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: Journal of Biological Chemistry

Article Title: Phosphorylation of Myosin Phosphatase Targeting Subunit 3 (MYPT3) and Regulation of Protein Phosphatase 1 by Protein Kinase A

doi: 10.1016/s0021-9258(19)84033-4

Figure Lengend Snippet: FIGURE1.In-gelkinaseassaydetectionofMYPT3kinase.A,overexpressedMYPT3canbephosphorylatedbyendogenouskinase(s).His6-taggedMYPT3was purified from COS7 cells, and 10 M [-32P]ATP was added and incubated at 30 °C for 30 min. The proteins were separated on SDS-PAGE, stained with GelCode Blue (Pierce), and dried for overnight autoradiography. C denotes the control lane (beads with untransfected cell lysate), and the MYPT3 band is indicated by arrowheads. A co-precipitated 37-kDa protein, marked by an asterisk, was identified as PP1c by mass spectrometry. B, in-gel kinase assays using GST or GST-MYPT3cross-linkedintothegelassubstrates.50 goflysates were loaded onto each lane. Arrowheadpoints to a 40-kDa band observed inthe GST-MYPT3 gel autoradiograph not present in the GST gel. C, effects of various kinase inhibitors. In-gel kinase assays of rat brain lysate or 0.5 units of bovine PKA were carriedoutinthepresenceof1mMkinaseinhibitororMe2SO(DMSO).D,phosphorylationofMYPT3inintactcells.InvitrophosphorylationofMYPT3wascarried out with 1 g of bacterially expressed GST-MYPT3 with and without GST-PKA and 1 mM ATP (left panel). For in vivo phosphorylation of MYPT3, HeLa cells were co-transfected with FLAG-MYPT3 and HA-PP1c constructs with or without Myc-PKA and treated as indicated. Proteins were separated on SDS-PAGE and blotted for phosphoprotein detection using a rabbit anti-phospho-PKA substrate antibody (Cell Signaling Technology); left panel shows IP products. Arrow- heads indicate positions of the full-length MYPT3 proteins; asterisks indicate endogenous proteins that are phosphorylated by PKA, and white asterisks indicate breakdown products of the bacterial GST fusion protein.

Article Snippet: All samples were separated on SDS-PAGE and transferred to polyvinylidene difluoride membranes for Western blotting with rabbit monoclonal anti-phospho-PKA substrate antibody (Cell Signaling Technology).

Techniques: Purification, Incubation, SDS Page, Staining, Autoradiography, Control, Mass Spectrometry, In Vivo, Phospho-proteomics, Transfection, Construct