Cells grown on coverslips were fixed with 3% paraformaldehyde solution at room temperature for 10 min and then permeabilized with 0.5% Triton X-100 at room temperature for 5 min. The cells were incubated with antibody against Aurora A (BD Biosciences, 610938), Aurora B (Santa Cruz, sc-25426), BubR1 (BD Biosciences, 612503), Pericentrin (Abcam, 28144) or CREST (ImmunoVision, HCT-0100) at 37 °C for 20 min and then incubated with corresponding secondary antibody at 37 °C for 20 min. For the staining with α-tubulin (Abcam, 18251) and Pericentrin antibodies, the cells were fixed with cold methanol at -20 °C for 20 min and then rehydrated in PBS three times. The cells were post-fixed with paraformaldehyde and permeabilized as described above. The nuclei were counterstained with Hoechst 33342. After a final wash with PBS, coverslips were mounted with antifade solution containing para-phenylenediamine and glycerol in PBS. The staining was determined using laser-scanning confocal microsope (LSM700, Carl Zeiss). Images are acquired using ZEN software (Carl Zeiss) [68 (link)].
Pericentrin
Manufactured by Abcam
Sourced in United States, United Kingdom
Pericentrin is a protein that is a key component of the centrosome, the main microtubule-organizing center of the cell. It plays a crucial role in the regulation of centrosome function and organization.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (4 mentions)
Ab4448, by Abcam (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Yl1 2, by Bio-Rad (2 mentions)
Mg132, by Merck Group (2 mentions)
Aurora b, by Santa Cruz Biotechnology (2 mentions)
Bubr1, by BD (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Confocal 810, by Zeiss (2 mentions)
Arl13b, by Proteintech (2 mentions)
Nedd1, by Abcam (2 mentions)
Plk1 clone 35 206, by Merck Group (2 mentions)
α tubulin, by Merck Group (2 mentions)
γh2ax, by Merck Group (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Phalloidin 568, by Thermo Fisher Scientific (2 mentions)
α tubulin, by Thermo Fisher Scientific (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
Lsm 510, by Zeiss (2 mentions)
34 protocols using pericentrin
1
Immunocytochemistry of mitotic proteins
2
Immunofluorescence Microscopy of Cytoskeletal Proteins
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Primary antibodies used were against glutamylated tubulin (1:800 [tissue sections] and 1:500 [cell lines]; mouse; GT335; AdipoGen), pericentrin (1:250; rabbit; ab4448; Abcam), centrin (1:500; rabbit; N-17; Santa Cruz Biotechnology, Inc.), centrin (1:500; mouse; 20H5; EMD Millipore), A647-conjugated centrin (1:500; mouse; 20H5; EMD Millipore), γ-tubulin (1:500; mouse; GTU88; Sigma-Aldrich), α-tubulin (1:50; rat; YL1/2; AbD Serotec), p53 (1:100; mouse; DO-1; EMD Millipore), E-cadherin (1:30; rabbit; Cell Signaling Technology), and CP110 (1:250; rabbit; Jiang et al., 2012 (link)). The secondary antibodies FITC, Cy5, and rhodamine red (1:50 [tissue sections] and 1:200 [cell lines]; Jackson ImmunoResearch Laboratories, Inc.) as well as Alexa Fluor 488 and 647 (1:500 [cell lines]; Thermo Fisher Scientific) were also used.
3
Immunofluorescence Staining of Cultured Cells
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Cultured cells were fixed with 2% formalin (Sigma) for 20 min at room temperature (RT), blocked with 1% BSA for 30 min, stained sequentially with primary antibodies against pericentrin (Abcam) and tubulin (Sigma) for 1 h at RT, and then incubated with secondary antibodies conjugated to Alexa-488 or −680 for 30 min. The nuclei were stained for 15 minutes with PBS containing 1 μg/ml of 4, 6-diamidino-2-phenylindole (DAPI, Sigma) before mounted with the anti-fade agent Prolong (Molecular Probes). Confocal analyses were performed with Olympus IX81 confocal microscopy systems.
4
Immunofluorescence Analysis of Endocytic Regulators
5
Immunofluorescence Staining of Cell Components
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The cells grown on coverslips were fixed with 3% paraformaldehyde solution at room temperature for 10 min and then permeabilized with 0.5% Triton X-100 at room temperature for 5 min. The cells were incubated with antibody against Aurora B (Santa Cruz Biotechnology; sc-25426), Aurora B-pT232 (Santa Cruz Biotechnology; sc-293127), PLK1 (Santa Cruz Biotechnology; sc-55504), PLK1-pT210 (Abcam; ab39068), Mad2 (Pierce; PA5-21594), BubR1 (BD Biosciences; 612503), histone H3-pS10 (Sigma; 06570), CCAR2 (Bethyl Laboratories; A300-434A), CREST (ImmunoVision, Springdale; HCT-0100), Pericentrin (Abcam; ab28144) or α-Tubulin (Invitrogen; PA5-29444, Sigma; T5168) at 37 °C for 20 min and then incubated with corresponding secondary antibody at 37 °C for 20 min. The nuclei were counterstained with Hoechst 33342 (Invitrogen; H21492). After a final wash with PBS, coverslips were mounted with antifade solution containing para-phenylenediamine and glycerol in PBS.
6
Immunofluorescence Assay for Cell Cycle Markers
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Cells were fixed in 3% paraformaldehyde, permeabilized with 0.1% TX100 and blocked in PBS with 0.01% saponin and 3% (essentially fatty acid free) BSA. Staining was performed using primary antibodies Lamin B1 (goat) (1:200; Santa Cruz, Texas, USA), Plk1 (Rb) (1:100; Abcam, Cambridge, UK), NuMA (Rb) (1:100; Abcam), Aurora B (Mo) (1:100; Abcam), Pericentrin (1:100; Abcam), Tubulin (Mo) (1:400; Sigma, Missouri, USA) and Actin (goat) (1:200; Santa Cruz) before being exposed to secondary Alexa fluor 488 donkey anti‐mouse (1:500) (Invitrogen, Massachusetts, USA), Alexa fluor 546 donkey anti‐rabbit (1:500) (Invitrogen) and/or Alexa fluor 488 anti‐rabbit (1:500) (Invitrogen) or Alexa fluor 546 donkey anti‐goat (1:500) antibodies. Imaging was performed using a 60×/1.4 DIC oil immersion objective on a LSM510 META confocal microscope (Zeiss, Germany).
7
Cdk1, Cdk5, and PCTAIRE1 Knockdown Protocol
8
Immunoblot and Immunofluorescence for Cell Cycle
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Immunoblot and immunofluorescence studies were performed as previously described (27 (link)). Antibodies employed for the immunoblot and immunofluorescence assays were: pericentrin (Abcam), cyclin E (Santa Cruz Biotechnology, Santa Cruz, CA), P~Rb and β-actin (Sigma, St. Louis, MO). Results are derived from three independent experiments.
9
Immunofluorescence Analysis of Primary Cilia
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Cells were cultured in 4-well chamber slides (LabTek). After serum starvation, cells were washed twice with PBS and fixed in PFA for 10 min and then permeabilized with 0.1% Triton-X-100 in PBS for 15 min at room temperature. Cells were then blocked in 10% goat serum for 1 h at room temperature. Primary antibodies were diluted in PBS containing 1% serum and incubated overnight at 4 °C. Antibodies used were as follows: ARL13B (Proteintech, 1:100); acetylated α-tubulin (Sigma T6793, 1:2000); pericentrin (Abcam, 1:2000). DAPI was used to stain the nuclei. Detection was performed with secondary Alexa Fluor 488/568 antibodies (Invitrogen, 1:1000). Images were captured on Zeiss Confocal 810. Images were collected with 1024 × 1024 pixel definition and Z-sections were taken at 0.5-µm step size. Max projections of the Z-stacks were used for primary cilium counting in ImageJ (NIH).
10
Pericyte Migration and Polarization Assay
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Cell-culture plates with inserts (catalog number: 81176) were purchased from ibidi GmbH (Munich, Germany). Around 1.5 × 104 cells (Pericytes and PCHIF2α OE) were seeded into either side of the inserts. Cells were synchronized using starvation media for 12 h before removal of the inserts. Serial images of the gap between Pericytes and PCHIF2α OE were taken for 10 h. Cell migration rate and orientation were calculated by comparing cells at 0 and 10 h. Pericentrin (1:1000; ab4448; Abcam) and rhodamine-phalloidin (1:400; Invitrogen) were used to stain the microtubule-organizing center (MTOC) and actin filaments, respectively. For the quantification of polarized cells, polarized cells were shown as percentage against the total cells in the image field. Total cells were counted by the DAPI-stained cells, and polarized cells were counted by the orientation of Pericentrin to its nucleus.
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