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. 2014 Oct 21;289(49):33904–33915. doi: 10.1074/jbc.M114.618405

FIGURE 4.

FIGURE 4.

FBXO22a is required for optimal SR activity in cells. A, HPLC analysis of endogenous d-serine production in A172 glioblastoma cells revealed a discrete d-serine peak in the culture media of cells transfected with non-silencing siRNA (Ctl), which decreased in cells transfected with siRNA to hFBXO22. Treatment with d-serine deaminase enzyme (DsdA) prior to HPLC analysis abolished the peak of d-serine, indicating that it corresponds to authentic d-serine. arb. units, arbitrary units. B, quantification of d-serine (d-Ser) in culture media of cells transfected with non-silencing siRNA (Ctl) or siRNA to hFBXO22. Values represent the mean ± S.E. of four experiments with different cell cultures. C, knockdown of endogenous rat FBXO22 in primary astrocyte cultures infected with lentivirus containing FBXO22 shRNA when compared with non-silencing shRNA. D, FBXO22 knockdown decreases d-serine production in primary astrocyte cultures. E and F, lack of effect of hFBXO22a (E and F) or hFBXO22b (F) on SR activity in vitro. A 30-fold molar excess of GST-hFBXO22 isoforms did not affect His-SR activity when compared with GST-α-synuclein or GST-CHIP controls. Values represent the mean ± S.E. of at least three experiments with different protein preparations. *, p < 0.05.