Journal: JCI insight

Article Title: Inhibition of phosphodiesterase 4D suppresses mTORC1 signaling and pancreatic cancer growth.

doi: 10.1172/jci.insight.158098

Figure Lengend Snippet: Figure 2. PDE4D promotes mTORC1 activity through Raptor Ser791 phosphorylation. (A and B) PDE4D inhibits Raptor Ser791 phosphorylation. (A) HA-tagged Raptor was coexpressed with FLAG-tagged PDE4D4 in HEK293A cells for 48 hours; cells were then treated with forskolin (10 μM) for 1 hour. Lysates were immunoprecipitated (IP) with anti-HA antibody and immunoblotted with anti-pPKA substrate (RRXS/T) antibody. (B) Same as A but with FLAG-tagged PDE4D6. (C) PDE4D controls Raptor Ser791 phosphorylation. FLAG-tagged PDE4D (WT), FLAG-tagged PDE4D catalytically inactive mutant (D620A), and HA-tagged Raptor were overexpressed. Cells were treated with forskolin (10 μM) for 1 hour. Lysates were immunoprecipitated with anti-HA antibody and immunoblotted with anti-pPKA substrate antibody. (D) PDE4D depletion enhances Raptor Ser791 phosphorylation. Cells with PDE4D shRNA were stimulated with forskolin (10 μM) for 1 hour. Lysates were immunoprecipitated with anti-Raptor antibody and immunoblotted for pPKA substrate. Arrow indicates PDE4D. (E) Chemical inhibition of PDE4D decreases mTORC1 activity. HEK293A cells were stimulated with rolipram, roflumilast, and GEBR- 7b for 2 hours and mTORC1 activity was analyzed. (F and G) Raptor Ser791 phosphorylation controls mTORC1 activity. (F) HEK293A cells expressing FLAG- tagged Raptor (WT) or FLAG-tagged Raptor S791A (phospho-defective) were treated with or without GEBR-7b (20 μg/mL) for 2 hours. mTORC1 activity was analyzed. (G) HA-tagged Raptor (WT) or HA-tagged Raptor S791A (phospho-defective) was overexpressed in PDE4D-depleted cells (shPDE4D). mTORC1 activity was analyzed. (H) PDE4D controls mTORC1 activity. Cells were transfected with FLAG-tagged PDE4D4 for 48 hours and stimulated with roflumi- last (50 μM) for 2 hours. mTORC1 activity was analyzed. (I and J) Pharmacologic inhibition of PDE4D enhances Raptor Ser791 phosphorylation. HEK293A cells were pretreated with either roflumilast (50 μM) (I) or GEBR-7b (20 μg/m) (J) for 1 hour and then treated with forskolin (10 μM) for 1 hour. Lysates were immunoprecipitated with anti-Raptor antibody and Raptor Ser791 phosphorylation was assessed. Immunoblots probed for Raptor, pCREB, CREB, FLAG, HA, S6K1, and β-actin are controls. WCL, whole-cell lysates.

Article Snippet: Antibodies against the following proteins were used: β-actin (catalog 3700), HA tag (catalog 3724), pULK1 (catalog 6888), ULK1 (catalog 8054), pS6K1 (catalog 9234), S6K1 (catalog 9202), p4EBP1 (catalog 2855), 4EBP1 (catalog 9452), pCREB (catalog 9198), CREB (catalog 9197), mTOR (catalog 2972), mLST8 (catalog 3274), PKA Catα (catalog 4782), PKA RIα (catalog 5675), pPKA substrate (RRXS/T; catalog 9621) (all Cell Signaling Technology); Raptor (Cell Signaling Technology, catalog 2280 and Bethyl Laboratories, catalog A300-553A), AKAP13 (Thermo Fisher Scientific, catalog PA554078), PKA RIIα (Bethyl Laboratories, catalog A301-670A-M), FLAG tag (Sigma-Aldrich, catalog F1804-50UG), PDE4A (Abcam, catalog ab200383), PDE4C (Proteintech, catalog 21754-1-AP), PDE4B (Thermo Fisher Scientific, catalog 40-1400), and PDE4D (Sigma-Aldrich, catalog ABS22; Bethyl Laboratories, catalog A303-744A; Proteintech, catalog 12918-1-AP; and Thermo Fisher Scientific, catalog PA5-21590).

Techniques: Activity Assay, Phospho-proteomics, Immunoprecipitation, Mutagenesis, shRNA, Inhibition, Expressing, Transfection, Western Blot

Journal: medRxiv

Article Title: GP73 is a glucogenic hormone regulating SARS-CoV-2-induced hyperglycemia

doi: 10.1101/2021.04.30.21256060

Figure Lengend Snippet: (A) Glucose production in PMHs treated with rmGP73 at the indicated concentrations for 2 h. (B) Glucose production in PMHs treated with 32 nM rmGP73 with or without anti-GP73 antibody for 2 h. (C-E) Gluconeogenesis gene expression (C), intracellular cAMP accumulation (D), and hepatocyte PKA activity (E) in PMHs treated with 32 nM rmGP73 for 2 h. (F-G) Immunoblotting analysis of phosphorylated PKA C subunit (PKA-C-α-p) levels and substrate (phosphoRRXT*T*-PKA substrate, RRXpS/T) levels in HepG2 (F) or HK2 (G) cells treated with 32 nM rmGP73 or IBMX for the indicated times. (H) Immunoblotting analysis of phosphorylated CREB (CREB-p) in the livers of mice treated with a single dose of i.v. rmGP73, glucagon, or both for 15 min (n = 3 in each group). (I) Immunoblotting analysis of phosphorylated AKT (AKT-p) in PMHs, HepG2, or L6 cells treated with the indicated concentrations of insulin in the presence or absence of GP73. Cell-based studies were performed independently at least three times with comparable results. The data are presented as the means ± SEMs. **, P < 0.01; and ***, P < 0.001; one-way or two-way ANOVA followed by Bonferroni’s post hoc test for A-C and two-tailed Student’s t-tests for D-E.

Article Snippet: Anti-phospho-Akt (Thr308, 13038, 1:1000 dilution), anti-Akt (9272, 1:1000 dilution), anti-phospho-PKA-C-α (Thr197, 5661, 1:1000 dilution), anti-phospho-PKA substrate (RRXpS/T, 9624, 1:1000 dilution), and anti-PKA-C-α (5842, 1:1000 dilution) antibodies were purchased from Cell Signaling Technology (Danvers, USA).

Techniques: Expressing, Activity Assay, Western Blot, Two Tailed Test

Journal: medRxiv

Article Title: GP73 is a glucogenic hormone regulating SARS-CoV-2-induced hyperglycemia

doi: 10.1101/2021.04.30.21256060

Figure Lengend Snippet: (A) QRT-PCR analysis of glucogenic gene expression in Huh-7 cells infected with SARS-CoV-2 (MOI 0.1) for 24 h with or without GP73 antibody (1 µg/mL). (B) Glucose levels in Huh-7 cells infected with SARS-CoV-2 (MOI 0.1) for 24 h in glucose-free medium with or without GP73 antibody (1 µg/mL). (C) Immunoblotting analysis of PKA-C-α phosphorylation and substrate levels in Huh-7 cells infected with SARS-CoV-2 (MOI 0.1) for 24 h with or without anti-GP73 antibody. (D) Glucose levels in WT and GP73 KO-1 Huh-7 cells infected with SARS-CoV-2 (MOI 0.1) for 24 h in glucose-free medium. (E) QRT-PCR analysis of glucogenic gene expression in WT and GP73 KO-1 cells infected with SARS-CoV-2 (MOI 0.1) for 24 h. (F-G) PKA enzymatic activity (F) or glucogenic gene expression (G) in Huh-7 cells cultured with serum from healthy controls or COVID-19 patients in the presence or absence of anti-GP73 antibody. (H) Schematic representation of the involvement of upregulated GP73 in SARS-CoV-2-induced gluconeogenesis exaggeration. Cell-based studies were performed independently at least three times with comparable results. The data are presented as the means ± SEMs. *, P < 0.05; **, P < 0.01; and ***, P < 0.001; one-way ANOVA followed by Bonferroni’s post hoc test.

Article Snippet: Anti-phospho-Akt (Thr308, 13038, 1:1000 dilution), anti-Akt (9272, 1:1000 dilution), anti-phospho-PKA-C-α (Thr197, 5661, 1:1000 dilution), anti-phospho-PKA substrate (RRXpS/T, 9624, 1:1000 dilution), and anti-PKA-C-α (5842, 1:1000 dilution) antibodies were purchased from Cell Signaling Technology (Danvers, USA).

Techniques: Quantitative RT-PCR, Expressing, Infection, Western Blot, Activity Assay, Cell Culture

Journal: medRxiv

Article Title: GP73 is a glucogenic hormone regulating SARS-CoV-2-induced hyperglycemia

doi: 10.1101/2021.04.30.21256060

Figure Lengend Snippet: (A-B) Immunoblotting analysis of PKA-C-α phosphorylation and substrate levels in HepG2 cells cultured with serum from healthy subjects, diabetic subjects, hepatocellular carcinoma (HCC) patients, asthmatic subjects (A) or serum from healthy or diabetic subjects with or without anti-GP73 antibody (B). (C-F) Fasting glucose level (C; n=6 in each group), hepatic and renal GP73 mRNA expression (D; n = 3 in each group), hepatic glucogenic gene expression (E; n = 3 in each group), and renal glucogenic gene expression (F; n = 3 in each group) in BKS and db/db mice. (G-H) Fasting blood glucose levels in db/db mice (G) or HFD+STZ-induced T2D mice (H) at the indicated times after anti-GP73 treatment (n=6 in each group). (I) QRT-PCR analysis of glucogenic gene expression in the livers of WT and GP73 KO adult mice fasted for 6 h (n=3 in each group). The data are the means ± SEM. **, P < 0.01, and ***, P < 0.001; two-tailed Student’s t-tests for C and one-way ANOVA followed by Bonferroni’s post hoc test for D-I.

Article Snippet: Anti-phospho-Akt (Thr308, 13038, 1:1000 dilution), anti-Akt (9272, 1:1000 dilution), anti-phospho-PKA-C-α (Thr197, 5661, 1:1000 dilution), anti-phospho-PKA substrate (RRXpS/T, 9624, 1:1000 dilution), and anti-PKA-C-α (5842, 1:1000 dilution) antibodies were purchased from Cell Signaling Technology (Danvers, USA).

Techniques: Western Blot, Cell Culture, Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: Molecules and Cells

Article Title: PKA regulates autophagy through lipolysis during fasting

doi: 10.1016/j.mocell.2024.100149

Figure Lengend Snippet: Protein kinase A (PKA) is activated during short-term fasting (STF) and suppresses AMPK-dependent autophagy. (A) Ratio of AMP/ATP levels from metabolomic analysis (STF [4 hours], long-term fasting [LTF] [8 hours]). (B) Western blot of protein extracts from worms during fasting (STF [4 hours], LTF [8 hours]). (C) Western blot of protein extracts from kin-1 RNAi worms during fasting (STF, 4 hours). (D) Quantification of PKA activity (pPKA substrates normalized to the fed group) and AMPK activity (pAMPK normalized to AMPK in each group). (E) Western blot of protein extracts from kin-2 RNAi worms during fasting (LTF, 8 hours). (F) Quantification of PKA activity (pPKA substrates normalized to the fed group) and AMPK activity (pAMPK normalized to AMPK in each group). (G) kin-1 RNAi worms showed increased GFP::LGG-1 puncta during STF ( n = 7). (H and I) Double RNAi knockdown with aak-2 decreased GFP::LGG-1 puncta during fasting (STF [4 hours], LTF [8 hours]) ( n = 7). (J) Upregulation of PKA activity by kin-2 RNAi resulted in increased FFA levels during STF (STF [4 hours], LTF [8 hours]). (K) Western blots of isoproterenol-treated (1 μM) 3T3-L1 adipocytes, PKA activity decreased after 12 hours of isoproterenol treatment, which is the time point at which CARS-only signals decrease ( B). (L) LC3-II/LC3-I ratio increased when PKA activity and CARS-only signals decreased. Data represent the mean ± SD; * P < .05, *** P < .001 vs fed and # P < .05, ## P < .01. ns, not significant.

Article Snippet: Antibodies against the pPKA substrate (9624S, Cell Signaling Technology; 1:1,000), GAPDH (LF-PA0018, LabFrontier; 1:1,000), pAMPK (2531, Cell Signaling Technology; 1:1,000), AMPK (2532, Cell Signaling Technology; 1,000), LC3B (NB100-2220, Novus Biologicals; 1:1,000), P62 (H00008878-M01, Novus Biologicals; 1:1,000), GFP (sc-9996, Santa Cruz Biotechnology; 1:1,000), actin (ab14128, Abcam; 1:2,000), and β-actin (A5316, Sigma-Aldrich; 1:2,000) were used for western blotting analysis.

Techniques: Western Blot, Activity Assay, Knockdown

Journal: International Journal of Molecular Sciences

Article Title: Caput Ligation Renders Immature Mouse Sperm Motile and Capable to Undergo cAMP-Dependent Phosphorylation

doi: 10.3390/ijms221910241

Figure Lengend Snippet: Changes to post-translational modifications in sperm after epididymal ligation. Sperm samples were collected from each experimental region 7-days post-ligation surgery: NL Caput, Lig Caput, NL Cauda, and Lig Cauda. Samples were capacitated for 40 min prior to protein extraction and separation by SDS-PAGE. ( A ) Western blot of phosphorylated protein kinase A substrates (pPKA substrates). n = 5. ( B ) Membranes were stripped and re-probed with an antibody against tyrosine phosphorylation (pY). n = 3. ( C ) Membranes were re-stripped and re-probed with anti β-tubulin antibody to evaluate equal loading. ( D ) Quantification of optical densitometry ratio between pPKA substrates and β-tubulin. n = 5. ( E ) Quantification of optical densitometry ratio between pY and β-tubulin. n = 3. Statistical significance comparing NL Caput vs. Lig Caput, Lig Caput vs. NL Cauda, and NL Cauda vs. Lig Cauda using an unpaired t -test in all graphs indicated; ns p > 0.05, * p < 0.05, ** p < 0.01.

Article Snippet: Western blotting was performed using the following antibodies: anti-pPKA substrates monoclonal antibody (clone 100G7) diluted 1:10,000 (Cell Signaling, # 9624, Danvers, MA, USA); monoclonal anti-pY antibody (clone 4G10) diluted 1:10,000 (Millipore, cat # 05-321, Burlington, MA); O-GlcNAc monoclonal antibody (clone 110.6) diluted 1:2000 (Cell Signaling, cat # 9875, Danvers, MA, USA); OGT polyclonal antibody 1:1000 (Cell Signaling, cat # 5368, Danvers, MA, USA).

Techniques: Ligation, Protein Extraction, SDS Page, Western Blot, Phospho-proteomics