Fig. 7. S and Rho-1/Brho in S2 cells. S2 cells were transiently transfected with actin5c (Ac5c) driving expression of the indicated constructs. 72 hours after transfection, cellular lysates and conditioned media were analyzed by western blot with antibody raised against Grk extracellular domain. mbGrk^myc was used to distinguish easily the membrane-bound Grk and cleaved Grk forms. (A) Cell lysates: secGrk proteins exhibited M_r of about 40-45 kDa (lane 2), while mbGrk^myc displayed Mr around 70 kDa (lane 3). Lysates from S2 cells expressing mbGrk^myc with Rho-1 or Brho^GFP (lanes 6 and 7); or mbGrk^myc with Rho-1 or (Brho^GFP) and S^myc (lanes 4 and 5) contained both the mbGrk^myc form and also a cleaved form of Grk that migrated with the secGrk species (lane 2). Lysates from cells expressing mbGrk^myc with S^myc contained only the larger mbGrk^myc species and not the cleaved form of Grk (lane 8). (B) Immunoprecipitates of conditioned media from the transfections described in A: no Grk was detected in media from cells expressing empty vector (lane 1), mbGrk^myc alone (lane 5), mbGrk^myc with Rho-1 or Brho^GFP (lanes 8 and 9, respectively), mbGrk^myc with S^myc (lane 10). The co-expression of mbGrk^myc with both S and Rho-1/Brho^GFP (lanes 6 and 7, respectively) leads to the accumulation of a soluble cleaved form of Grk in the medium that migrates with secGrk (lane 4). As control, secGrk and mbGrk^myc lysates were loaded on the same gel (lanes 2 and 3, respectively). We note that cleaved Grk released in the medium is slightly higher that the engineered secGrk. (C) S and Brho intracellular localization: S2 cells were transfected with Ac5c-Brho^GFP (C1-3) or Ac5c-S^myc (C4-6), both shown in green. The cells were also stained (all shown in red) with phalloidin to mark the plasma membrane (C1 and C4), anti-Drosophila Golgi mAb (C2 and C5) or anti-KDEL to mark the ER (C3 and C6). Brho is localized in discrete vesicles that colocalize with the Golgi apparatus (C2) and not the ER (C3). S is mostly expressed in a peri-plasma membrane pattern (C4). We note that some of S colocalizes with the ER (C6), however, none co-localizes with the Golgi (C5).

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