Alexa fluor 488 or alexa fluor 568 conjugated secondary antibody

Ad-Fhit-infected cells were seeded on coverslips, grown overnight, and fixed with 4% paraformaldehyde in PBS for 15 min. Following permeabilization with 0.5% Triton X-100 in PBS for 10 min at room temperature, cells were then blocked with 3% BSA in PBS for 30 min at room temperature and incubated overnight at 4°C with primary antibodies. After three 5-min washes in PBS, cells were incubated for 1 h at room temperature with Alexa Fluor 488- or Alexa Fluor 568-conjugated secondary antibodies (Invitrogen). Then, cells were counterstained with Hoechst 33342 for 5 min for nuclear staining, washed twice in PBS, and mounted on glass slides using Vectashield (Vector Laboratories; Burlingame, CA, USA). Cells were visualized using a Zeiss LSM 700 laser scanning microscope (Carl Zeiss; Oberkochen, Germany). Quantification of proteins in the acquired images was performed by using ImageJ software (NIH Image; Bethesda, MD, USA).

293T cells attached to poly-D-lysine- and collagen-coated coverslips were transfected with the indicated plasmids. After 48 h, cells were washed once with PBS and subsequently fixed with PBS containing 4% paraformaldehyde at room temperature for 20 min. Cells washed with PBS were blocked for 1 h at room temperature in blocking solution (PBS containing 0.1% Triton X-100, 10% normal goat serum, and 1% bovine serum albumin) and subsequently incubated with blocking solution containing antibodies against HA (1:150 dilution, Biolegend, San Diego, CA) and/or EE (EMD Millipore, Billerica, MA) at room temperature for 1 h. Cells were washed four times for 7 min each and incubated with Alexa Fluor 488- or Alexa Fluor 568-conjugated secondary antibodies (1500; Invitrogen) for 30 min. Following four additional washes for 7 min, cells were mounted with VECTASHIELD (Vector Laboratories, Burlingame, CA) and observed under a Zeiss LSM 700 confocal microscope with 40 × 1.2 numerical aperture objective (Carl Zeiss, Oberkochen, Germany). Pearson correlation coefficient (PCC) was calculated to quantify colocalization between PAR4-Venus and RGS-HA proteins in the presence of Gα. We assigned four square-shaped regions per cell to the cell membrane. PCC was calculated by IMAGE J software (National Institutes of Health, Bethesda, MD) between the stacks of images from two channels.

Staged embryos were fixed in 4% paraformaldehyde overnight at 4°C, cryoprotected with 30% sucrose in phosphate-buffered saline (PBS) and embedded in Optimal Cutting Temperature tissue freezing medium (Triangle Biomedical Sciences, Durham, USA) for cryosectioning. 10 µm (immunohistochemistry) and 16 µm (in situ hybridization) transverse sections were collected. For immunohistochemistry, antigen retrieval was performed by boiling the slides in sodium citrate buffer (10 mM sodium citrate and 0.05% Tween 20, pH 6.0) for 20 min in a vegetable steamer. Furthermore, the sections were washed in PBS and incubated for 30 min with an Image iT™ FX signal enhancer (Molecular Probes, USA). Antigen was first recognized by primary antibodies during overnight incubation at 4°C and then visualized using Alexa Fluor 488- or Alexa Fluor 568-conjugated secondary antibodies (Molecular Probes, USA). Cell nuclei were counterstained with DAPI (1 : 1000) for 10 min (Sigma, USA). Slides were washed with PBS and mounted with Vectashield (Vector, USA). Images were taken with a Zeiss Axio Observer fluorescent microscope (Zeiss, Germany). The primary antibodies used: p27^Kip1 (Santa Cruz Biotechnology, sc-528, 1 : 100), p57^Kip2 (Abcam, ab75974, 1 : 100), H3K27me3 (Millipore, 07-449, 1 : 100) and SC-35 nuclear speckles (Abcam, ab11826, 1 : 200).