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. 2015 Feb 3;107(3):dju502. doi: 10.1093/jnci/dju502

Figure 3.

Figure 3.

CypD phosphorylation. A) Mitochondrial extracts from LN229 cells were immunoprecipitated (IP) with IgG or an antibody to CypD, and pellets were analyzed by Western blotting. B) Recombinant GST-CypD or GST was incubated with mitochondrial extracts of LN229 cells, and bound proteins were analyzed by western blotting. C) The indicated recombinant proteins were incubated with recombinant active Akt2 or vehicle in a kinase assay, and radioactive proteins were visualized by autoradiography. GSK3β was a control Akt substrate. D) LN229 cells with stable CypD knockdown were reconstituted with the indicated CypD cDNAs, immunoprecipitated with IgG or an antibody to CypD, and pellets were analyzed with an antibody to phosphorylated Ser (pSer) by western blotting. E) LN229 cells with stable shRNA knockdown of CypD were transfected with the indicated FLAG-tagged CypD cDNAs, treated with vehicle or PX-866, and immunoprecipitated with anti-FLAG-M2 gel followed by western blotting with anti-pSer antibody. The position of full-length or mature CypD band is shown. F) CypD+/+ or CypD-/- mouse embryonic fibroblasts (MEFs) were transfected with wild-type or CypD mutant cDNAs and analyzed for peptidyl prolyl cis,trans isomerase (PPIase) activity. PPIase-defective CypD H168Q mutant was used as a control. Mean ± SD. G and H) LN229 cells with stable CypD knockdown were transfected with the indicated CypD cDNAs, and isolated mitochondrial extracts were analyzed by western blotting (G) or HK-II activity (H). Mean ± SD of replicates from a representative experiment out of at least two independent determinations.**P = .001 by two-sided unpaired t test. I and J) LN229 cells with stable shRNA knockdown (KD) of CypD (I) or CypD-/- MEFs (J) were transfected with the indicated CypD cDNAs and analyzed for cell viability by a 3-(4,5 dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. Mean ± SD of replicates from a representative experiment out of at least two independent determinations. ***P < .001 by two-sided unpaired t test. CB = Coomassie blue staining; IgGL = Ig light chain; IP = immunoprecipitated; MW = molecular weight markers; PPIase = peptidyl prolyl cis,trans isomerase; Veh = vehicle; WT = wild-type.