites used for CRISPR/Cas9-mediated DNA cleavage. ( B ) Immunoblot to detect CEP135 (upper panel) or SSX2IP (lower panel) in cell clones after KO of the respective genes. Please note that the SSX2IP antibody tended to reveal unspecific signals in some immunoblots (see A). Clones wdr8 ko308, ssx2ip ko2-13, and cep138 ko8 were used for all further experiments. ( C ) Immunofluorescence to detect CEP135, WDR8, or SSX2IP in human RPE-1 wildtype (WT) and representative cep135 , wdr8 , or ssx2ip KO cell lines (see for all cell clones). ( D ) Cell proliferation in WT and wdr8 , ssx2ip , and cep135 RPE-1 KO cell lines. A total of 10 5 cells (3.5 cm cm 2 dish) were seeded and counted at indicated times (20 h, 32 h, 44 h, and 56 h). ( E , F ) DNA content analysis using propidium iodide (PI) staining to analyze cell-cycle progression in WT and wdr8 , ssx2ip , or cep135 RPE-1 KO cell lines. The indicated range shows cells in S/G 2 /M-phase. ( F ) Quantification of the fraction of cells in S/G 2 /M-phase. The graph shows average values from independent experiments ± S.D.; the p -value was calculated using a two-tailed Student’s t -test. * indicates p < 0.05, **** p < 0.001.

Article Snippet: Antibodies against human (hs) WDR8 were generated in rabbits against the N-terminal (MNFSEVFKLSSLLCK) and C-terminal (ETEAVVGTACRQLGGHT) peptides (Seramun Diagnostica GmbH, Heidesee, Germany) and used for immunofluorescence (1:50).

Techniques: Generated, CRISPR, Western Blot, Clone Assay, Immunofluorescence, Staining, Two Tailed Test

Journal: Cells

Article Title: Mitotic Maturation Compensates for Premature Centrosome Splitting and PCM Loss in Human cep135 Knockout Cells

doi: 10.3390/cells11071189

Figure Lengend Snippet: Protein expression and centrosomal localization of CEP135, WDR8, and SSX2IP are interdependent in RPE-1 cell lines. ( A ) Immunoblotting to show expression levels of PCM1, CEP135, SSX2IP, and γ-tubulin. α-Tubulin was used as a control for total protein loaded. Please note that the SSX2IP antibody tended to reveal unspecific signals in some immunoblots. ( B – E ) Centrosomal CEP135 ( B , C ) and SSX2IP ( D , E ) signals in WT and KO cells as indicated. cep135 KO cells frequently displayed separated centrosomes with two distinct γ-tubulin signals (split pair). Representative immunofluorescence images ( C , E , both with γ-tubulin signals) and quantifications ( B , D ). The ImageJ threshold feature was used to identify the region of the CEP135 signal in ( B ). A square (8.6 µm in xy ) was drawn with the γ-tubulin signal in the center in ( D ). ( B , D ) Graphs show mean values (fat colored/black dots) of single experiments and individual data points (transparent dots). The added intensity of the two separated γ-tubulin signals (“split pair”) in cep135 KO cells was quantified in ( D ). Three or four independent experiments were performed with more than 500 ( B ) or 210 ( D ) cells measured in each experiment. Errors bars show the SD from means; p -values were calculated with a two-tailed Student’s t -test. * indicates p < 0.05, **** p < 0.001. The surface plots represent sums (ImageJ z -projections) of all images. ( C , E ) Scale bars: 5 µm ( C ) or 2 µm ( E ).

Article Snippet: Antibodies against human (hs) WDR8 were generated in rabbits against the N-terminal (MNFSEVFKLSSLLCK) and C-terminal (ETEAVVGTACRQLGGHT) peptides (Seramun Diagnostica GmbH, Heidesee, Germany) and used for immunofluorescence (1:50).

Techniques: Expressing, Western Blot, Control, Immunofluorescence, Two Tailed Test

Journal: Cells

Article Title: Mitotic Maturation Compensates for Premature Centrosome Splitting and PCM Loss in Human cep135 Knockout Cells

doi: 10.3390/cells11071189

Figure Lengend Snippet: cep135 KO cells show premature centrosome splitting and diminished PCM. ( A ) γ-Tubulin was stained to identify cells with premature split centrosomes and reduced PCM in WT and cep135 KO cells. Scale bar: 20 µm. ( B ) Quantification of centrosome splitting by more than 1 µm at different cell-cycle stages in cep135 , wdr8 , and ssx2ip KO cell lines. ( C ) Quantification of centrosome distance in WT and cep135 KO cells at G 1 (48 h starvation) before and after nocodazole treatment that greatly increased centrosome distances in cep135 KO cells. ( D , E ) Quantification of the centrosomal γ-tubulin ( D ) and CDK5RAP2 ( E ) signals in WT, wdr8 , ssx2ip , and cep135 KO cells. cep135 KO cells displaying two distinct γ-tubulin signals were quantified separately either using summed intensities of both centrosomes (split-pair) or as separated values for each of the two individual centrosomes (split single). ( D ) Cells in G 0/1 and S-phase. ( E ) G 0/1 cells. ( B – E ) Graphs show mean values (fat colored/black dots) of single experiments and individual data points (transparent dots). Around 300 cells were measured for each cell line. Three independent experiments were performed. Error bars show the SD. The p -value was calculated with a two-tailed Student’s t -test. ** indicates p < 0.01, *** p < 0.005, **** p < 0.001. ( F ) Representative images showing γ-tubulin and CDK5RAP2 signals in WT and cep135 KO cells. Scale bar: 10 µm or 2 µm (for enlarged areas).

Article Snippet: Antibodies against human (hs) WDR8 were generated in rabbits against the N-terminal (MNFSEVFKLSSLLCK) and C-terminal (ETEAVVGTACRQLGGHT) peptides (Seramun Diagnostica GmbH, Heidesee, Germany) and used for immunofluorescence (1:50).

Techniques: Staining, Two Tailed Test