For immunofluorescence experiments in cell lines, cells were cultured on coverslips until confluent and starved for 48 h. To quantify ciliary GLI2 and GPR161 levels, cells were treated with 500 nM SAG or DMSO for 24 h after 24 h of serum starvation. Cells were fixed with 4% PFA for 10 min at room temperature. After blocking with 5% normal donkey serum, the cells were incubated with primary antibody solutions for 1 h at room temperature followed by treatment with secondary antibodies for 30 min along with DAPI. Primary antibodies used were against GPR161 (1:200, custom-made)21 (link), acetylated α-tubulin (mAb 6-11B-1, Sigma; 1:2000), GLI2 (1:500, gift from Jonathan Eggenschwiler)91 (link), pericentrin (611814, BD Biosciences; 1:500). Coverslips were mounted with Fluoromount-G and images were acquired with a Zeiss AxioImager.Z1 microscope using a 40x oil immersion objective lens.
Protocol detail
Quantifying Ciliary Protein Levels in Cell Lines
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Antibodies
Antibody
Cell lines
Cells
Ciliary
Dapi
Dmso
Donkey
Immersion
Immunofluorescence
Lens
Microscope
Pericentrin
Serum
α tubulin
Corresponding Organization : The University of Texas Southwestern Medical Center
Other organizations : Washington University in St. Louis, Mercy Research, Stanford University, Boston VA Research Institute, Harvard University, Tyrolean Cancer Research Institute, Universität Innsbruck
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Variable analysis
independent variables
- Treatment with 500 nM SAG or DMSO for 24 h
- Ciliary GLI2 levels
- Ciliary GPR161 levels
- Cell lines cultured on coverslips until confluent
- Cells starved for 48 h
- Cells fixed with 4% PFA for 10 min at room temperature
- Blocking with 5% normal donkey serum
- Primary antibody incubation for 1 h at room temperature
- Secondary antibody incubation for 30 min
- DAPI staining
- Primary antibodies used: GPR161 (1:200), acetylated α-tubulin (1:2000), GLI2 (1:500), pericentrin (1:500)
- Coverslips mounted with Fluoromount-G
- Images acquired with Zeiss AxioImager.Z1 microscope using 40x oil immersion objective lens
- Positive control: None specified
- Negative control: DMSO treatment
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