phospho darpp 32 trh 75  (Cell Signaling Technology Inc)

Journal: Current Research in Neurobiology

Article Title: Δ9-Tetrahydrocannabinol does not upregulate an aversive dopamine receptor mechanism in adolescent brain unlike in adults

doi: 10.1016/j.crneur.2023.100107

Figure Lengend Snippet: Downstream effectors of D1-D2 dopamine receptor heteromer after chronic THC administration. Homogenates from dorsal striatum (dStr) and nucleus accumbens (NAc) of adult and adolescent rats chronically treated with vehicle (Veh) or THC were subjected to Western blot using antibodies for A) CaMKII⍺ and phospho-CaMKII⍺ (pCamKII⍺), B) GSK3⍺β and phospho-GSK3⍺β (pGSK3⍺β), C) BDNF and its receptor TrkB, and phosphorylated TrkB (pTrkB), D) phosphorylated Thr75-DARPP-32 (pThr75) E) and prodynorphin (Pdyn) and its receptor KOR and phosphorylated KOR (pKOR). F) GluA1 phosphorylated at serine845 (pS845-GluA1). GAPDH and β-actin antibodies were used as loading controls. Representative images are shown. Results are expressed as percent of control, mean ± SEM, with statistical significance measured by Student's t-test (*p < 0.05, **p < 0.01).

Article Snippet: Primary antibodies and dilutions used were: phospho-CaMKIIα (Cell Signaling Technologies, 12,726; 1:1000), CaMKIIα (Cell Signaling Technologies, 3362; 1:1000), phospho-GSK3αβ (Cell Signaling Technologies, 9331; 1:1000), GSK3αβ (Cell Signaling Technologies, 5676; 1:1000), phospho-TrkB (Sigma, ABN1381; 1:500), TrkB (Cell Signaling Technologies, 4607; 1:1000), phospho-Thr75-DARPP-32 (Cell Signaling Technologies, 2301 S; 1:1000), prodynorphin (Neuromics, GP10110; 1:700), kappa opioid receptor (Abcam, ab183825; 1:1000), kappa opioid receptor phospho-Ser369 (SAB Bio; 12,227), phospho-Ser845-GluA1 (Millipore, 1:1000), AB5849; phospho-CB1R (Abcam, ab186428; 1:1000), GAPDH (Sigma, G8795; 1:10,000) and β-actin (Abgent, AM1021B; 1:1000).

Techniques: Western Blot, Control

Journal: Current Research in Neurobiology

Article Title: Δ9-Tetrahydrocannabinol does not upregulate an aversive dopamine receptor mechanism in adolescent brain unlike in adults

doi: 10.1016/j.crneur.2023.100107

Figure Lengend Snippet: Proposed mechanism for D1-D2 heteromer-induced negative emotionality in adults, absent in adolescents Chronic THC in adult but not adolescent rat and monkeys upregulates the dopamine D1-D2 heteromer in nucleus accumbens medium spiny neurons (MSNs) with increased expression of downstream markers phospho-CaMKII, Thr75-DARPP-32 and dynorphin leading to anxiety- and depression-like aversive behaviour. In adolescents, lack of D1-D2 heteromer upregulation conceivably increases the aversion threshold and rewarding effects of THC predominate such that there is little anxiety- and depression-like behaviour and potentially greater propensity for drug abuse.

Article Snippet: Primary antibodies and dilutions used were: phospho-CaMKIIα (Cell Signaling Technologies, 12,726; 1:1000), CaMKIIα (Cell Signaling Technologies, 3362; 1:1000), phospho-GSK3αβ (Cell Signaling Technologies, 9331; 1:1000), GSK3αβ (Cell Signaling Technologies, 5676; 1:1000), phospho-TrkB (Sigma, ABN1381; 1:500), TrkB (Cell Signaling Technologies, 4607; 1:1000), phospho-Thr75-DARPP-32 (Cell Signaling Technologies, 2301 S; 1:1000), prodynorphin (Neuromics, GP10110; 1:700), kappa opioid receptor (Abcam, ab183825; 1:1000), kappa opioid receptor phospho-Ser369 (SAB Bio; 12,227), phospho-Ser845-GluA1 (Millipore, 1:1000), AB5849; phospho-CB1R (Abcam, ab186428; 1:1000), GAPDH (Sigma, G8795; 1:10,000) and β-actin (Abgent, AM1021B; 1:1000).

Techniques: Expressing

Journal: NPJ Precision Oncology

Article Title: IKKα promotes lung adenocarcinoma growth through ERK signaling activation via DARPP-32-mediated inhibition of PP1 activity

doi: 10.1038/s41698-023-00370-3

Figure Lengend Snippet: a Human A549 cell lines stably overexpressing FLAG-tagged human DARPP-32 isoforms (DARPP-32 and t-DARPP) were lysed and subjected to immunoprecipitation using anti-FLAG antibody–conjugated agarose beads. Immunoprecipitated lysates were used to perform nonradioactive in vitro kinase assays following incubation with commercially available active IKKα protein. At the end, the reaction mixtures were subjected to immunoblotting using antibodies against DARPP-32 phosphorylated on Thr-34 or Thr-75 and total DARPP-32 protein. b , c Human HCC827, PC9, and H1975 lung adenocarcinoma cell lines retrovirally transduced with either FLAG-tagged human DARPP-32 ( b ) or t-DARPP ( c ) cDNA plasmids were lysed, immunoprecipitated, incubated with active IKKα protein, and subjected to western blotting using anti-phospho (Thr-34 or Thr-75) DARPP-32 and anti-DARPP-32 antibodies. Data from one experimental replicate are shown. The experiments were repeated three times independently; each circle in a bar represents one experiment. Error bars indicate SEM. * P < 0.05; ns not significant.

Article Snippet: Primary antibodies (1 μg/μl) identifying two different phosphorylated sites on DARPP-32 (T34: cat no. 12438; dilution 1:1000; and T75: cat no. 2301; dilution 1:1000), phosphorylated PP1α (T320; cat no. 2581; dilution 1:1000), IKKα (cat no. 2682; dilution 1:1000), phosphorylated p44/42 MAPK (T202/Y204; cat no. 4370; dilution 1:1000), and total p44/42 MAPK (cat no. 4695; dilution 1:1000) were purchased from CST.

Techniques: Stable Transfection, Immunoprecipitation, In Vitro, Incubation, Western Blot, Transduction

Journal: NPJ Precision Oncology

Article Title: IKKα promotes lung adenocarcinoma growth through ERK signaling activation via DARPP-32-mediated inhibition of PP1 activity

doi: 10.1038/s41698-023-00370-3

Figure Lengend Snippet: a , b Human lung cancer cells, HCC827 ( a ) and H1650 ( b ), transfected with GFP (control), constitutively active IKKα, or kinase-dead IKKα were lysed using 1× RIPA buffer supplemented with protease and phosphatase inhibitors. Equal amounts of proteins were separated with 4–20% SDS-PAGE and transferred to polyvinyl difluoride membranes. Antigen-coated membranes were incubated overnight with primary antibodies against IKKα, phosphorylated DARPP-32 (Thr-34), total DARPP-32, phosphorylated PP1α (Thr320), total PP1α, phosphorylated ERK (Thr202/Tyr204), total ERK, and α-tubulin (loading control). c , d Vehicle (DMSO)- or calyculin A-treated human HCC827 ( c ) and H1650 ( d ) cells were lysed with 1× RIPA buffer and subjected to immunoblotting using anti-phospho PP1α (Thr320), -total PP1α, -phospho ERK (Thr202/Tyr204), -total ERK, -DARPP-32, and -α-tubulin (loading control) antibodies. Chemiluminescence signals were detected after incubating membranes with HRP-tagged secondary antibodies. Representative images from one experiment are shown, but results were validated by performing three independent biological repeats. Bar graphs at the right show quantification of the results from the three western blotting experiments. Error bars indicate SEM. * P < 0.05; ns not significant.

Article Snippet: Primary antibodies (1 μg/μl) identifying two different phosphorylated sites on DARPP-32 (T34: cat no. 12438; dilution 1:1000; and T75: cat no. 2301; dilution 1:1000), phosphorylated PP1α (T320; cat no. 2581; dilution 1:1000), IKKα (cat no. 2682; dilution 1:1000), phosphorylated p44/42 MAPK (T202/Y204; cat no. 4370; dilution 1:1000), and total p44/42 MAPK (cat no. 4695; dilution 1:1000) were purchased from CST.

Techniques: Transfection, Control, SDS Page, Incubation, Western Blot

Journal: NPJ Precision Oncology

Article Title: IKKα promotes lung adenocarcinoma growth through ERK signaling activation via DARPP-32-mediated inhibition of PP1 activity

doi: 10.1038/s41698-023-00370-3

Figure Lengend Snippet: a Representative images of HCC827, PC9, and H1650 cells transduced with lentivirus encoding either LacZ shRNA or IKKα shRNAs forming colonies on soft-agar cell culture dishes 1 to 2 weeks after plating. b Human NSCLC HCC827, PC9, and H1650 cells transduced with lentivirus designed to silence LacZ (control) or IKKα protein expression were subjected to soft-agar colony formation assays to determine anchorage-independent cell growth. ImageJ was used to count colonies on the cell culture dishes after 1 to 2 weeks of incubation, and the number of counted colonies was plotted. Each circle on a graph represents an independent experiment. Soft-agar colony formation experiments were repeated at least six times. Error bars indicate SEM ( n = 6). Scale bar 200 µm. A value of P ≤ 0.05 was considered significant, one-way ANOVA followed by Dunnett’s test. c , d Lysates from HCC827 ( c ) and PC9 ( d ) cells transduced with either LacZ shRNA or IKKα shRNA were subjected to immunoblotting with primary antibodies against IKKα, phosphorylated DARPP-32 (Thr-34), total DARPP-32, phosphorylated PP1α (Thr320), total PP1α, phosphorylated ERK (Thr202/Tyr204), total ERK, and α-tubulin (loading control). Bar graphs at the right show values obtained from the densitometric quantification of the results from three western blotting experiments. * P < 0.05; Two-tailed unpaired t -test.

Article Snippet: Primary antibodies (1 μg/μl) identifying two different phosphorylated sites on DARPP-32 (T34: cat no. 12438; dilution 1:1000; and T75: cat no. 2301; dilution 1:1000), phosphorylated PP1α (T320; cat no. 2581; dilution 1:1000), IKKα (cat no. 2682; dilution 1:1000), phosphorylated p44/42 MAPK (T202/Y204; cat no. 4370; dilution 1:1000), and total p44/42 MAPK (cat no. 4695; dilution 1:1000) were purchased from CST.

Techniques: Transduction, shRNA, Cell Culture, Control, Expressing, Incubation, Western Blot, Two Tailed Test

Journal: NPJ Precision Oncology

Article Title: IKKα promotes lung adenocarcinoma growth through ERK signaling activation via DARPP-32-mediated inhibition of PP1 activity

doi: 10.1038/s41698-023-00370-3

Figure Lengend Snippet: a Human lung cancer HCC827 cells treated with calyculin A (50 nM) for the indicated times were lysed using 1× RIPA buffer supplemented with protease and phosphatase inhibitors. An equal amount of protein was separated with 4–20% SDS-PAGE and transferred to polyvinyl difluoride membranes. Antibody-reactive protein bands were visualized after overnight incubation of primary antibodies against phosphorylated PP1α (Thr320), total PP1α, PARP-I, phosphorylated ERK (Thr202/Tyr204), total ERK, and α-tubulin (loading control). b Human NSCLC HCC827, PC9, and H1650 cells were incubated with vehicle (DMSO) or calyculin A (50 nM) for 15 min prior to plating. Representative images indicate cell colonies grown on the soft-agar cell culture dishes after 2 weeks of incubation. Scale bar 200 µm. c Bar graphs show the average count of cell colonies observed after 2 weeks of incubation. Bar graphs indicate mean ± SEM ( n = 9). A value of P ≤ 0.05 was considered significant, two-tailed unpaired t -test.

Article Snippet: Primary antibodies (1 μg/μl) identifying two different phosphorylated sites on DARPP-32 (T34: cat no. 12438; dilution 1:1000; and T75: cat no. 2301; dilution 1:1000), phosphorylated PP1α (T320; cat no. 2581; dilution 1:1000), IKKα (cat no. 2682; dilution 1:1000), phosphorylated p44/42 MAPK (T202/Y204; cat no. 4370; dilution 1:1000), and total p44/42 MAPK (cat no. 4695; dilution 1:1000) were purchased from CST.

Techniques: SDS Page, Incubation, Control, Cell Culture, Two Tailed Test

Journal: NPJ Precision Oncology

Article Title: IKKα promotes lung adenocarcinoma growth through ERK signaling activation via DARPP-32-mediated inhibition of PP1 activity

doi: 10.1038/s41698-023-00370-3

Figure Lengend Snippet: a Luciferase-labeled IKKα-depleted human HCC827 cells were orthotopically injected into the left thorax of SCID mice and imaged for luminescence on the indicated days. Total luminescence intensity (photon count) was calculated using molecular imaging software and plotted as a line graph. Error bars are shown as dotted lines indicating SEM. A value of P ≤ 0.05 was considered significant, two-way ANOVA followed by Sidak’s test. b Images of anesthetized mice were captured to detect luminescence signals on the indicated days. c Tumor tissue lysates obtained from either LacZ shRNA- or IKKα shRNA-transduced human HCC827 cells-derived xenografts were subjected to immunoblotting using primary antibodies against IKKα, phosphorylated PP1α (Thr320), total PP1α, phosphorylated ERK (Thr202/Tyr204), total ERK, and α-tubulin (loading control). Bar graphs show densitometric quantification values of the results from three western blotting experiments. * P < 0.05; Two-tailed unpaired t -test. d Overall morphological evaluation was performed on formalin‐fixed, paraffin‐embedded lung tissues ( n = 5 mice per group) obtained from human HCC827 cells-derived lung tumor xenograft mice model using hematoxylin and eosin (H&E) dye. Scale bar 50 µm.

Article Snippet: Primary antibodies (1 μg/μl) identifying two different phosphorylated sites on DARPP-32 (T34: cat no. 12438; dilution 1:1000; and T75: cat no. 2301; dilution 1:1000), phosphorylated PP1α (T320; cat no. 2581; dilution 1:1000), IKKα (cat no. 2682; dilution 1:1000), phosphorylated p44/42 MAPK (T202/Y204; cat no. 4370; dilution 1:1000), and total p44/42 MAPK (cat no. 4695; dilution 1:1000) were purchased from CST.

Techniques: Luciferase, Labeling, Injection, Imaging, Software, shRNA, Derivative Assay, Western Blot, Control, Two Tailed Test, Formalin-fixed Paraffin-Embedded

Journal: PLoS ONE

Article Title: Adaptive Gene Regulation in the Striatum of RGS9-Deficient Mice

doi: 10.1371/journal.pone.0092605

Figure Lengend Snippet: ( a ) Total DARPP32 from striatal homogenates of 3-month-old wt and RGS9-deficient mice was measured by Western blot quantification and normalized to actin. ( b ) DARPP32 phosphorylation at Thr34 and Thr75 is given relative to total DARPP32. Representative Western blots are shown, quantification was performed with n = 6–8 per genotype.

Article Snippet: The primary antibodies were: anti-RGS9 (Santa Cruz Biotechnology Inc., Dallas, USA), anti-phosphoDARPP32 (Thr34) and (Thr75) (Cell Signaling, Boston, USA), anti-DARPP32 (Cell Signaling, Boston, USA), anti-phospho-p44/42 MAPK (Cell Signaling, Boston, USA), anti-MAPK (Zymed Laboratories Inc., San Francisco, USA), anti-CaMKIIβ (ProteinTech Group Inc., Chicago, USA), anti-phospho-Thr286 CaMKII (PhosphoSolutions, Aurora, USA), anti-CaMKIIγ (Santa Cruz Biotechnology Inc., Dallas, USA), anti-GluR2 (Santa Cruz Biotechnology Inc., Dallas, USA) and anti-actin (Molecular Probes, Eugene, USA).

Techniques: Western Blot