C6 cells were transiently transfected with GLT-1, N4K-R, C7K-R or 11R cDNAs. (a) Na⁺-dependent L-[³H]-glutamate transport was measured as described in the Materials and Methods. A summary of results of four independent experiments performed in triplicate (mean ± S.E.) is shown. Data are expressed as a percentage of activity observed in naïve cells (control). *** indicates a p<0.001 compared to naïve control (one-way ANOVA with Bonferroni post-hoc analysis). (b) C6 cells were treated with vehicle (DMSO) or PMA (100 nM) for 30 min. Following treatment, transporters were immunoprecipitated with GLT-1 antibody. Immunoprecipitates were analyzed by Western blot. Representative Western blot probed with ubiquitin antibody (n=3). Arrows point to ubiquitin immunoreactive signal. Note the lack of ubiquitin signal in the 11R samples. Western blots were stripped and reprobed with GLT-1 antibody.

(a) Schematic representation of transporters with increasing numbers of carboxyl-terminal Lys-Arg (K-R) mutations in GLT-1. (b) C6 cells were transiently transfected with carboxyl-terminal K-R mutant cDNAs and treated with vehicle (DMSO) or PMA (100 nM) for 2 h. Cells were lysed and then analyzed by Western blot. Representative Western blot probed with GLT-1 and actin antibodies. Arrows point to transporter monomers and multimers. All lanes are from different parts of the same gel. (c) Summary of results of three independent experiments (mean ± SE). The effects of PMA were examined by one-sample t-tests; # indicates a p<0.05 compared to the corresponding vehicle control; ## indicates a p<0.01 compared to the corresponding vehicle control (one-sample t-test). PMA had no significant effect on C6K-R expression compared to control (one-sample t-test).

(a) C6 cells were transiently transfected with GLT-1 or GLT-1 (K-R) mutant transporter cDNAs and treated with vehicle (DMSO) or PMA (100 nM) for 2 h. Cell lysates were analyzed by Western blot. Representative Western blots probed with GLT-1 and actin antibodies. Arrows point to transporter monomers and multimers. (b) Summary of results of a minimum of three independent experiments (mean ± SE). The effects of PMA were examined by one-sample t-tests; # indicates a p<0.05 compared to the corresponding vehicle control; ### indicates a p<0.001 compared to the corresponding vehicle control (one-sample t-test). PMA had no significant effect on C7K-R or 11R expression compared to their vehicle controls (one-sample t-test). The effects of PMA on the different constructs were compared by one-way ANOVA with Bonferroni post-hoc analysis; ** indicates a p<0.01 compared to % change in lysate expression of GLT-1 due to PMA (one-way ANOVA with Bonferroni post-hoc analysis).

(a) C6 cells were transiently transfected with GLT-1, N4K-R(517R), N4K-R(526R), N4K-R(517,526R), N4K-R(517,526,550R), or 11R cDNAs and treated with vehicle (DMSO) or PMA (100 nM) for 30 min. Following treatment, transporters were immunoprecipitated with GLT-1 antibody, as described in the Materials and Methods. Immunoprecipitates were analyzed by Western blot. Representative Western blot probed with ubiquitin antibody (n=3). Arrows point to ubiquitin immunoreactive signal. Western blots were stripped and reprobed with GLT-1 to confirm immunoprecipitation of transporters from all samples. Arrows point to transporter monomers and multimers. (b) C6 cells were transiently transfected with GLT-1, GLT-1(517R), GLT-1(526R), GLT-1(550R), GLT-1(517,526R), GLT-1(517,526,550R), or 11R cDNAs and treated with vehicle (DMSO) or PMA (100 nM) for 2 h. Following treatment, cells were lysed, and lysates were analyzed by Western blot. Arrows point to transporter monomers and multimers. Summary of results of a minimum of six independent experiments (mean ± SE). The effects of PMA were examined by one-sample t-tests; # indicates a p<0.05 compared to the corresponding vehicle control; ## indicates a p<0.01 compared to the corresponding vehicle control; ### indicates a p<0.001 compared to the corresponding vehicle control (one-sample t-test). PMA had no significant effect on 11R expression compared to control (one-sample t-test).

(a) C6 cells were transiently transfected with GLT-1 cDNA. After 16-20 h, cells were pre-treated with Bis II (10 μM) for 5 min and then with vehicle (DMSO) or PMA (100 nM) for 2 h. In addition, GLT-1-transfected C6 cells were pre-treated with Bis II (10 μM) for 5 min before either vehicle or PMA treatment. Cell lysates were analyzed by Western blot. Representative Western blots probed with GLT-1 and actin antibodies. Summary of results of three independent experiments (mean ± SE). The effects of PMA were examined by ANOVA; ## indicates a p<0.01 compared to the vehicle control. (b) C6 cells were transiently transfected with GLT-1 or amino-terminal tagged Flag-GLT-1 cDNAs and treated with vehicle (DMSO) or PMA (100 nM) for 2 h. Cell lysates were analyzed by Western blot. Representative Western blots probed with GLT-1 and actin antibodies. The lower panel represents the summary of results of three independent experiments (mean ± SE) with the levels of GLT-1 expressed as % of that observed Flag-GLT-1 transfected cells that were treated with vehicle. The effects of PMA were examined by one-way ANOVA; # indicates a p<0.05 compared to the corresponding vehicle control (one-sample t-test). (c) These same samples were probed with an anti-Flag antibody. Summary of results of three independent experiments (mean ± SE). The effects of PMA were examined by one-sample t-tests; ## indicates a p<0.01 compared to the vehicle control (one-sample t-test). (d) C6 cells were transiently transfected with GLT-1 cDNA and treated with vehicle (DMSO) or PMA (100 nM) for 2 h or 5 min followed by washout and a 2 h incubation without PMA. Cell lysates were analyzed by Western blot. Representative Western blots probed with GLT-1 and actin antibodies and summary of results of three independent experiments (mean ± SE). The effects of PMA were examined by one-sample t-tests; # indicates a p<0.05 compared to the corresponding vehicle control (one-sample t-test).

C6 cells were transiently transfected with 11R, 11R(517K), 11R(526K), 11R(550K), 11R(517,526K), 11R(517,526,550K), or GLT-1 cDNAs. (a) C6 cells were treated with vehicle or PMA for 30 min, immunoprecipitated with GLT-1 antibody, and analyzed by Western blot, as described in the Materials and Methods. Representative Western blot probed with ubiquitin antibody (n=3). Arrows point to ubiquitin immunoreactive signal. Western blots were stripped and reprobed with GLT-1 to confirm immunoprecipitation of transporters. Arrows point to transporter monomers and multimers. (b) C6 cells were treated with vehicle or PMA for 2 h, lysed, and analyzed by Western blot. Arrows point to transporter monomers and multimers. Summary of results of six independent experiments (mean ± SE). The effects of PMA were examined by one-sample t-tests; # indicates a p<0.05 compared to the corresponding vehicle control; ## indicates a p<0.01 compared to the corresponding vehicle control; ### indicates a p<0.001 compared to the corresponding vehicle control (one-sample t-test). PMA had no significant effect on 11R, 11R(517K), or 11R(550K) expression compared to control (one-sample t-test), although there was a trend towards decreased expression for 11R(517K) and 11R(550K). The effects of PMA on the different constructs were compared by one-way ANOVA with Bonferroni post-hoc analysis. All 11R(R-K) mutants displayed significantly different % changes in response to PMA compared to 11R. * indicates a p<0.05 compared to % change due to PMA for 11R; ** indicates a p<0.01 compared to % change due to PMA for 11R; *** indicates a p<0.001 compared to % change due to PMA for 11R (one-way ANOVA with Bonferroni post-hoc analysis).

(a) Untransfected or GLT-1 transfected C6 cells were treated with vehicle (DMSO) or PMA (100 nM) for 30 min at 37°C. In addition, GLT-1-transfected C6 cells were pre-treated with Bis II (10 μM) for 5 min before either vehicle or PMA treatment. Following treatment, cells were lysed, and proteins were immunoprecipitated with control rabbit IgG or anti-GLT-1 antibody at 4°C, as described in the Materials and Methods. Immunoprecipitates were analyzed by Western blot. Representative Western blot probed with ubiquitin antibody, showing the effects of PKC activation or inhibition on ubiquitination of GLT-1 (n=3). Arrows point to ubiquitin immunoreactive signal. (b) The Western blot was stripped and reprobed with GLT-1 antibody to test for equal immunoprecipitation of GLT-1 in samples. (c) GLT-1 transfected cells were treated with PMA for varying amounts of time (min). Representative Western blot showing the time-course of ubiquitination of GLT-1 (n=2). (d) Primary neuron/astrocyte co-cultures were treated with vehicle or PMA for 30 min. GLT-1 was immunoprecipitated following treatment. Immunoprecipitates were analyzed by Western blot and probed with ubiquitin antibody. Representative Western blot showing ubiquitination of GLT-1 after PMA treatment (3 out of 5 experiments displayed ubiquitin immunoreactivity). The blot was stripped and reprobed for GLT-1.

C6 cells were transiently transfected with GLT-1 or GLT-1 (K-R) mutant transporter cDNAs and treated with vehicle (DMSO) or PMA (100 nM) for 30 min. Following treatment, cell surface proteins were biotinylated and batch extracted with avidin beads at 4°C, as described in the Materials and Methods. Cellular fractions were analyzed by Western blot. (a) Representative Western blot for GLT-1 samples probed with GLT-1 and actin antibodies, showing decreased cell surface expression of GLT-1 after PMA treatment. (b) Representative Western blot for N4K-R samples probed with GLT-1 and actin antibodies, showing decreased cell surface expression of N4K-R after PMA treatment. (c) Representative Western blot for C7K-R samples probed with GLT-1 and actin antibodies. In this experiment most of the transporter migrated as a multimer. (d) Representative Western blot for 11R samples probed with GLT-1 and actin antibodies. In this experiment most of the transporter migrated as a multimer. (e) Summary of results of five independent experiments (mean ± SE). Immunoreactivity observed with PMA treatment was expressed as a percentage of that observed with vehicle treatment (control). The effects of PMA were examined by one-sample t-tests; # indicates a p<0.05 compared to the corresponding vehicle control (one-sample t-test). PMA had no significant effect on C7K-R or 11R cell surface expression (one-sample t-test). The percentage of biotinylated actin in untreated controls was 3% or less for all constructs and did not change with treatment.