Journal: Translational Psychiatry

Article Title: Paternal methamphetamine exposure induces higher sensitivity to methamphetamine in male offspring through driving ADRB1 on CaMKII-positive neurons in mPFC

doi: 10.1038/s41398-023-02624-x

Figure Lengend Snippet: a Levels of c-Fos protein. b The c-Fos immunostaining. Scale bar, 500 μm /100 μm. c Density of dendritic spine. Scale bar, 50 μm /10 μm. d Levels of β-arestin2 and PKA protein. e Levels of ERK1/2, p-ERK1/2, ERK1/2/p-ERK1/2, CREB, p-CREB, p-CREB/ CREB and ΔFosB protein. F1-METH-Ctrl, METH-sired male F1 mice injected with Ctrl virus. F1-METH-KD, METH-sired male F1 mice injected with KD virus. The data are presented as the Mean ± SD. N.S., P > 0.05. * P < 0.05, ** P < 0.01 vs F1-METH-Ctrl.

Article Snippet: The transferred membranes were blocked with 5% non-fat dry milk and 0.1% Tween 20 in TBST buffer for 2 h at room temperature, then subsequently incubated with the following primary antibodies: Rabbit anti-c-Fos (1:1000, RRID:AB_224721, Cell Signaling Technology, USA), Rabbit anti-GAPDH (1:10000, RRID:AB_2651132, Bioworld Technology, USA), Rabbit anti-ADRB1 (1:1000, RRID:AB_10885544, Bioss, China), Rabbit anti-ARRB2 (1:1000, BA3767, Boster, China), Rabbit anti-ERK1/2 (1:1000, BM4326, Boster, China), Rabbit anti-phospho-ERK1/2 (1:1000, BM4156, Boster, China), Rabbit anti-ΔFosB (1:1000, bsm-52071R, Bioss, China), Rabbit anti-PKA (1:1000, bs-0520R, Bioss, China), Rabbit anti-CREB (1:1000, RRID:AB_2800317, Cell Signaling Technology, USA), Rabbit anti-phospho-CREB (1:1000, RRID:AB_2561044, Cell Signaling Technology, USA).

Techniques: Immunostaining, Injection, Virus

Journal: The Journal of Biological Chemistry

Article Title: The Prostaglandin E 2 Receptor, EP2, Stimulates Keratinocyte Proliferation in Mouse Skin by G Protein-dependent and ?-Arrestin1-dependent Signaling Pathways *

doi: 10.1074/jbc.M110.117689

Figure Lengend Snippet: Butaprost induced β-arrestin1-Src complex formation. A, butaprost increased β-arrestin1-Src complex formation. Mice were sacrificed at 2 h after 50 or 100 nmol of butaprost treatment. Skin lysates (200 μg) were immunoprecipitated (IP; non-IP, not immunoprecipitated) with a monoclonal anti-Src antibody, and β-arrestin1 and Src were detected by Western blotting. B, butaprost increased the formation of a β-arrestin1-Src complex. Skin lysates (200 μg) were immunoprecipitated with a polyclonal anti-β-arrestin1 antibody and subjected to Western blot analysis using a monoclonal Src antibody. In A and B, nonspecific IgG light chain served as a control for protein loading and membrane transfer. The number above each lane shows the relative intensities of the bands to IgG light chain. C, butaprost induced phosphorylation of Src and EGFR and increased the β-arrestin1-Src complex in WT but not in β-arrestin1−/− mice. WT and β-arrestin1−/− mice were treated for the indicated times with 100 nmol of butaprost. Src and EGFR were immunoprecipitated from protein lysates (200 μg) and subjected to Western blot (WB) analysis using an anti-p-Tyr or an anti-β-arrestin1 antibody. D, butaprost induced phosphorylation of ERK1/2, Akt, and STAT3 in WT but not in β-arrestin1−/− mice. WT and β-arrestin1−/− mice were treated for the indicated times with 100 nmol of butaprost. Skin lysates were immunoblotted with the indicated antibodies. In C and D, the number above each lane shows the mean-fold intensity as determined by densitometry. In A–D, the data presented are representative of two independent experiments. E, β-arrestin1 deficiency suppressed butaprost-induced keratinocyte proliferation. WT and β-arrestin1−/− mice were pretreated topically with acetone or 100 nmol of butaprost and sacrificed 24 h following butaprost treatment. *, p < 0.05 versus mice treated with acetone alone. **, p < 0.05 versus butaprost-treated WT mice. BrdU-stained cells were counted in five skin sections from each of five mice. Data are expressed as the mean ± S.D. (n = 5) of the number of stained cells per 100 basal cells in each group.

Article Snippet: The membranes were incubated overnight at 4 °C with 1:500–1,000 dilution of the following antibodies: p-Akt (Ser 473 ), Akt, p-ERK1/2 (Thr 202 /Tyr 204 ), ERK1/2, p-STAT3 (Tyr 705 ), STAT3, p-GSK3β (Ser 9 ), GSK3β, p-CREB (Ser 133 ), and CREB and p-(Ser/Thr) PKA substrate polyclonal antibodies (Cell Signaling Technology); EGFR and Src (Santa Cruz Biotechnology); β-arrestin1 (Abcam, Cambridge, MA); and phosphotyrosine (Upstate Biotech Millipore, Lake Placid, NY).

Techniques: Immunoprecipitation, Western Blot, Staining

Journal: bioRxiv

Article Title: Abnormal Lipid Metabolism and Altered Neuronal Support by Astrocytes Lacking Akap11 , a Risk Gene for Schizophrenia and Bipolar Disorder

doi: 10.1101/2025.04.25.650548

Figure Lengend Snippet: (A) RNA levels of protein kinase A subunits remain unchanged in Akap11 +/- and Akap11 -/- astrocytes compared to Akap11 +/+ controls. (B) Immunoblot analysis of lysates from embryonic cortical neurons and cortical brain tissues of Akap11 +/+ , Akap11 +/- , and Akap11 -/- mice using the indicated antibodies. (C) Quantification of the results from (B) with protein levels normalized to β-Actin. Data are presented as mean ± SEM (n ≥ 2 mice/genotype). Statistical significance was determined by one-way ANOVA. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant. (D) Lysates from control (si-Ctrl) and Akap11 knock down (si-Akap11) cultured rat hippocampal neurons were analyzed by immunoblotting with the indicated antibodies. (E) Lysates from WT and AKAP11 knockout (KO) HEK293T cells were analyzed by immunoblotting with the indicated antibodies. (F) Akap11 +/+ , Akap11 +/- , and Akap11 -/- astrocytes were subjected to nutrient starvation in EBSS media for the indicated time points, followed by immunoblotting with the indicated antibodies. (G) Quantification of the results from ( F ), with protein levels normalized to β-Actin and then to the time 0 value. Data are from three independent experiments. (H) Akap11 +/+ , Akap11 +/- , and Akap11 -/- astrocytes were subjected to nutrient starvation in the presence of Baf A1, followed by immunostaining with anti-PKA R1-α, anti-P62, and anti-GFAP antibodies. Scale bar, 10 μm.

Article Snippet: Commercial antibodies included: anti-PKA Cα (Cell Signaling Technology, #4782), anti-PKA R1α (Cell Signaling Technology, #5675), anti-VAPA (Proteintech, 15275-1-AP), anti-VAPB (Proteintech, 14477-1-AP), anti-ABCD3/PMP70 (Invitrogen, PA1-650), anti-ATAD3A/3B (Proteintech, 16610-1-AP), anti-PLIN2 (Proteintech, 15294-1-AP), anti-CANX/Calnexin (Abcam, ab22595), anti-LAMP2 (DSHB, H4B4), anti-GFP (Abcam, ab13970), anti-β-actin (Abcam, ab8224), and anti-GAPDH (Cell Signaling Technology, #3683).

Techniques: Western Blot, Control, Knockdown, Cell Culture, Knock-Out, Immunostaining

Journal: bioRxiv

Article Title: Specific degrader for fusion oncokinase kills tumors and is augmented by bimodal degrader-siRNA

doi: 10.1101/2025.04.24.650501

Figure Lengend Snippet: A) Levels of PRKACA and DNAJB1::PRKACA were monitored in Huh7.5 cells after induction of PKI 1-52 SPOP (24h) in the presence or absence of MG132. B) PRKACA and DNAJB1::PRKACA were monitored in Huh7.5 cells after induction of PKI 1-52 SPOP(24h). Cells were incubated with bortezomib (BZ) for 18 h or 12 h before harvesting. C, D, E) Dependence on E1 ubiquitination. To test the dependence of degradation on E1 activity, PKI 1-52 SPOP was induced in Huh7.5 cells for either 24 or 48h. Then the E1 inhibitor, Tak-243, was added for a duration of either 8hr or 16 hr prior to harvesting, or simultaneously with harvesting. C) Level of PRKACA and DNAJB1::PRKACA were monitored by Western blot. D) Quantification of the levels of DNAJB1::PRKACA, normalized to GAPDH from panel C. E) The level of Nano-luc-DNAJB1::PRKACA was monitored using luminescence in the absence and presence of Tak-243, at different time points subsequent to Dox induction and normalized to the no Dox counterpart.

Article Snippet: Blots were probed with the primary antibodies against PRKACA (Cell Signaling Technology Cat# 5842, RRID:AB_10706172, 1:3000), GAPDH (GeneTex Cat# GTX627408, RRID:AB_11174761, 1:5000) or Ubiquitin (Sigma-Aldrich Cat# 05-944, RRID: AB_3668631, 1:2000), for overnight at 4°C in blocking buffer, followed by the secondary antibodies anti-mouse (Sigma-Aldrich Cat# A9917, RRID:AB_258476, 1:10,000) IgG and goat anti-rabbit IgG (Sigma-Aldrich Cat# A0545, RRID:AB_257896, 1:10,000-1:25,000) for 1 hour at room temperature.

Techniques: Incubation, Activity Assay, Western Blot

Journal: bioRxiv

Article Title: Specific degrader for fusion oncokinase kills tumors and is augmented by bimodal degrader-siRNA

doi: 10.1101/2025.04.24.650501

Figure Lengend Snippet: PKI 1-52 SPOP similarly inhibits PRKACA and DNAJB1::PRKACA. Kinase activity was quantified at different concentrations of PKI 1-52 SPOP (n=3). B, C) The effect of different PKI-E3 ligases on abundance of PRKACA and DNAJB1::PRKACA in Huh7.5 cells. B) Western blot assay of the effects of expression of PKI-CHIP, PKI-SPOP, PKI-VHL, βTrCP-PKI, FBW7-PKI and SKP2-PKI. Each was analyzed in the absence (-) and presence (+) of 1μg/ml doxycycline (Dox), which induces the expression of the PKI-E3. (Quantification of DNAJB1::PRKACA and PRKACA bands in B , normalized by GAPDH are in Supplement 2). C) Fraction of DNAJB1::PRKACA or PRKACA remaining after adding dox. D, E) Switching order of PKI and E3 . Huh7.5 cells were transduced with PKI 1-25 SPOP or SPOP-PKI 1-25 . D) The levels of DNAJB1::PRKACA and PRKACA were assayed by Western blot with an antibody that recognizes the shared carboxyl terminus. E) The ratio of each protein +/- dox plotted for each degrader. F, G, H, I) Degradation of DNAJB1::PRKACA requires lysine in the amino terminus. F) Western blot analysis showing degradation of DNAJB1::PRKACA by increasing concentration of Dox and F) abolition of degradation when all 7 lysines of its DNAJB1 domain are mutated to arginines (DNAJB1(KR)::PRKACA). H) Quantification of DNAJB1::PRKACA and I) DNAJB1(KR)::PRKACA (right), normalized by the level of GAPDH from western blots in A. J) PKI 1-52 -SPOP degrades Nluc-ATP1B::PRKACA. Increasing induction of degrader by Dox increased the selective degradation of Nluc-ATP1B::PRKACA with no effect on PRKACA as assessed by Western blot K, L) Degradation upon direct targeting to proteasome . PKI was expressed as a fusion to a peptide that binds to the PSMD2 subunit of the proteasome. Upon induction by Dox, there is a selective degradation of DNAJB1::PRKACA relative to PRKACA as seen by K) Western Blot and L) Quantification: the ratio of each protein +/- dox was plotted for each degrader. Quantification is from three independent experiments. M) Dynamics of exon 1 of the fusion kinases and PRKACA. Molecular dynamic simulations of DNAJB1::PRKACA (left) PRKACA (top), and ATP1B1::PRKACA (right) were used to calculate the radius of gyration of the amino terminus of each kinase (center bottom). Lysines in the amino terminal domains (blue) are marked by a red sphere. A space filling model of the MD simulations is in Supplement S3. The error bars are the standard deviation of three independent 1µs simulations for each kinase.

Article Snippet: Blots were probed with the primary antibodies against PRKACA (Cell Signaling Technology Cat# 5842, RRID:AB_10706172, 1:3000), GAPDH (GeneTex Cat# GTX627408, RRID:AB_11174761, 1:5000) or Ubiquitin (Sigma-Aldrich Cat# 05-944, RRID: AB_3668631, 1:2000), for overnight at 4°C in blocking buffer, followed by the secondary antibodies anti-mouse (Sigma-Aldrich Cat# A9917, RRID:AB_258476, 1:10,000) IgG and goat anti-rabbit IgG (Sigma-Aldrich Cat# A0545, RRID:AB_257896, 1:10,000-1:25,000) for 1 hour at room temperature.

Techniques: Activity Assay, Western Blot, Expressing, Transduction, Concentration Assay, Standard Deviation

Journal: bioRxiv

Article Title: Specific degrader for fusion oncokinase kills tumors and is augmented by bimodal degrader-siRNA

doi: 10.1101/2025.04.24.650501

Figure Lengend Snippet: Quantification of DNAJB1::PRKACA and PRKACA bands from , normalized by GAPDH

Article Snippet: Blots were probed with the primary antibodies against PRKACA (Cell Signaling Technology Cat# 5842, RRID:AB_10706172, 1:3000), GAPDH (GeneTex Cat# GTX627408, RRID:AB_11174761, 1:5000) or Ubiquitin (Sigma-Aldrich Cat# 05-944, RRID: AB_3668631, 1:2000), for overnight at 4°C in blocking buffer, followed by the secondary antibodies anti-mouse (Sigma-Aldrich Cat# A9917, RRID:AB_258476, 1:10,000) IgG and goat anti-rabbit IgG (Sigma-Aldrich Cat# A0545, RRID:AB_257896, 1:10,000-1:25,000) for 1 hour at room temperature.

Techniques:

Journal: bioRxiv

Article Title: Specific degrader for fusion oncokinase kills tumors and is augmented by bimodal degrader-siRNA

doi: 10.1101/2025.04.24.650501

Figure Lengend Snippet: A) Western Blot analysis of FLC cells expressing PKI 1-25 SPOP or PKI 1-25 SPOP mut . B)Effect of PKI-SPOP on immunofluorescence of PRKACA and DNAJB1::PRKACA in FLC cells . A subset of FLC-1 cells were transiently transfected with PKI-SPOP. An antibody against the carboxyl-terminus shows PRKACA and DNAJB1::PRKACA (red) and FLAG signal (from Flag-PKI-SPOP in yellow) and DAPI (cyan). C) Effect of PKI-SPOP on growth of FLC tumors as PDX in mice . Tumor volume over time for PDX of FLC-2 PDX transduced with inducible PKI 1-25 SPOP, and tet regulator. D) PKI-SPOP reduces FLC tumor size. Extracted PDX from mice in C. E) Expression of PKI-SPOP has no adverse effects on weight of the mice. F) Efficacy of AAV in FLC cells. Titration of efficacy of transduction by AAV strains AAV2, AAV3ST and AAV MP5 to FLC cells monitored by GFP fluorescence. Delivery of AAV encoding PKI 1-25 SPOP or βPKI-SPOP (G, H, I, J, K). G) AAV with PKI 1-25 SPOP reduces tumor growth. Effect of AAV delivery on growth of PDX of FLC-3, assessed by volume. H) AAV delivery does not affect weight of mice. I-K) AAV delivery on mouse physiology. ALT, AST and TBIL levels in the serum at the end of the study. Dotted line marks the upper limit of normal values in NSG mice.

Article Snippet: Blots were probed with the primary antibodies against PRKACA (Cell Signaling Technology Cat# 5842, RRID:AB_10706172, 1:3000), GAPDH (GeneTex Cat# GTX627408, RRID:AB_11174761, 1:5000) or Ubiquitin (Sigma-Aldrich Cat# 05-944, RRID: AB_3668631, 1:2000), for overnight at 4°C in blocking buffer, followed by the secondary antibodies anti-mouse (Sigma-Aldrich Cat# A9917, RRID:AB_258476, 1:10,000) IgG and goat anti-rabbit IgG (Sigma-Aldrich Cat# A0545, RRID:AB_257896, 1:10,000-1:25,000) for 1 hour at room temperature.

Techniques: Western Blot, Expressing, Immunofluorescence, Transfection, Transduction, Titration, Fluorescence

Journal: Journal of Virology

Article Title: Small Hepatitis B Virus Surface Antigen Promotes Hepatic Gluconeogenesis via Enhancing Glucagon/cAMP/Protein Kinase A/CREB Signaling

doi: 10.1128/jvi.01020-22

Figure Lengend Snippet: SHBs elevates cAMP levels by regulating AC. (A) cAMP levels in the liver of LSL-SHBs and LSL-SHBs/Alb-Cre mice fasted for 16 h ( n = 6 to 7 mice/group). (B to E) Effect of AC inhibitor SQ22536 or PDE inhibitor IBMX on cAMP levels (B), glucose production (C), gluconeogenic gene expression (D), CREB phosphorylation, and PKA activity (E) in Ad-SHBs-infected mouse primary hepatocytes and in Huh7-SHBs cells. Cells were treated with SQ22536 (500 μM) or IBMX (500 μM) for 1 h and then with glucagon (100 nM) stimulation or FSK (10 μM) for 15 min (B), 6 h (C), 2 h (D) and 30 min (E). (F) Huh7 cells were transfected with pcDNA 3.1-SHBs-Flag for 48 h, and then co-IP assays were performed to determine the interaction between SHBs and PKA subunits. The immunoprecipitated complexes with anti-Flag antibody were subjected to immunoblotting with PKA Cα, PKA RIα/β, PKA RIIα, or PKA RIIβ antibody. The glycosylated (gp) and nonglycosylated (p) forms of SHBs were indicated. Arrowhead points to phospho-CREB. Data are presented as means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, no significant difference.

Article Snippet: The primary antibodies used in this studies included anti-Flag (F1804; Sigma-Aldrich), anti-SHBs (DMABT-51328MH; Creative-Diagnostics, Shirley, NY), anti-CREB (9197S; Cell Signaling Technology [CST], Beverly, MA), anti-phospho-CREB (9198S; CST), anti-phospho-PKA substrate (9624S; CST), anti-PKA Cα (4782S; CST), anti-PKA RIα/β (3927; CST), anti-β-Actin (3700; CST), anti-PKA RIIα (MAB8000; R&D Systems, Minneapolis, MN), anti-PKA RIIβ (PAB18374; Abnova Inc., Walnut, CA), and anti-AC1 (LS-C314759; LifeSpan Biosciences, Seattle, WA).

Techniques: Gene Expression, Phospho-proteomics, Activity Assay, Infection, Transfection, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot