Journal: bioRxiv

Article Title: Loss of hepatic aldolase B activates Akt and promotes hepatocellular carcinogenesis by destabilizing Aldob/Akt/PP2A protein complex

doi: 10.1101/2020.06.09.141986

Figure Lengend Snippet: (A and B) IB analysis of anti-Flag IP and WCL derived from Huh7 cells transfected with the indicated constructs. (C) GST pull-down assays were performed with WCL derived from Flag-PP2AC transfected Huh7 cells and recombinant proteins GST-Akt1 and WT-Aldob or Aldob-R304A mutant. GST was used as the negative control. (D) In vitro PP2A dephosphorylation assays were performed with phosphorylated Myc-Akt1 and active PP2A in the presence or absence of recombinant WT-Aldob or Aldob-R304A mutant (top). The relative signal intensities of pT308-Akt and pS473-Akt were quantified (bottom). (E) IB analysis of WCL derived from Huh7 cells transfected with the indicated constructs. Resulting cells were treated 4 hours with okadaic acid (OA) at 100 nM. (F) IB analysis of endogenous anti-Aldob and anti-Akt1 IP and WCL derived from 2 pairs of human HCC samples. IgG was used as the negative control.

Article Snippet: Then immunoprecipitated phospho-Akt were reacted with recombinant active PP2A (BPS Bioscience, 30056) at 30 °C for indicated time periods in the presence or absence of recombinant WT-Aldob or Aldob-R304A mutant.

Techniques: Derivative Assay, Transfection, Construct, Recombinant, Mutagenesis, Negative Control, In Vitro, De-Phosphorylation Assay

Journal: bioRxiv

Article Title: Loss of hepatic aldolase B activates Akt and promotes hepatocellular carcinogenesis by destabilizing Aldob/Akt/PP2A protein complex

doi: 10.1101/2020.06.09.141986

Figure Lengend Snippet: In normal cells, Aldob interacts with Akt in an Akt kinase activity-dependent manner, which promotes phosphatase PP2A binding to p-Akt and results in p-Akt dephosphorylation. Aldob is involved in the intracellular homeostasis of Akt phosphorylation through modulating substrate/phosphatase interactions. In Aldob-deficient cancer cells, downregulation of Aldob releases its negative regulation of Akt activity, leading to constitutive activation of oncogenic Akt signaling which facilitates cell cycle progression and increases metabolic flux to glycolysis and TCA cycle for cancer cell proliferation and tumorigenesis. Targeting hyperactive Akt through directly inhibiting Akt activity or reactivating PP2A phosphatase activity may be a viable therapeutic approach to treat Aldob-deficient human cancers. Bold arrows represent up-regulation of indicated signaling events in response to the absence of Aldob.

Article Snippet: Then immunoprecipitated phospho-Akt were reacted with recombinant active PP2A (BPS Bioscience, 30056) at 30 °C for indicated time periods in the presence or absence of recombinant WT-Aldob or Aldob-R304A mutant.

Techniques: Activity Assay, Binding Assay, De-Phosphorylation Assay, Activation Assay

Journal:

Article Title: Long Term Synaptic Depression That Is Associated with GluR1 Dephosphorylation but Not ?-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Internalization *

doi: 10.1074/jbc.M803431200

Figure Lengend Snippet: Neither GluR1 nor GluR2/3 total surface expression is changed 45 min following cLTD treatment, as determined by cross-linking assay. A, representative blots of X-linked (25.0 μg of total protein loaded) and un-X-linked (5.0 μg of total protein loaded) samples for control and cLTD conditions probed with antibodies specific for GluR1 (left group) and GluR2/3 (right group). B, representative blots of un-X-linked and X-linked (5.0μg of total protein loaded for both) samples probed with antibodies specific for PP2Aα (top bands) and synapsin (bottom bands). C and D, graphs of cumulative data comparing X-linked control and X-linked cLTD samples probed with antibodies specific for GluR1 (C) and GluR2/3 (D)(n = 8). E and F, data from C and D normalized to the transferrin receptor. G and H, graphs of cumulative data comparing the percent surface expression (see “Experimental Procedures” for explanation of calculation) between control and cLTD conditions for GluR1 (G) and GluR2/3 (H)(n = 8). I and J, graphs of cumulative data comparing un-X-linked and X-linked samples probed with antibodies specific for PP2Aα (I) and synapsin (J)(n = 8). No significant differences were observed for any of the comparisons as determined by Student's paired t test. Values represent the mean ± S.E.

Article Snippet: Primary antibodies were as follows: GluR1 and GluR2/3 (Chemicon) used at 1:3000 in BSA, phospho-Ser-845 and phospho-Ser-831 (PhosphoSolutions) used at 1:1000 in BSA, PP2Aα (BD Pharmingen) used at 1:5000 in milk, synapsin, NR2B, and NR1 (PhosphoSolutions) used at 1:2000 in BSA, the α subunit of calcium/calmodulin-dependent protein kinase II (αCaMKII) (BD Pharmingen) used at 1:3000 in BSA, phospho-Thr-286 (PhosphoSolutions) used at 1:3000 in milk, PSD-95 (Affinity Bioreagents) used at 1:5000 in BSA, and transferrin receptor (Zymed Laboratories Inc.) used at 1:1000 in milk.

Techniques: Expressing