Supplementary Materials Supplemental Materials supp_26_4_622__index. play a key role in cell interactions with extracellular matrix (Gieger 0.001 compared with WT, c 0.001 compared SGI-110 (Guadecitabine) with DKO. (C) Expression of arrestins in DKO and WT cells was detected by Western blot. Purified bovine arrestin-2 and arrestin-3 (0.2 ng/lane) were run for comparison. (D, E) DKO cells were retrovirally infected with Ha-tagged arrestin-2 (Arr2), arrestin-2-7 (Arr27), arrestin-3 (Arr3), arrestin-3-7 (Arr37), or GFP as a control (DKO and WT). Cells were plated on FN and PDL. Arrestin-expressing cells were stained for actin and HA (E), and control cells were stained for actin and GFP (D). Level bar, 10 m. (F) Western blots showing the expression of HA-arrestins and GFP. GAPDH is used as a loading control. (G) Cell size was measured on FN and analyzed as explained for B. # 0.001 DKO from all other conditions, * 0.001, ** 0.01, * 0.05 to WT. Data are from 37C82 cells/condition from three or four experiments. (H) Cell size was measured on PDL from 29C54 cells in three experiments and analyzed as in B. # 0.001 for DKO from all other conditions, * 0.001 from WT. To confirm that the absence of arrestin-2/3 is responsible for the morphological phenotype of DKO cells, we tested whether retroviral expression of arrestin-2 or arrestin-3 rescues them. To ensure that infection did not impact cell morphology, we used cells infected with green fluorescent protein (GFP) as controls (Physique 1D). Cells plated on FN or PDL were SGI-110 (Guadecitabine) stained for hemagglutinin (HA)-tagged arrestins and actin filaments (Physique 1E). The expression of either of the nonvisual arrestins (Physique 1F) reduces DKO cell size nearly back to WT on SGI-110 (Guadecitabine) FN and PDL. Cells expressing arrestin-3 are closer to WT, whereas the rescue by arrestin-2 is usually partial (Physique 1, G and H). Thus each nonvisual arrestin significantly affects cell distributing. Single- Rabbit polyclonal to annexinA5 arrestin-2 or -3Cknockout cells do not reach the size of SGI-110 (Guadecitabine) DKO MEFs and behave like WT MEFs on PDL, further supporting this notion (Physique 1, ACC). The best-characterized function of arrestins is usually their high-affinity binding to active phosphorylated GPCRs (Gurevich and Gurevich, 2006b ). To test whether arrestin interactions with GPCRs play a role in cell distributing, we used receptor bindingCdeficient arrestin mutants with a 7-residue deletion in the interdomain hinge (7; Hanson 0.001, ** 0.01. Means SD from three experiments. (C) Adhesion of DKO cells expressing arrestin-2 + GFP, arrestin-3 SGI-110 (Guadecitabine) + GFP, or GFP alone (controls). Cells were plated on 0.32 g/ml FN. Means SD from 24 data points in three experiments. *** 0.001 compared with DKO. (D) Cells were plated in Transwell chambers coated with 0.32 g/ml FN and allowed to migrate for 4 h. Cells were counted in six fields/chamber in each of four impartial experiments. The data were analyzed by one-way ANOVA with cell type as the main factor, *** 0.001. Insets, representative membranes postmigration. (E) Migration of DKO cells expressing arrestin-2 and GFP or arrestin-3 and GFP, or cells expressing GFP only (DKO and WT). Means SD from 5 fields/chamber from three impartial experiments performed in duplicate analyzed by one-way ANOVA with cell type as the main factor. *** 0.001 compared with WT. DKO-Arr2, ## 0.01, and DKO-Arr3, # 0.05, compared with DKO. (F) Arrestin expression in DKO cells was decided using arrestin-2C or arrestin-3Cspecific antibodies, with corresponding purified bovine arrestins (0.1 ng/lane) run as standards. To determine whether the reversal of DKO morphology by arrestin-2 or -3 rescues enhanced adhesion and motility deficit, were infected DKO cells with arrestin-2 or -3 in constructs that drive GFP coexpression, with controls expressing only GFP. Cells were sorted for GFP expression (Physique 2F) and used in adhesion and Transwell migration assays. Of interest, arrestin-3 but not arrestin-2 reduces the adhesion of DKO cells, although not to WT level (Physique 2C). Similarly,.