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: 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

Journal: Pharmaceutics

Article Title: Formulation of Liver-Specific PLGA-DY-635 Nanoparticles Loaded with the Protein Kinase C Inhibitor Bisindolylmaleimide I

doi: 10.3390/pharmaceutics12111110

Figure Lengend Snippet: Protein kinase C inhibition by hepatocyte-directed PLGA-DY-635 (BIM-I) NPs. ( A ) DY-635 HepG2 pretreated with DMSO, free or encapsulated or BIM-I (200 nmol L −1 ) for 30 min and stimulated with PMA for 15 min. pPKC substrates were detected by Western blotting from total protein lysates and loading was evaluated by Coomassie staining of the gel. ( B ) Representative images from intravital microscopy of PLGA-DY-635 (BIM-I) NPs in the liver. Hepatocytes are detected through their strong NADPH auto-fluorescence and liver sinusoids can be identified by their negative staining (black). Lower panel depicts a zoom area (green square) of the upper panel. Some DY-635 accumulation in Kupffer cells (asterisks) was observed. DY-635-stained bile canaliculi (arrowheads) indicated the uptake and degradation of the NPs as well as elimination of DY-635. Scale bar: 0.1 mm (upper panel) and 0.025 mm (lower panel).

Article Snippet: PVDF membranes were incubated in 5% bovine serum albumin (BSA) in TBS-Tween for 1 h, followed by 1 h of phospho-PKC (pPKC) substrate antibody (Cell Signaling Technologies, 6967S, RRID:AB_10949977 diluted 1:1000 in 5% BSA in TBS-Tween).

Techniques: Inhibition, Western Blot, Staining, Intravital Microscopy, Fluorescence, Negative Staining

Journal: Communications biology

Article Title: ZNF185 prevents stress fiber formation through the inhibition of RhoA in endothelial cells.

doi: 10.1038/s42003-023-04416-x

Figure Lengend Snippet: Fig. 1 Identification of ZNF185 as a PKA substrate in HUVECs. a Several PKA substrates are phosphorylated using forskolin in HUVECs. Forskolin (10 µM) was added to HUVECs for 1 h. pPKA substrates are indicated by arrows. Representative blots are shown (n = 3). b Representative silver-staining of the immunoprecipitated PKA substrates in HUVECs. Forskolin (10 µM) was added to the HUVECs for 1 h. pPKA substrates indicated by asterisks were immunoprecipitated by a pPKA substrate antibody (n = 3). The band indicated by a red asterisk was ZNF185 (Supplementary Fig. 1). c Overexpressed ZNF185 was phosphorylated by forskolin in HUVECs. Stable cell lines of HUVECs overexpressing Myc-ZNF185-HA were generated. One hour after the administration of forskolin (10 µM), anti-Myc beads were used for immunoprecipitation. Densitometric analysis of pZNF185. The black arrows indicate overexpressed ZNF185 and the arrowhead indicates endogenous ZNF185. The red arrows indicate phosphorylated ZNF185. *p < 0.05 (n = 3). d Overexpressed ZNF185 was phosphorylated by forskolin within 5 min. Time course of ZNF185 phosphorylation after the administration of forskolin (10 µM) is shown. Anti-Myc beads were used for immunoprecipitation. Densitometric analysis of pZNF185. The arrows indicate overexpressed ZNF185. **p < 0.01 (n = 3). e Overexpressed pZNF185 is localized to the membrane region. Immunofluorescence staining of pPKA (green) and Myc (magenta) in HUVECs overexpressing Myc-ZNF185-HA is shown. pZNF185 accumulates at the membrane region after the administration of forskolin (10 µM) for 1 h. Arrowheads indicate the cell boundaries. Arrows indicate pZNF185 localized to the membrane region. VE-cadherin was used to stain the cell membrane. Representative images are shown (n = 3). Scale bars, 10 µm. Data are presented as the mean ± standard error.

Article Snippet: Anti-pPKA substrate antibody (Cell Signaling Technology) was bound to protein G magnetic beads (Invitrogen Corporation) and the cell lysates were purified according to the manufacturer’s instructions.

Techniques: Silver Staining, Immunoprecipitation, Stable Transfection, Generated, Phospho-proteomics, Membrane, Staining