c terminus gipc  (TaKaRa)

Journal: Oncology Reports

Article Title: Berberine exerts its antineoplastic effects by reversing the Warburg effect via downregulation of the Akt/mTOR/GLUT1 signaling pathway

doi: 10.3892/or.2021.8204

Figure Lengend Snippet: Ubc9 and GIPC mediate the post-translational modification and cytoplasmic retention of GLUT1, respectively. MCF7 cells were pretreated with the (A) proteasome inhibitor MG-132 or (B) lysosomal inhibitor, leupeptin, and the expression levels of GLUT1 were analyzed using western blotting. Flag-tagged GLUT1, His-tagged Ubc9 and His-tagged GIPC were constructed, and the interactions between (C) Ubc9 and GLUT1 and (D) GIPC and GLUT1 were analyzed using co-immunoprecipitation. Data are presented as the mean ± SD; n=3. **P<0.01 and ## P<0.01. Ubc9, ubiquitin conjugating enzyme E2 I; GIPC, Gα-interacting protein-interacting protein at the C-terminus; GLUT1, glucose transporter 1; Con, control; His, histidine; WCL, whole cell lysate.

Article Snippet: The open reading frames (ORFs) of GLUT1, ubiquitin conjugating enzyme E2 I (Ubc9) and Gα-interacting protein-interacting protein at the C-terminus (GIPC) were amplified with PrimerSTAR ® Max DNA Polymerase (cat. no. R045A; Takara Bio, Inc.) using cDNA as a template.

Techniques: Modification, Expressing, Western Blot, Construct, Immunoprecipitation

Journal: Oncology Reports

Article Title: Berberine exerts its antineoplastic effects by reversing the Warburg effect via downregulation of the Akt/mTOR/GLUT1 signaling pathway

doi: 10.3892/or.2021.8204

Figure Lengend Snippet: Proposed underlying mechanism of the antineoplastic effects of BBR, which involves the reversal of the Warburg effect via downregulating the Akt/mTOR/GLUT1 signaling pathway. Treatment with BBR downregulates the expression levels of p-Akt in cancer cells, which in turn downregulates the levels of its downstream signaling protein, p-mTOR. Subsequently, the binding between GIPC and GLUT1 is weakened, which results in the cytoplasmic retention of GLUT1. The weakened binding of GIPC with GLUT1 also strengthens the binding between Ubc9 and GLUT1, which leads to the post-translational degradation of GLUT1 and further diminishes the glucose transport function of GLUT1. Consequently, the glucose uptake capacity of cancer cells and ATP synthesis are decreased, therefore the Warburg effect of cancer cells is reversed, which contributes to the antineoplastic activity of BBR. BBR, berberine; Ubc9, ubiquitin conjugating enzyme E2 I; GIPC, Gα-interacting protein-interacting protein at the C-terminus; GLUT1, glucose transporter 1; p-, phosphorylated.

Article Snippet: The open reading frames (ORFs) of GLUT1, ubiquitin conjugating enzyme E2 I (Ubc9) and Gα-interacting protein-interacting protein at the C-terminus (GIPC) were amplified with PrimerSTAR ® Max DNA Polymerase (cat. no. R045A; Takara Bio, Inc.) using cDNA as a template.

Techniques: Expressing, Binding Assay, Activity Assay

Journal: Antioxidants

Article Title: Comparative Proteomics Unveils LRRFIP1 as a New Player in the DAPK1 Interactome of Neurons Exposed to Oxygen and Glucose Deprivation

doi: 10.3390/antiox9121202

Figure Lengend Snippet: Several DAPK1 interactors are CTD-NR2B binding partners. ( A ) WB and ( B ) quantification of the effect of OGD on the co-immunoprecipitation of DAPK1 with the well-known DAPK1 interactors NR2B, CAMKII and GIPC ( t test, n = 4–5 co-immunoprecipitations from independent primary neuron culture preparations). ( C ) WB showing the effect of MCAO on the NR2B band levels. ( D ) Effect of MCAO on 170 kDa, 115 kDa and total NR2B levels (paired t test, n = 9 rats, comparing ipsilateral (ipsi, ischemic) brain hemisphere with the contralateral (contra, control) one). The results are shown as the mean and SEM. * p < 0.05, *** p < 0.005 vs. respective control in all graphs.

Article Snippet: The following primary antibodies were used: goat anti-GAIP interacting protein at C terminus (GIPC) (1:250, sc-25658, RRID:AB_640993) and mouse anti-Ca 2+ /calmodulin-dependent protein kinase II (CAMKII) (1:200, sc-32288, RRID:AB_634551) were from Santa Cruz Biotechnology; mouse anti-phospho-DAPK1 at Ser308 (pDAPK1) (clone DKPS308, 1:50, D4941, RRID:AB_476906), rabbit anti-DAPK1 (1:400, D1319, RRID:AB_1078622), mouse anti-DAPK1 (clone DAPK-55, 1:400, D2178, RRID:AB_259206), rabbit anti-actin (1:500, A2066, RRID:AB_476693) and mouse anti-alpha-tubulin (1:5000, T6074, RRID:AB_477582) were from Sigma-Aldrich; anti-NMDAR 2B subunit (against the N-terminal extracellular region) (1:100, Millipore, MAB5782, RRID:AB_827428), rabbit anti-LRRFIP1 (1:400, Aviva Systems Biology, ARP38506_P050, RRID:AB_10570968); rabbit IgG isotype control (10500C, AB_2532981) and mouse IgG1 isotype control (MOPC-21) (MA1–10407, RRID:AB_2536775) were from Thermo Fisher Scientific; rabbit anti-glutathione peroxidase 4 (GPX4) (1:1000, ab125066, RRID:AB_10973901) was from Abcam.

Techniques: Binding Assay, Immunoprecipitation, Control