Journal: Nature communications

Article Title: α–SNAP inhibits AMPK signaling to reduce mitochondrial biogenesis and dephosphorylates Thr172 in AMPKα in vitro

doi: 10.1038/ncomms2565

Figure Lengend Snippet: ( a ) Coomassie blue staining of purified wild type (WT) α-SNAP, mutants M105I, L294A and 1-285 were purified, used in assays for phosphatase activity. ( b and c ) 32 P labeled-GST-AMPKα1-392 recombinant protein was used as substrate of α-SNAP phosphatase. α-SNAP (2, 5, 10 and 50 ng) was incubated with 32 P-GST-AMPKα-1-392 ( b ) 32 P-GST-AMPKα-1-392 recovered by trichloroacetic acid precipitation was resolved in SDS-PAGE and detected by phosphor imager and GST-AMPKα-1-392 and α-SNAP were examined by immunoblotting ( c ) 32 P phosphate release was measured by scintillation counting and specific activity of different amounts of α-SNAP calculated. ( d ) Phosphorylated FLAG-AMPKα2 was purified from activated HEK293T cells and incubated for 30 min with GST (as control, CON), or α-SNAP WT, M105I, L294A or 1-285 (0.5 μg each). Phosphorylated T172 of AMPKα2 was analyzed by immunoblotting. ( e ) Raptor and Akt were expressed in HEK293T cells, purified by FLAG or MYC immunoprecipitation and incubated with GST (as control, CON), or α-SNAP WT, M105I, L294A or 1-285 (0.5 μg each). Phosphorylation of Akt T308 and Raptor S792 were analyzed by immunoblotting.

Article Snippet: The antibodies were obtained from the following sources: AMPKα1/2, β1/2, pAMPKα T172, pACC S79, pRaptor S792, pS6K T389, cleaved PARP, NSF and COXIV from Cell Signaling Technology, FLAG, FLAG M2 bead, HA and β-actin from Sigma, α-SNAP from Exalpha Biologicals, pAkt T308 from Merck, CV-α and CIII-core2 from Mitosciences.

Techniques: Staining, Purification, Activity Assay, Labeling, Recombinant, Incubation, TCA Precipitation, SDS Page, Western Blot, Control, Immunoprecipitation, Phospho-proteomics

Journal: Frontiers in Cell and Developmental Biology

Article Title: Sustained AMPK Activation and Proline Metabolism Play Critical Roles in the Survival of Matrix-Deprived Transformed Cells

doi: 10.3389/fcell.2021.771366

Figure Lengend Snippet: +ST cells display increased survival and sustained AMPK activity under matrix-deprived conditions as compared to −ST cells: (A) −ST and +ST cells grown in a 96-well ultra-low attachment plate that prevents cell-attachment were subjected to MTT assay at specified time periods. Absorbance of formazan crystals dissolved in DMSO was monitored for 4 days; n = 4. Error bars represent mean ± SEM. (B) −ST and +ST cells subjected to conditions of attachment (Att) and matrix-deprivation (Det) for 24 h, were harvested for immunoblot analyses for the specified proteins. Graphs represent densitometric quantification of phospho-proteins to total proteins normalized to α -tubulin and relative to the attached condition for each cell line; error bars represent mean ± SEM; n = 3. (C) −ST and +ST cells subjected to matrix-deprivation (Det) for 2, 4 and 6 days were harvested for immunoblot analyses for the specified proteins. Adherent/attached cells (Att) were used as controls; n = 3.

Article Snippet: Primary antibodies used in the study are against pAMPK α (T172), pACC (S79), pRaptor (S792), AMPK, ACC, Raptor, AMPKα1, AMPKα2 (Cell Signaling Technology), and α Tubulin (Calbiochem).

Techniques: Activity Assay, Cell Attachment Assay, MTT Assay, Western Blot

Journal: Nature communications

Article Title: α–SNAP inhibits AMPK signaling to reduce mitochondrial biogenesis and dephosphorylates Thr172 in AMPKα in vitro

doi: 10.1038/ncomms2565

Figure Lengend Snippet: ( a ) Oxygen consumption rate (OCR, pmol/min) in HEK293T cells measured by a Seahorse Analyzer. Lentiviral shRNA infected cells are indicated and double knockdown was performed as Fig. 2d . Basal measurements were normalized for protein concentrations and the mean ±SEM from 4 independent experiments are plotted. Student's t test was used to calculate significance. ( b ) Seahorse continuous measurement of OCR. ( c ) HEK293T cells were infected with lentiviral shRNA targeting GFP (control, CON) or α-SNAP (SNAP sh1 and sh2). Mitochondrial proteins were detected by immunoblotting: CV-α, ATP synthase subunit alpha; CIII-core2, Complex III subunit Core 2; COXIV, cytochrome c oxidase subunit IV. ( d and e ) Citrate synthase activity as a measure of mitochondrial content was assayed and normalized relative to controls. Data represents mean ±SEM (n = 4) and p values from Student's t-test of significance are indicated. ( d ) Cells prepared as c . e, α-SNAP and LKB1 were knocked down as described in Fig. 2d .

Article Snippet: The antibodies were obtained from the following sources: AMPKα1/2, β1/2, pAMPKα T172, pACC S79, pRaptor S792, pS6K T389, cleaved PARP, NSF and COXIV from Cell Signaling Technology, FLAG, FLAG M2 bead, HA and β-actin from Sigma, α-SNAP from Exalpha Biologicals, pAkt T308 from Merck, CV-α and CIII-core2 from Mitosciences.

Techniques: shRNA, Infection, Knockdown, Control, Western Blot, Activity Assay

Journal: PLoS ONE

Article Title: Therapeutic Trial of Metformin and Bortezomib in a Mouse Model of Tuberous Sclerosis Complex (TSC)

doi: 10.1371/journal.pone.0031900

Figure Lengend Snippet: Tsc2 +− A/J strain mice were treated with vehicle, rapamycin, bortezomib or metformin for 1 week, and kidney lysates were prepared for immunoblot analysis. A) Blot strips were incubated with the antibodies against pACC-S79, AKT, pS6-S235/236, pS6-S240/244 and S6. B) Blot strips incubated with antibodies against pRaptor-S792, GRP78 and AKT.

Article Snippet: Antibodies used for Western blotting were as follows: pACC (S79, Millipore,Temecula, CA); Akt (Santa Cruz Biotechnology, Santa Cruz, CA); GRP78/BiP (Enzo, Farmingdale, NY; formerly Stressgen); and pIκBα (S32/36), pS6 (S240/244), pS6 (S235/236), total S6 and pRaptor (S792) (all from Cell Signaling Technology, Bedford, MA).

Techniques: Western Blot, Incubation

Journal: PLoS ONE

Article Title: Therapeutic Trial of Metformin and Bortezomib in a Mouse Model of Tuberous Sclerosis Complex (TSC)

doi: 10.1371/journal.pone.0031900

Figure Lengend Snippet: Tsc2 +/− A/J strain mice of ages 9–10 months were treated with drugs for 1 week, and kidneys prepared for histology. Sections were prepared and stained using pS6-S235/236 (red), pACC-S79 (red), pIκBα-S32/34 (red) and GRP78 (red) antibodies. The bottom panel shows apoptosis assessed by TUNEL method. Representative sections are shown. All images shown are taken at 100× magnification. Insets show portions of the tumor at higher magnification (400×).

Article Snippet: Antibodies used for Western blotting were as follows: pACC (S79, Millipore,Temecula, CA); Akt (Santa Cruz Biotechnology, Santa Cruz, CA); GRP78/BiP (Enzo, Farmingdale, NY; formerly Stressgen); and pIκBα (S32/36), pS6 (S240/244), pS6 (S235/236), total S6 and pRaptor (S792) (all from Cell Signaling Technology, Bedford, MA).

Techniques: Staining, TUNEL Assay

Journal: Nature communications

Article Title: α–SNAP inhibits AMPK signaling to reduce mitochondrial biogenesis and dephosphorylates Thr172 in AMPKα in vitro

doi: 10.1038/ncomms2565

Figure Lengend Snippet: ( a ) Coomassie blue staining of purified wild type (WT) α-SNAP, mutants M105I, L294A and 1-285 were purified, used in assays for phosphatase activity. ( b and c ) 32 P labeled-GST-AMPKα1-392 recombinant protein was used as substrate of α-SNAP phosphatase. α-SNAP (2, 5, 10 and 50 ng) was incubated with 32 P-GST-AMPKα-1-392 ( b ) 32 P-GST-AMPKα-1-392 recovered by trichloroacetic acid precipitation was resolved in SDS-PAGE and detected by phosphor imager and GST-AMPKα-1-392 and α-SNAP were examined by immunoblotting ( c ) 32 P phosphate release was measured by scintillation counting and specific activity of different amounts of α-SNAP calculated. ( d ) Phosphorylated FLAG-AMPKα2 was purified from activated HEK293T cells and incubated for 30 min with GST (as control, CON), or α-SNAP WT, M105I, L294A or 1-285 (0.5 μg each). Phosphorylated T172 of AMPKα2 was analyzed by immunoblotting. ( e ) Raptor and Akt were expressed in HEK293T cells, purified by FLAG or MYC immunoprecipitation and incubated with GST (as control, CON), or α-SNAP WT, M105I, L294A or 1-285 (0.5 μg each). Phosphorylation of Akt T308 and Raptor S792 were analyzed by immunoblotting.

Article Snippet: The antibodies were obtained from the following sources: AMPKα1/2, β1/2, pAMPKα T172, pACC S79, pRaptor S792, pS6K T389, cleaved PARP, NSF and COXIV from Cell Signaling Technology, FLAG, FLAG M2 bead, HA and β-actin from Sigma, α-SNAP from Exalpha Biologicals, pAkt T308 from Merck, CV-α and CIII-core2 from Mitosciences.

Techniques: Staining, Purification, Activity Assay, Labeling, Recombinant, Incubation, TCA Precipitation, SDS Page, Western Blot, Immunoprecipitation