Rin protein induces neurite outgrowth in PC12 cells. (A) Empty vector–transfected PC12 cells were left untreated or stimulated with 50 ng/ml NGF for 44 h. Cells were fixed with 3% PFA-PBS and were visualized under a phase contrasted bright field microscopy. (B) PC12 cells were transfected with an expression vector encoding Myc-tagged wild-type Rin or Myc-tagged constitutively active RinQ78L protein. After 48 h, transfected cells were fixed, permeabilized, and processed for immunofluorescence with an anti-Myc antibody. Transfected cells were visualized with a Cy3-labeled anti–mouse secondary antibody. (C) PC12 cells were left untreated or stimulated with 50 ng/ml NGF for 2 d. Other PC12 cells were transfected with an expression vector encoding Myc-tagged wild-type Rin or Myc-tagged RinQ78L protein and were maintained for 5 d. Cells were washed with an ice-cold PBS buffer and lysed with an ice-cold lysis buffer. Cell lysates were cleared by centrifugation and Western blotting was performed as described in the Materials and methods, using an antineurofilament antibody, an anti-MAPK 1/2 antibody, and an anti-Myc antibody. Data are representative of three independent experiments, which gave essentially identical results.

Dominant negative Rac/Cdc42 inhibits the Rin-mediated neurite outgrowth in PC12 cells. (A and B) Cells were transfected with an HA-tagged wild-type Rin vector and a Myc-tagged dominant negative RacS17N/Cdc42S17N/RhoT19N vector either alone or in pairs. After 48 h, cells were fixed and immunostained with an anti-HA antibody and an anti-Myc antibody. Columns and vertical bars denote the mean ± SEM, respectively (n = 3). In the merge of A, HA-Rin staining is shown in green (using FITC-labeled anti–rat secondary antibody), whereas Myc-Rac staining is shown in red (using Cy3- labeled anti–mouse secondary antibody).

CaM association is necessary for Rin-induced neurite outgrowth in PC12 cells. (A, B) PC12 cells were transfected with Myc-tagged expression vectors encoding several Rin proteins and incubated with vehicle, 50 or 100 μM W13 (CaM antagonist) or W12 (inactive analogue of W13). After 48 h, cells were fixed and immunostained with an anti-Myc antibody. Columns and vertical bars denote the mean ± SEM, respectively (n = 3). Asterisks indicate P < 0.05. (C) Comparison of the COOH-terminal amino acid sequences (amino acids 191–217) of wild-type Rin protein, RinΔ18, RinC-4, and RinC-7. The residues of the mutant proteins that are different from those of the wild-type Rin protein are italicized. (D) Cos-7 cells were transfected with Myc-tagged expression vectors encoding several Rin proteins. After 48 h, cells were lysed, cleared, and Rin proteins were pulled down with CaM-conjugated agarose beads at 4°C for 2 h as described in the Materials and methods. Bound Myc-Rin protein was visualized with an anti-Myc antibody. Data are representative of three independent experiments, which gave essentially identical results. (E) PC12 cells were transfected with Myc-tagged expression vectors encoding several Rin mutant proteins. After 48 h, cells were fixed and immunostained with an anti-Myc antibody. Columns and vertical bars denote the mean ± SEM, respectively (n = 3).

Endogenous Rin protein is involved in calcium-mediated neurite outgrowth in PC12 cells. (A) The expression of Rin and GAPDH (constitutively expressed gene) at the mRNA level was detected by performing RT-PCR from total RNA isolated from Cos-7, PC12, and primary dissociated neuronal cells from P3 mouse brain, as described in the Materials and methods. (B) PC12 cells were transfected with an HA-tagged wild-type Rin vector and a Myc-tagged vector encoding RinG29V-C-7 mutant protein either alone or in pairs. After 48 h, cells were fixed and immunostained with an anti-HA antibody and an anti-Myc antibody. In the merge, HA-Rin staining is shown in green (using FITC-labeled anti–rat secondary antibody), whereas Myc-Rin staining is shown in red (using Cy3-labeled anti–mouse secondary antibody). (C) PC12 cells were transfected with an empty vector or a Myc-tagged RinG29V-C-7 expression vector. After 4 h, transfected cells were stimulated with vehicle or 50 ng/ml NGF for 44 h. Cells were fixed and immunostained with an anti-Myc antibody. Columns and vertical bars denote the mean ± SEM, respectively (n = 3). (D) PC12 cells were transfected with an empty vector or RinG29V-C-7 expression vector. After 48 h, they were stimulated with vehicle or 50 ng/ml NGF for 5 min. Cells were lysed and cleared, followed by a Rac/Cdc42 pull-down assay (Rac PD and Cdc42 PD). Bound endogenous Rac proteins (arrow) and Cdc42 proteins were visualized by Western blotting. Constitutively active Myc-Rac (positive control) is indicated by asterisk. Data are representative of three independent experiments, which gave essentially identical results. (E) PC12 cells were transfected with an empty vector or a Myc-tagged RinG29V-C-7 expression vector. After 4 h, transfected cells were pretreated with vehicle or the L-type calcium channel blocker nitrendipine (final 10 μM)/diltiazem (final 50 μM) for 30 min and stimulated with 50 μM forskolin alone or 50 μM forskolin plus 50 mM KCl for 44 h. Cells were fixed and immunostained with an anti-Myc antibody. Columns and vertical bars denote the mean ± SEM, respectively (n = 3). Asterisks indicate P < 0.05. (F) PC12 cells were transfected with 1 μg of Myc-tagged wild-type Rin vector and indicated amounts of siRNA. After 48 h, cells were lysed, cleared and followed by Western blotting as described in the Materials and methods. (G) PC12 cells were transfected with 1 μg of pGFP-C1 vector and indicated amounts of siRNA. After 4 h, transfected cells were stimulated with 50 μM forskolin alone or 50 μM forskolin plus 50 mM KCl for 44 h. Cells were fixed and counted as described in the Materials and methods. Columns and vertical bars denote the mean ± SEM, respectively (n = 3). The asterisk indicates P < 0.05. (H) PC12 cells were transfected with 1 μg of Myc-tagged RacS17N/Cdc42S17N and 1 μg of pGFP-C1 vector plus 10 μg of Rin-specific siRNA either alone or in pairs. After 4 h, transfected cells were stimulated with 50 μM forskolin alone or 50 μM forskolin plus 50 mM KCl for 44 h. Cells were fixed and counted as described in the Materials and methods. Columns and vertical bars denote the mean ± SEM, respectively (n = 3). Asterisks indicate P < 0.05.

MAPK is not involved in the Rin-signaling pathway in PC12 cells. (A) PC12 cells were serum starved for 24 h and left untreated or stimulated with 50 ng/ml NGF or 0.5 μg/ml ionomycin for 5 min. Other PC12 cells were transfected with an expression vector encoding Myc-tagged wild-type Rin or Myc-tagged RinQ78L protein and serum starved for 24 h. Cell lysates were subjected to Western blotting as described in the Materials and methods, using an antiphospho-MAPK antibody, an anti-MAPK 1/2 antibody and an anti-Myc antibody. Data are representative of three independent experiments, which gave essentially identical results. (B) Serum-starved PC12 cells were preincubated with vehicle or various amounts of MAPK kinase inhibitor U0126 for 48 h at 37°C, followed by 50 ng/ml NGF for 5 min. Cell lysates were subjected to Western blotting as described in the Materials and methods. Data are representative of three independent experiments, which gave essentially identical results. (C) Rin-transfected cells or 50 ng/ml NGF-stimulated cells were treated with vehicle or 7.5 μM U0126 for 48 h at 37°C. Cells were fixed and visualized with an anti-Myc antibody. Cells with neurites exceeding one cell body diameter in length were counted as a ratio of the total number of transfected cells. Columns and vertical bars denote the mean ± SEM, respectively (n = 3). Asterisk indicates P < 0.05 as compared with the control value.

Rin activates endogenous Rac/Cdc42 in PC12 cells. Cells were transfected with an empty vector or Myc-tagged expression vectors encoding several Rin proteins. After 48 h, cells were lysed and cleared, followed by Rac/Cdc42 pull-down assay, as described in the Materials and methods. Bound endogenous Rac proteins (arrow) and Cdc42 proteins were visualized by Western blotting using an anti-Rac antibody and anti-Cdc42 antibody, respectively. Lane 1, empty vector; lane 2, wild-type Myc-Rin, lane 3, Myc-RinQ78L; lane 4, Myc-RinΔ18; lane 5, Myc-RinC-4; lane 6, Myc-RinC-7; lane 7, empty vector with 50 ng/ml NGF for 3 min; lane 8, constitutively active Myc-Rac (positive control, asterisk). Data are representative of three independent experiments, which gave essentially identical results.

Rin activates endogenous Rho in PC12 cells. (A) PC12 cells were transfected with an empty vector or Myc-tagged Rin vector. After 48 h, cells were stimulated with vehicle or 10 μM LPA for 1 min. Cells were lysed and cleared, followed by Rho activation assay, as described in the Materials and methods. Lane 1, empty vector; lane 2, wild-type Myc-Rin, lane 3, Myc-RinQ78L; lane 4, Myc-RinΔ18; lane 5, Myc-RinC-4; lane 6, Myc-RinC-7; lane 7, empty vector with 10 μM LPA for 1 min. Data are representative of three independent experiments, which gave essentially identical results. (B) PC12 cells were transfected with indicated amounts of siRNA of RhoA or control siRNA. After 48 h, cells were lysed, cleared and followed by Western blotting as described in the Materials and methods. (C) PC12 cells were transfected with a Myc-tagged wild-type Rin vector and a siRNA of RhoA either alone or in pairs. After 48 h, cells were fixed and observed as described in the Materials and methods.