GABABRs increase GS stability by reducing its ubiquitination and proteasomal degradation.
A, COS-7 cells expressing GS or GS/R2 were labeled for 30 min with 100 μCi/ml [35S]methionine and incubated for 8 h. The level of 35S-labeled GS remains at 0, 4, and 8 h was measured using immunoprecipitation followed by SDS-PAGE (top lefthand panel). After quantification using a phospho-imager the amount of 35S-labeled GS was normalized to 0 time as shown in the lefthand panel Data represent mean ± S.E., (paired t test; p < 0.05, n = 3). B, cells expressing GS, GS/R2, or control non-transfected cells (NT) were subject to immunoprecipitated with GS antibody. Precipitated material was subject to SDS-PAGE and immunoblotted with GS and UB antibodies. The ratios of UB:GS immunoreactivity were determined and normalized to values seen in cells expressing GS alone. Data represent mean ± S.E., (unpaired t test; p < 0.01, n = 4). C, cells expressing GS and GS/R2 were treated with vehicle (−) or lactacystin (+) for 20 μm for 8 h. Lysates were subject to immunoblotting with R2, GS, and actin antibodies and GS levels were normalized to those seen in vehicle-treated controls (dotted line). Data represent mean ± S.E. (p < 0.01, paired t test, n = 4). *, significantly different from control, (p < 0.05); **, p < 0.01.